Category Archives: Genome

Researchers have assembled the genome and 3D structures of a 52,000-year-old woolly mammoth. It is the first time cell-specific gene activity has been measured in ancient DNA.

The researchers say they discovered an entirely new type of fossil which has superbly preserved ancient remains which led to the first ever 3D assembly of a chromosome.

This was possible because the mammoths remains freeze-dried shortly after it died. Its DNA was preserved in a glass-like state.

The woolly mammoth was discovered in Siberia in 2018. The research is published in the journal Cell.

This is a new type of fossil, and its scale dwarfs that of individual ancient DNA fragmentsa million times more sequence, says corresponding author Erez Lieberman Aiden, Director of the Center for Genome Architecture at Baylor College of Medicine.

Being able to reconstruct the fossilised chromosomes provides insight into how the mammoths genome was organised in its living cells and which genes were active in the skin tissue.

Using modern elephants as a baseline, the scientists were able to put the giant DNA puzzle together. It led them to create the first ever ancient karyotype an individuals complete set of chromosomes. It revealed woolly mammoths have the same number of chromosomes as todays African and Asian elephants 28.

The mammoths genomic structure was reconstructed using DNA extracted from the skin on the animals ear.

A method called Hi-C was used to detect sections of DNA likely to be in close proximity to and interact with each other.

Imagine you have a puzzle that has 3 billion pieces, but you dont have the picture of the final puzzle to work from, says corresponding author Marc A. Marti-Renom, a Catalan Institution for Research and Advanced Studies research professor. Hi-C allows you to have an approximation of that picture before you start putting the puzzle pieces together.

Bringing the DNA together allowed the researchers to see which genes were active and which were turned off in the skin cells.

The mammoths skin showed unique gene activation patterns compared to its closest living relative, the Asian elephant. It is possible these genes control its woolly-ness and cold tolerance.

These results have obvious consequences for contemporary efforts aimed at woolly mammoth de-extinction, says corresponding author M. Thomas Gilbert from the University of Copenhagen and the Norwegian University of Science and Technology.

The method used could also be applied to other ancient DNA specimens including other mammoths and mummified people from Egypt and other parts of the world.

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First 3D assembly of woolly mammoth chromosome thanks to freeze-dried skin - Cosmos

A team of researchers has used cut-and-paste mobile genetic elements (MGEs) from the insert sequence (IS)110 family and clues from noncoding (nc)RNA to determine that large-scale genome design could be a possibility through a new class of guide RNAs.

The potential breakthrough came from asking whether ncRNA might assist recombinase in recognizing the target DNA site or the donor DNA (that is, the IS110 element itself), according to Drs. Matthew Durrant and Nicholas Perry of the Arc Institute in Palo Alto, CA. Together, Durrant, a computational biologist and senior scientist at Arc, and Perry, a PhD graduate student at the University of California (UC), Berkeley, led the experimental study with Dr. Patrick Hsu at Arc's Patrick Hsu lab.

Aided by cryo-electron microscopy analysis and nanopore sequencing, the study used Escherichia coli (E. coli) for its large, circular molecule of DNA chromosome and small, circular molecule plasmids.

In June, researchers confirmed a mechanism for a programmable target loop that allows the user to specify any desired genomic target sequence and any donor DNA molecule to be inserted. The development, detailed in the journal Nature, could eventually lead to a new genome editing method that sidesteps CRISPR DNA-cutting techniques, according to Arc.

The key, researchers discovered, lies in a new class of guide RNA, called "bridge RNA," that connects target and donor DNA and enables recombination by the IS621 recombinase. IS621, which resides in the IS110 family and is native to some strains of E. coli, as well as five closely related orthologues, was a central focus of this research, according to Durrant and colleagues.

"The bridge RNA system is a fundamentally new mechanism for genome design," said Hsu, senior author of the study and an Arc Institute core investigator and UC Berkeley assistant professor of bioengineering, in "Genomes by Design," an Arc blog post. "Bridge recombination can universally modify genetic material through sequence-specific insertion, excision, inversion, and more, enabling a word processor for the living genome beyond CRISPR."

Arc describes the discovery as a compact and entirely new type of programmable molecular system.

First, the team constructed a custom sequence database of bacterial isolate and metagenomic sequences by aggregating publicly available sequence databases.

As explained in Nature, the work investigated the potential presence of an IS110-encoded ncRNA by focusing on IS621. Researchers also evaluated the ncRNA consensus secondary structure across 103 diverse orthologues.

Durrant and colleagues found that ncRNA is necessary for in vitro recombination, and that the four components (ncRNA, recombinase, target DNA, and donor DNA) are sufficient to produce the expected recombination product. In addition, the base-pairing mechanism of target and donor recognition by the bridge RNA suggested programmability.

To assess programmability, the team designed an E. coli selection screen linking thousands of barcoded pairs of DNA targets and bridge RNAs on a single plasmid. This step helped to assess mismatch tolerance and reprogramming rules of bridge RNAs. They reprogrammed bridge RNAs to target sequences found only once in the E. coli genome.

"Altogether, these experiments provide evidence of the robust capability of IS621 to specifically insert multi-kilobase cargos into the genome, and offer further insights into the mechanisms of recombination," Durrant and colleagues wrote.

"The system can go far beyond its natural role that inserts the IS110 element itself, instead enabling insertion of any desirable genetic cargo like a functional copy of a faulty, disease-causing gene into any genomic location," Arc explained, adding that Hsu and colleagues demonstrated over 60% insertion efficiency of a desired gene in E. coli with over 94% specificity for the correct genomic location.

According to Arc, the Hsu lab found that when IS110 excises itself from a genome, the non-coding DNA ends are joined together to produce an RNA molecule the bridge RNA that folds into two loops. One loop binds to the IS110 element itself, while the other loop binds to the target DNA where the element will be inserted.

"We demonstrate that the target-binding and donor-binding loops can be independently reprogrammed to direct sequence-specific recombination between two DNA molecules," the researchers explained in Nature. "The bridge RNA that we discovered in this work is the first example, to our knowledge, of a bispecific guide molecule that encodes modular regions of specificity for both the target and the donor DNA, coordinating these two DNA sequences in close proximity to catalyse efficient recombination."

Arc Institute operates in collaboration with Stanford University, UC Berkeley, and the University of California, San Francisco, according to information on the institute's website. The bridge RNA study included collaborators Hiroshi Nishimasu and Masahiro Hiraizumi at the University of Tokyo.

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Researchers discover new class of guide RNA for genome editing - LabPulse

Crowdfund Capital Advisors (CCA) has distributed its new Crowdfunding Genome, an ecosystem report and visualization of the crowdfunding industry. The report includes the hot spots in the US, and the Phoenix/Scottsdale region is in the top spot, beating out the traditional startup ecosystem of the San Francisco Bay area.

The Arizona community earned the leading position due to metrics such asgrowth in valuation, repeat issuers, robust investment climate, supportive community, and innovative spirit.

CCA explains that entrepreneurs in Phoenix/Scottsdale have successfully leveraged crowdfunding (Reg CF) to fund their ventures, which the firm believes makes it a model for other regions to emulate.

Calling Phoenix/Scottsdale the top startup ecosystem, Sherwood Neiss, Principal at CCA, believes the regions success is a testament to its vibrant entrepreneurial community.

It is another proof point that startups need not be located in Silicon Valley to prosper, said Neiss.

Neiss told CI that Phoenix/Scottsdales continuous use of investment crowdfunding is a key factor in its rise to the top of the list.

Local issuers dont just use it for one or two rounds; they see it as an ongoing tool for capital formation. Startups in the region are adept at raising capital, achieving their goals such as increasing revenues and hitting key milestones, and then returning for follow-on capital at higher valuations. This strategic approach, combined with Phoenix/Scottsdales leading number of deals per population, has made it a standout ecosystem for pre-IPO startups.

Recent challenges in San Francisco have led to the exit of both established and early-stage firms, and Arizona has emerged as one of the beneficiaries of this exodus. Apparently, these entrepreneurs are also tapping into online capital formation to fund their firms.

At this same time, California is no slouch and continues to reign as the top state when it comes to innovation and access to capital.

Neiss says the states ecosystem enables unparalleled opportunities for entrepreneurs to scale and succeed.

It is exciting to see the broad application of investment crowdfunding for California entrepreneurs.

Other trends gleaned from the data include a biotech boom as more ventures in the biotechnology sector raise funds. There is also a sustainability surge. Tech, including Fintech and AI, continues to be a popular sector of investment crowdfunding.

Neiss said the Crowdfunding Genome helps provide a data-driven understanding of the diverse startup ecosystem in the US.

Traditional metrics often overlook the unique dynamics of crowdfunding and its impact on early-stage ventures. By leveraging our proprietary investment crowdfunding data and advanced analytics, our goal is to offer valuable insights that empower entrepreneurs, investors, and policymakers to make informed decisions and continue to foster innovation.

The report is available on the CCA website.

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Crowdfund Capital Advisors Reveals "Crowdfunding Genome" - Phoenix/Scottsdale Top Hub, Beats Bay Area - Crowdfund Insider

Reuters file photo Corn plants affected by leafhoppers are seen in a National Institute of Agricultural Technology experimental field, in Marcos Juarez, Cordoba, Argentina on April 20.

Reuters

16:36 JST,July 11, 2024

BUENOS AIRES (Reuters) An Argentine scientific institute has cracked the genome of the leafhopper, the insect which carries the bacteria responsible for wiping out vast swathes of the South American nations latest corn crop, the government said on June 25.

The development, which determined the Dalbulus maidis genetic makeup, will serve future efforts to fight off the leafhopper, according to the government statement.

Experts argue that the leafhopper population has surged in recent months largely due to the lack of frosts during last years Southern Hemisphere winter, which likely would have killed off the insect.

The tiny bug, which sucks sap out of plants, transmits bacteria that produce stunt disease in corn, causing the key grains crop to grow ears with loose or missing kernels.

In the 2023-24 season, the Rosario Grains Exchange expects local farmers to harvest 47.5 million metric tons of corn, about a fifth less than originally estimated due to losses caused by the leafhopper.

This research will help us understand the biology and evolution of the insect, which in turn will help predict and mitigate future outbreaks, the statement said, adding that the scientific advance could also lead to the development of new varieties of leafhopper-resistant genetically modified corn.

Agricultural analysts have said that farmers will likely plant smaller corn fields in the 2024-25 season due to the pest, although fall and winter frosts should improve prospects for the crop.

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Argentina Cracks Genome of Leafhopper to Defend Crop - The Japan News

Laurenti, E. & Gottgens, B. From haematopoietic stem cells to complex differentiation landscapes. Nature 553, 418426 (2018).

Article CAS PubMed PubMed Central Google Scholar

Bauer, T. R. Jr. et al. Correction of the disease phenotype in canine leukocyte adhesion deficiency using ex vivo hematopoietic stem cell gene therapy. Blood 108, 33133320 (2006).

Article CAS PubMed PubMed Central Google Scholar

Blaese, R. M. et al. T lymphocyte-directed gene therapy for ADA-SCID: initial trial results after 4years. Science 270, 475480 (1995).

Article CAS PubMed Google Scholar

Boztug, K. et al. Stem-cell gene therapy for the Wiskott-Aldrich syndrome. N. Engl. J. Med. 363, 19181927 (2010).

Article CAS PubMed PubMed Central Google Scholar

Cowan, M. J. et al. Early outcome of a phase I/II clinical trial (NCT03538899) of gene-corrected autologous CD34+ hematopoietic cells and low-exposure busulfan in newly diagnosed patients with Artemis-deficient severe combined immunodeficiency (ART-SCID). Biol. Blood Marrow Transpl. 26, S88S89 (2020).

Article Google Scholar

Gaspar, H. B. et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 364, 21812187 (2004).

Article CAS PubMed Google Scholar

Kanter, J. et al. Biologic and clinical efficacy of LentiGlobin for sickle cell disease. N. Engl. J. Med. 386, 617628 (2022).

Article CAS PubMed Google Scholar

Kohn, L. A. & Kohn, D. B. Gene therapies for primary immune deficiencies. Front. Immunol. 12, 648951 (2021).

Article CAS PubMed PubMed Central Google Scholar

Kondo, M. et al. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu Rev. Immunol. 21, 759806 (2003).

Article CAS PubMed Google Scholar

Locatelli, F. et al. Betibeglogene autotemcel gene therapy for non-0/0 genotype -thalassemia. N. Engl. J. Med. 386, 415427 (2022).

Article CAS PubMed Google Scholar

Malech, H. L. et al. Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease. Proc. Natl Acad. Sci. USA 94, 1213312138 (1997).

Article CAS PubMed PubMed Central Google Scholar

Morgan, R. A., Gray, D., Lomova, A. & Kohn, D. B. Hematopoietic stem cell gene therapy: progress and lessons learned. Cell Stem Cell 21, 574590 (2017).

Article CAS PubMed PubMed Central Google Scholar

Sago, C. D. et al. Nanoparticles that deliver RNA to bone marrow identified by in vivo directed evolution. J. Am. Chem. Soc. 140, 1709517105 (2018).

Article CAS PubMed PubMed Central Google Scholar

Shi, D., Toyonaga, S. & Anderson, D. G. In vivo RNA delivery to hematopoietic stem and progenitor cells via targeted lipid nanoparticles. Nano Lett. 23, 29382944 (2023).

Article CAS PubMed PubMed Central Google Scholar

Sou, K., Goins, B., Oyajobi, B. O., Travi, B. L. & Phillips, W. T. Bone marrow-targeted liposomal carriers. Expert Opin. Drug Deliv. 8, 317328 (2011).

Article CAS PubMed PubMed Central Google Scholar

Sou, K., Klipper, R., Goins, B., Tsuchida, E. & Phillips, W. T. Circulation kinetics and organ distribution of Hb-vesicles developed as a red blood cell substitute. J. Pharmacol. Exp. Ther. 312, 702709 (2005).

Article CAS PubMed Google Scholar

Xue, L. et al. Rational design of bisphosphonate lipid-like materials for mRNA delivery to the bone microenvironment. J. Am. Chem. Soc. 144, 99269937 (2022).

Article CAS PubMed Google Scholar

Boulais, P. E. & Frenette, P. S. Making sense of hematopoietic stem cell niches. Blood 125, 26212629 (2015).

Article CAS PubMed PubMed Central Google Scholar

Ikonomi, N., Kuhlwein, S. D., Schwab, J. D. & Kestler, H. A. Awakening the HSC: dynamic modeling of HSC maintenance unravels regulation of the TP53 pathway and quiescence. Front. Physiol. 11, 848 (2020).

Article PubMed PubMed Central Google Scholar

Li, J. Quiescence regulators for hematopoietic stem cell. Exp. Hematol. 39, 511520 (2011).

Article PubMed Google Scholar

Man, Y., Yao, X., Yang, T. & Wang, Y. Hematopoietic stem cell niche during homeostasis, malignancy, and bone marrow transplantation. Front. Cell Dev. Biol. 9, 621214 (2021).

Article PubMed PubMed Central Google Scholar

Nakamura-Ishizu, A., Takizawa, H. & Suda, T. The analysis, roles and regulation of quiescence in hematopoietic stem cells. Development 141, 46564666 (2014).

Article CAS PubMed Google Scholar

Eppert, K. et al. Stem cell gene expression programs influence clinical outcome in human leukemia. Nat. Med. 17, 10861093 (2011).

Article CAS PubMed Google Scholar

Lapidot, T. et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367, 645648 (1994).

Article CAS PubMed Google Scholar

Mandal, T., Beck, M., Kirsten, N., Linden, M. & Buske, C. Targeting murine leukemic stem cells by antibody functionalized mesoporous silica nanoparticles. Sci. Rep. 8, 989 (2018).

Article PubMed PubMed Central Google Scholar

Pei, S. & Jordan, C. T. How close are we to targeting the leukemia stem cell? Best Pract. Res. Clin. Haematol. 25, 415418 (2012).

Article CAS PubMed Google Scholar

Li, C. et al. Prophylactic in vivo hematopoietic stem cell gene therapy with an immune checkpoint inhibitor reverses tumor growth in syngeneic mouse tumor models. Cancer Res. 80, 549560 (2020).

Article CAS PubMed Google Scholar

Li, C. et al. In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal globin in beta-YAC mice. Blood Adv. 5, 11221135 (2021).

Article CAS PubMed PubMed Central Google Scholar

Li, C. et al. In vivo HSC gene therapy using a bi-modular HDAd5/35++ vector cures sickle cell disease in a mouse model. Mol. Ther. 29, 822837 (2021).

Article CAS PubMed Google Scholar

Li, C. et al. Safe and efficient in vivo hematopoietic stem cell transduction in nonhuman primates using HDAd5/35++ vectors. Mol. Ther. Methods Clin. Dev. 24, 127141 (2022).

Article PubMed Google Scholar

Psatha, N. et al. Enhanced HbF reactivation by multiplex mutagenesis of thalassemic CD34+ cells in vitro and in vivo. Blood 138, 15401553 (2021).

Article CAS PubMed PubMed Central Google Scholar

Muruve, D. A., Barnes, M. J., Stillman, I. E. & Libermann, T. A. Adenoviral gene therapy leads to rapid induction of multiple chemokines and acute neutrophil-dependent hepatic injury in vivo. Hum. Gene Ther. 10, 965976 (1999).

Article CAS PubMed Google Scholar

Sweeney, C. L. & De Ravin, S. S. The promise of in vivo HSC prime editing. Blood 141, 20392040 (2023).

Article CAS PubMed Google Scholar

Worgall, S., Wolff, G., Falck-Pedersen, E. & Crystal, R. G. Innate immune mechanisms dominate elimination of adenoviral vectors following in vivo administration. Hum. Gene Ther. 8, 3744 (1997).

Article CAS PubMed Google Scholar

Lek, A. et al. Death after high-dose rAAV9 gene therapy in a patient with Duchennes muscular dystrophy. N. Engl. J. Med. 389, 12031210 (2023).

Article CAS PubMed Google Scholar

Hou, X., Zaks, T., Langer, R. & Dong, Y. Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 6, 10781094 (2021).

Article CAS PubMed PubMed Central Google Scholar

Cheng, Q. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat. Nanotechnol. 15, 313320 (2020).

Article CAS PubMed PubMed Central Google Scholar

Dilliard, S. A., Cheng, Q. & Siegwart, D. J. On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles. Proc. Natl Acad. Sci. USA 118, e2109256118 (2021).

Article CAS PubMed PubMed Central Google Scholar

Dilliard, S. A. & Siegwart, D. J. Passive, active and endogenous organ-targeted lipid and polymer nanoparticles for delivery of genetic drugs. Nat. Rev. Mater. 8, 282300 (2023).

Article CAS PubMed PubMed Central Google Scholar

Farbiak, L. et al. All-in-one dendrimer-based lipid nanoparticles enable precise HDR-mediated gene editing in vivo. Adv. Mater. 33, e2006619 (2021).

Article PubMed PubMed Central Google Scholar

Liu, S. et al. Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing. Nat. Mater. 20, 701710 (2021).

Article CAS PubMed PubMed Central Google Scholar

Liu, S. et al. Zwitterionic phospholipidation of cationic polymers facilitates systemic mRNA delivery to spleen and lymph nodes. J. Am. Chem. Soc. 143, 2132121330 (2021).

Article CAS PubMed PubMed Central Google Scholar

Wang, X. et al. Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery. Nat. Protoc. 18, 265291 (2023).

Article CAS PubMed Google Scholar

Wei, T., Cheng, Q., Min, Y. L., Olson, E. N. & Siegwart, D. J. Systemic nanoparticle delivery of CRISPR-Cas9 ribonucleoproteins for effective tissue specific genome editing. Nat. Commun. 11, 3232 (2020).

Article CAS PubMed PubMed Central Google Scholar

Zhang, D. et al. Enhancing CRISPR/Cas gene editing through modulating cellular mechanical properties for cancer therapy. Nat. Nanotechnol. 17, 777787 (2022).

Article CAS PubMed PubMed Central Google Scholar

Wu, L. C. et al. Correction of sickle cell disease by homologous recombination in embryonic stem cells. Blood 108, 11831188 (2006).

Article CAS PubMed PubMed Central Google Scholar

Metais, J. Y. et al. Genome editing of HBG1 and HBG2 to induce fetal hemoglobin. Blood Adv. 3, 33793392 (2019).

Article PubMed PubMed Central Google Scholar

Newby, G. A. et al. Base editing of haematopoietic stem cells rescues sickle cell disease in mice. Nature 595, 295302 (2021).

Article CAS PubMed PubMed Central Google Scholar

Stavropoulou, V., Peters, A. & Schwaller, J. Aggressive leukemia driven by MLL-AF9. Mol. Cell Oncol. 5, e1241854 (2018).

Article PubMed Google Scholar

Hou, X. et al. Vitamin lipid nanoparticles enable adoptive macrophage transfer for the treatment of multidrug-resistant bacterial sepsis. Nat. Nanotechnol. 15, 4146 (2020).

Article CAS PubMed PubMed Central Google Scholar

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Bone-marrow-homing lipid nanoparticles for genome editing in diseased and malignant haematopoietic stem cells - Nature.com

Human endogenous retroviruses are remnants of viral genes in the human genome

NICOLLE R. FULLER/SCIENCE PHOTO LIBRARY

Ancient viruses embedded in human DNA millions of years ago may play a role in raising peoples risks of depression, schizophrenia and bipolar disorder. The viral genes have unusual activity levels in people who have a higher genetic risk of experiencing these mental health conditions, a study has found.

Retroviruses are a large group of viruses whose life cycle involves inserting their genetic information into the DNA of their host. Remnants of such viral genes can be seen in the DNA of many animal species, and these sequences are called human endogenous retroviruses (HERVs) when found in the human genome.

Until recently, most HERVs were thought to remain dormant and were called fossil viruses. But previous studies have suggested that some of the viral genes may in fact be active although not producing infectious viruses and may even play a role in neurological conditions, such as multiple sclerosis.

In a new study, Timothy Powell at Kings College London and his colleagues investigated the possible role of these viral remnants in mental health conditions by looking at viral proteins in nearly 800 brains that had been donated for medical research.

While the team didnt record if any of the donors had been diagnosed with mental health conditions while alive, they found that some genetic variants that are known to raise the risks of depression, schizophrenia and bipolar disorder were associated with altered activity of genes from five different HERVs.

It wasnt possible to estimate how much of a raised risk was linked with the unusual viral gene activity, but it is probably in the range of a few per cent, as most human gene variants affect psychiatric risk by such small amounts, says Powell.

The findings also dont necessarily mean that the altered HERV activity is causing the conditions it could be a consequence, he says.

The methods used look pretty robust, says Rachael Tarlinton at the University of Nottingham in the UK. The results are likely real that these things are markers of these diseases, but [the researchers] are rightly cautious in saying they dont know what that means.

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Ancient viruses in the human genome linked to mental health conditions - New Scientist

Hundreds of genetic variants can nudge someones risk of breast cancer up or down or towards a particular subtype. The studies identifying those gene variants, though, have largely involved people with European ancestry and thus give a less accurate picture of breast cancer risk for people who are not white.

Thats beginning to change. Last week, researchers published a genome-wide association study on breast cancer in roughly 40,000 people of African descent in Nature Genetics, marking a leap forward in scientists knowledge of breast cancer genetics in people of African ancestry.

Before we started this study in 2016, there were just several thousand cases for Black Americans. It was a very small number, said Wei Zheng, the studys senior investigator and a cancer epidemiologist at Vanderbilt University. This study combined data from dozens of other studies and included genetic data for thousands of new participants, making it the largest combined breast cancer genetics study done with people with African ancestry.

Specifically, the study compiled data from about 30 different studies investigating breast cancer in African or African American people. About 18,000 of them had breast cancer, while the other 22,000 were healthy controls, and investigators were able to scour their genetic data for specific variations that seemed closely related to breast cancer. The statistical power that comes with such numbers enabled the team to make two key advances.

First, the team found 12 loci, or locations in the genome, that showed a significant association with breast cancer. Of those, the team identified variants of three genes that appear to increase the risk of triple negative breast cancer, one of the most aggressive subtypes. Since everyone has two copies or alleles of each gene, that means someone could have anywhere between one and six risk-related alleles of these three genes. Those who had all six risk-related alleles had roughly double the chance of getting triple negative breast cancer than those who only had three.

That could provide a foothold for scientists to begin predicting who might get this aggressive form of breast cancer, and it might offer an opportunity to better understand the biology of triple negative breast cancer by highlighting genes that seem to be important. Finally, we have enough data to drill down to estrogen negative and triple negative breast cancer, which are twice as common in the African American population as any other population, said Julie Palmer, an author of the study and a cancer researcher at Boston University.

The other advance came when the researchers used the data to build a breast cancer risk prediction model for people with African ancestry. Such models take into account hundreds of different genetic variants that can slightly push breast cancer risk up, adding them all up into a polygenic risk score.

In the past, these scores always performed better for white people than Black people, mainly because theres so much more research done in people with European ancestry a combined total of more than 100,000 participants for breast cancer. Polygenic risk scores have had an AUC, a measure of the models performance, of about 0.63 for people with European ancestry compared to 0.58 for the African ancestry population. When researchers combined the data from this study into their new model, however, that figure rose to 0.60. That equates to the model being able to correctly distinguish between someone whos likely to get breast cancer and someone who isnt about 60% of the time.

Even if this work is validated in other studies, as it still needs to be, that figure is not too useful for most individuals. An astute observer might note an AUC of 0.63 is only passably better than a coin toss. Thats an indication polygenic risk scores dont perform as well overall as scientists would like even at their best. When polygenic risk scores are combined with other breast cancer risk factors, like age of first childbirth or breast density, were still not very good at predicting breast cancer, Palmer said.

But research is continually improving on that. The hope is, one day, these scores will help scientists build tools that can reliably distinguish people who are more likely to get breast cancer and thus might have more to gain by beginning screening earlier or more frequently. Or, conversely, they could help weed out people who arent likely to get breast cancer and could thus screen less. If you dont need it, then why do it? said Laura Fejerman, a cancer researcher and epidemiologist at the University of California, Davis.

Polygenic risk scores might already be able to provide some of that context for a small minority of people, Fejerman added. For the 1% of people with the highest polygenic risk, their lifetime risk was a little bit above 30%, Fejerman said. That could be an argument for them to screen more often, even if they had no other risk factors. If you learn that, you might be more on top of your screening than most people who maybe let three years pass. So, if you could get the highest-risk women to screen every year, that would be a big benefit.

Without datasets in non-European ancestry populations, other racial demographic groups could be left out of that progress. In that sense, this new paper is definitely a big step forward for achieving racial equity, said Swati Biswas, a statistician and cancer researcher at the University of Texas at Dallas who did not work on the study.

In particular, the data are needed if scientists ever hope to create a unified polygenic risk score that works for everyone. At the moment, many models rely on racial categorization Black people use an African ancestry model; white people use a European model. But using such models in clinical practice isnt optimal, pointed out Jennifer James, a sociologist who studies breast cancer and bioethics at the University of California, San Francisco.

Imagine someone whose ancestry is 5% African and 95% European, but who also happened to inherit breast cancer risk alleles that were only found in the African ancestry population. That would mean the African ancestry polygenic risk model might work better for them, even if they didnt identify as Black themselves. You could be 1% Black, but the one thing you got was that allele, James said. We need to move towards a unified polygenic risk score.

That still wont be enough to end the breast cancer mortality gap between Black and white people, even if scientists created a perfectly accurate polygenic risk model, James added. Thats because part of the reason for the disparity has to do with the health care system writ large, not subtle biological differences across populations.

We know Black women have a longer time to diagnosis, longer time from diagnosis to treatment, James said. If everyone had equal access to healthcare, that would do more to close gaps in mortality than tweaking prediction models. I want when someone finds a lump in their breast or needs a mammogram, they have equal access to care.

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Study finds gene variants tied to breast cancer risk in Black women - STAT

Numerical simulations capture experimentally observed features of chromatin organization

We have developed a mathematical model to capture dynamic chromatin organization in the nucleus, in terms of its compaction into the heterochromatic phase or decompaction into the euchromatic phase (Fig.1a). We treat the meso-scale genomic organization as a dynamic, far-from-equilibrium process, governed by the energetics of phase-separation in conjunction with the kinetics of epigenetic reactions and the formation of chromatin loops aided by supercoiled DNA extrusion through cohesin due to RNAPII-mediated transcription. The model ingredients are depicted schematically in Fig.1a. We begin by defining the energetics of the chromatin distribution in terms of the entropic-enthalpic balance of chromatin-chromatin interactions, the chromatin-lamina interactions as well as the penalty on the formation of phase boundaries via Eq. (6) (refer Methods, and Supplementary Section S1.2 in the SI). The gradients in the free-energy landscape, defined as the chemical potential (refer Supplementary Eq. (S3)), drive the dynamic evolution of chromatin towards the two energy wells corresponding to the euchromatin and heterochromatin phases via Eq. (7a, b) (refer Methods, Supplementary Section (S1.4) in the SI). Interconversion of the two phases of chromatin can occur via (a) epigenetic regulation of histone acetylation and methylation (Fig.1b), and (b) supercoiling-driven extrusion of chromatin loops from heterochromatin into euchromatin along the phase boundaries (Eq. (7b)) as shown in Fig.1c.

a Schematic of a portion of a nucleus showing the multiple mechanisms involved in chromatin organization such as chromatin-chromatin interactions, the chromatin-lamina interactions and epigenetic regulation. Additionally, extrusion of chromatin loops due to DNA supercoiling which is increased by transcriptional activity also plays a role in meso-scale genomic organization. While this may occur within either chromatin phases (red circle), we further explore the role of chromatin loop extrusion at the heterochromatin-euchromatin interface (black circle). b The model captures the chromatin-chromatin interaction energetics via a double well free energy description as shown in the contour plot. The two wells correspond to the heterochromatin (red circle) and euchromatin phases (blue circle). Any initial configuration (light blue circle) spontaneously decomposes into these wells at steady state. The dynamics of this transition are governed by diffusion and reaction kinetics comprising of epigenetic regulation and kinetics of supercoiling-driven chromatin extrusion (red box inset). c Loading of cohesin assisted by NIPBL/MAU2 initiates the formation of chromatin loops. Cohesin can also be dynamically unloaded via unloading factors viz. WAPL/PDS5. Active processes such as RNAPII mediated transcription further drive the extrusion of trapped DNA, supercoiling it into chromatin loops.

The process of phase separation is initiated by adding a random perturbation to the initially uniform chromatin configuration (as shown in Fig.2a, left panel) which captures the intrinsic intranuclear heterogeneities. As the simulation progresses heterochromatin domains (in red, center panel of Fig.2a) spontaneously nucleate and grow. The evolution ultimately stabilizes resulting in a steady state (right panel of Fig.2a) with a quasi-periodic distribution of stable domains of heterochromatin rich phase (({phi }_{h}={phi }_{h}^{max })) in red and euchromatin rich phase (left({phi }_{h}=0right)) in blue. Each of these domains are nearly circular (see Supplementary Section S2 of SI for a discussion on non-circular lamellar domains) with characteristic sizes. Concomitantly, heterochromatin domains localized to the nuclear lamina (called LADs) of comparable sizes appear in our simulations (Fig.2a).

a Visualization of the chromatin organization obtained from the simulations. The initial chromatin organization is a homogenous distribution with a small perturbation added, resulting in nucleation of heterochromatin domains (center panel) which grow into heterochromatin domains of characteristic sizes at a steady state. b Super-resolution visualizations of chromatin organization observed in-vivo via STORM imaging of HeLa nuclei (left panel, scale bar 3m, data previously reported in ref. 19, n=19 nuclei) and ChromSTEM imaging of BJ fibroblast nuclei (right panel, scale bar 1m, n=1 nucleus) show that chromatin organization in nucleus is characterized by interspersed heterochromatic domains of comparable sizes. c The smooth boundaries of the chromatin packing domains as seen in ChromSTEM observations are captured by the model. d Numerically predicted trend of sizes of heterochromatin domains as the transcription-mediated chromatin extrusion rate increases. e Schematic diagrams of the step-by step events (events i through vi) involved in the nucleation, growth and stabilization of heterochromatin domains at a steady state. f Plot of theoretically evaluated growth rate of heterochromatin domains with (red) and without (blue) reactions. Reactions give rise to a stable domain radius. In the absence of reactions, no stable heterochromatin domain length scales are observed. g The evaluation of stable radius (blue) and stable LAD thickness (red) as transcription mediated surface reactions are changed. Here, the relative radius is defined as the steady state radius relative to its value when transcription is zero, i.e., relative radius = ({widetilde{R}}_{d}^{{SS}}/{widetilde{R}}_{d}^{{SS}}|{Gamma }_{a}=0). The relative LAD thickness is similarly defined.

The meso-scale distribution of chromatin throughout the nucleus predicted by the mathematical model presents a striking qualitative similarity with the experimentally observed distribution of DNA in the nucleus using ChromSTEM, and STORM as reported previously19 (Fig.2b). Domains of compacted chromatin with a characteristic size are observed via a high histone density distinguished from regions of low histone density (Fig.2b). Lastly, the preferential accumulation of heterochromatin domains along the nuclear periphery seen via STORM imaging (Fig.2b), again with similar size scale, is also in excellent agreement with the experiments.

When defining the free energy density of chromatin organization in the nucleus (see Supplementary Eq. (S1) in SI), we penalized the formation of sharp interfaces via an interface penalty (eta), defined as the energy cost associated with the formation of the interfaces between heterochromatin and euchromatin phases. As we show in the SI (Supplementary Section S1.5), the energy penalty (eta) results in the formation of a smooth rather than a sharp interface between the heterochromatin and the euchromatin phases. Numerical simulations of chromatin organization exhibit such smooth interfaces around chromatin domains, as shown in the zoomed in image in Fig.2c (right panel). The width of the interface (delta) is controlled by the competition between the interfacial and bulk energy contributions (refer Supplementary Section S1.5).

Smooth chromatin phase boundaries are indeed observed in-vivo via Chrom-STEM imaging (Supplementary Section S1.11). We characterized the 3D chromatin density around individual heterochromatin domains in a BJ fibroblast nucleus using Chrom-STEM (Fig.2c, left panel; Supplementary Fig.S5). We estimated the average chromatin density within concentric circles emerging from the center of individual domains to the periphery (Fig.2c, Supplementary Fig.S5). The chromatin density was highest at the core of the domain and dropped slowly from the center of the domain to the periphery. The smooth decrease in radial density indicates that the chromatin domain boundaries are not abrupt (Fig.2c), in agreement with the numerical simulations.

We next investigate how the size scaling of the heterochromatin domains is regulated by the epigenetic reactions acetylation and methylation of histones and supercoiling-driven chromatin extrusion which together can lead to interconversion between heterochromatin and euchromatin. First, we see that in the absence of the epigenetic reactions and chromatin extrusion multiple domains of a characteristic size are not obtained as shown in Supplementary Fig.S10 (detailed discussion in Supplementary Section S5). In this case, although nucleation of multiple heterochromatin domains occurs even without reactions (Supplementary Fig.S10a), all of them merge into a single large cluster driven by Ostwald ripening so as to minimize the interface formation.

The model also predicts that the size of the heterochromatin domains in the interior and periphery can be regulated by the epigenetic reaction rates of acetylation and methylation as shown in Supplementary Fig.S6 (Supplementary Section S2). We see that as methylation increases the size of the interior domains increases too. On the other hand, increase in acetylation results in the formation of smaller heterochromatin domains. The trends followed by the domains towards the interior of the nucleus are replicated by the LADs as well. Lastly, we identify that the size scales of the domains the domain radii in the interior of the nucleus and the LAD thickness along its periphery depend on the level of transcription governed supercoiling-driven chromatin extrusion rate ({widetilde{Gamma }}_{a}) (Fig.2d, Supplementary Fig.S6). We note that, as the transcription (({widetilde{Gamma }}_{a})) is increased, the sizes of the heterochromatin domains decrease, both in the interior as well as at the periphery. At the same time, we also note that as chromatin extrusion rate is increased, the average volume fraction of heterochromatin (left({bar{phi }}_{h}right)) in the nucleus decreases, while that of euchromatin (left({bar{phi }}_{e}right)) increases.

Next, we theoretically predict an explicit dependence of the sizes of interior heterochromatic domains and LADs on epigenetic and transcription reactions and the diffusion kinetics of the epigenetic marks.

Intuitively, in the presence of more repressive methylation the overall heterochromatin content in the nucleus should increase, while in higher histone acetylation conditions the overall euchromatin content will increase. Thus, the epigenetic reactions can independently determine the average volume fractions of each form of chromatin, thereby breaking the detailed balance condition where the free energies of each phase determine their relative abundance in a thermodynamic equilibrium. A mathematical relation between the average volume fraction of each chromatin phase and the epigenetic reaction parameters can be determined by averaging the chromatin evolution equation (Eq. (7b)) at a steady state (i.e. (frac{partial {{{{{{rm{phi }}}}}}}_{{{{{{rm{d}}}}}}}}{partial widetilde{t}}=0)). In the absence of transcription driven chromatin extrusion (i.e. ({widetilde{Gamma }}_{a}=0)), we see that the epigenetic kinetics regulates the average heterochromatin content of the nucleus as, ({bar{phi }}_{h}approx frac{{widetilde{Gamma }}_{{me}},left(1-{bar{phi }}_{n}right)}{{widetilde{Gamma }}_{{me}}+1}) (Supplementary Eq. (S23), refer Supplementary Section S3 for more details).

The presence of transcription-mediated loop extrusion kinetics (i.e., ({widetilde{Gamma }}_{a}, ne , 0) in Eq. (7b)) further augments the deviation from thermodynamic equilibrium (i.e., the breaking of detail balance) via surface reactions that actively extrude DNA at the interface of heterochromatic domains. In the presence of transcription, the average heterochromatin (and euchromatin) content in the nucleus becomes (refer Supplementary Eq. (S22)),

$$begin{array}{c}{bar{phi }}_{h}, approx , frac{{widetilde{Gamma }}_{{me}},left(1-{bar{phi }}_{n}right)}{{widetilde{Gamma }}_{{me}}+1+kappa {widetilde{Gamma }}_{a}},,{bar{phi }}_{e}, approx , frac{left(1+kappa {widetilde{Gamma }}_{a}right),left(1-{bar{phi }}_{n}right)}{{widetilde{Gamma }}_{{me}}+1+kappa {widetilde{Gamma }}_{a}},end{array}$$

(1)

where (kappa) is a function of ({{{{{{rm{phi }}}}}}}_{{{{{{rm{h}}}}}}}^{max }), volume fraction change across the interface (Delta phi), and the length of the interface between the two chromatin phases (refer Supplementary Section S3 for derivation). Since supercoiling-mediated chromatin extrusion converts the tightly packed heterochromatin into low density transcriptionally active euchromatin phase, as extrusion rate ({widetilde{Gamma }}_{a}) increases, the average heterochromatin content decreases.

Thus, the overall mean chromatin composition of the nucleus (left({bar{phi }}_{h},{bar{phi }}_{e}right)) is determined by the reaction kinetics of epigenetic regulation along with transcription. The reaction kinetics alone would drive a homogenous chromatin organization with (left({bar{phi }}_{h},{bar{phi }}_{e}right)). On the (left({phi }_{d},{phi }_{n}right)) phase space we see that the average composition (shown as a light blue circle in Fig.1b) determined by reactions is energetically unfavorable it does not lie in the energy wells and hence must evolve in time.

Next, we show that the average composition of the two chromatin phases, shown in Fig.2e(i), plays a key role in the emergence of the characteristic sizes of the heterochromatin domains. To illustrate this, we first observe that the mean chromatin composition (left({bar{phi }}_{h},{bar{phi }}_{e}right)) lies in neither of the energy wells as shown in Fig.1b (light blue circle) and is thus energetically unfavorable. The need to reduce the total free energy in the nucleus drives the system to phase separate by nucleating heterochromatin domains (Fig.2e(iii)) corresponding to the red energy well labeled heterochromatin in Fig.1b surrounded by euchromatin domains corresponding to the dark blue energy well labeled euchromatin. The events entailing the individual steps in the nucleation and growth of a single droplet of heterochromatin due to phase separation, as shown in Fig.2e, are as follows:

Due to phase separation, the heterochromatin volume fraction immediately outside the droplet is ({phi }_{h}=0) corresponding to the euchromatic energy well. Far away from the droplet, the mean composition (left({bar{phi }}_{h},{bar{phi }}_{e}right)) remains undisturbed. The resulting spatial gradient in the chromatin composition (blue curve in Fig.2e(iv)) sets up a diffusive flux of heterochromatin into the droplet, allowing it to grow.

On the other hand, within the heterochromatin droplet (with ({phi }_{h}={phi }_{h}^{max })) histone acetylation reactions will allow conversion of heterochromatin inside the droplet into euchromatin outside. Active supercoiling-mediated chromatin loop extrusion further adds to the heterochromatin outflux. Together loop extrusion and acetylation oppose the diffusive influx of heterochromatin and thereby reduce the size of the droplet (Fig.2e).

Based on the above observations, the rate at which the nucleated heterochromatin droplet grows can be written in terms of the balance of reaction-diffusion gradient driven influx and acetylation and transcription driven outflux of heterochromatin as (refer Supplementary Section S4, Supplementary Eq. (S25)),

$$4pi {widetilde{R}}_{d}^{2}frac{d{widetilde{R}}_{d}}{dwidetilde{t}}=,underbrace{{4pi {widetilde{R}}_{d}{bar{phi }}_{h}}}_{{{{{{mathrm{inwards}}}}}} , {{{{{mathrm{diffusion}}}}}}} - underbrace{{frac{4}{3}pi {widetilde{R}}_{d}^{3}{phi }_{h}^{max }}}_{begin{array}{c}{{{{{mathrm{Acetylation}}}}}} , {{{{{mathrm{working}}}}}}\ {{{{{mathrm{against}}}}}} , {{{{{mathrm{inwards}}}}}}\ {{{{{mathrm{diffusion}}}}}}end{array}}-underbrace{{4pi {widetilde{R}}_{d}^{2}frac{delta }{2}{widetilde{Gamma }}_{a}{phi }_{h}^{max }}}_{begin{array}{c}{{{{{mathrm{Chromatin}}}}}} , {{{{{mathrm{extrusion}}}}}} , {{{{{mathrm{working}}}}}}\ {{{{{mathrm{against}}}}}} , {{{{{mathrm{inwards}}}}}}\ {{{{{mathrm{diffusion}}}}}}end{array}}$$

(2)

where (delta) is the rescaled width of the interface, which is in turn related to the length scale obtained via the competition between the interfacial energy and chromatin-chromatin interaction (refer Supplementary Section S1.5). The resulting evolution of the droplet growth rate (left(d{widetilde{R}}_{d}/dwidetilde{t}right)) as the radius of the droplet increases is shown in Fig.2e. Notice the two fixed points (Fig.2f, labeled critical and stable radius) where (d{widetilde{R}}_{d}/dwidetilde{t}=0). Beyond the critical radius the domains grow in size.

The second fixed point (stable radius) corresponds to the rescaled steady state (i.e., (d{widetilde{R}}_{d}/dwidetilde{t}=0)) heterochromatin domain size as determined by the active epigenetic and the transcriptional regulation in tandem with passive diffusion, and can be written as (derivation shown in Supplementary Section S4, Supplementary Eq. (S27)),

$$begin{array}{c}{widetilde{R}}_{d}^{{ss}}=-frac{3{widetilde{Gamma }}_{a}delta }{4}+sqrt{{left(frac{3{widetilde{Gamma }}_{a}delta }{4}right)}^{2}+frac{3}{{phi }_{h}^{max }}frac{{widetilde{Gamma }}_{{me}}left(1-{bar{phi }}_{n}right)}{1+{widetilde{Gamma }}_{{me}}+kappa {widetilde{Gamma }}_{a},}}.end{array}$$

(3)

From Eq. (3), we observe that the steady state droplet radius (left({widetilde{R}}_{d}^{{ss}}right)) depends on both diffusion and reaction kinetics. With increase in methylation, ({widetilde{R}}_{d}^{{ss}}) increases implying bigger heterochromatin domains. On the other hand, with increase in either the acetylation or transcription-mediated loop extrusion the steady state radius decreases. The quantitative dependence of the steady state radius on transcriptional kinetics is shown in Fig.2g (blue solid line). Note that the steady state radius shown in Fig.2g is normalized relative to the steady state radius with no transcription. Thus, our theory predicts an increase in the sizes of compacted chromatin domains in the interior of the nucleus upon inhibition of transcription.

The size dependence of chromatin domains along the nuclear periphery can be similarly determined by the balance of reaction, transcription, and diffusion kinetics for the LADs. The affinity of chromatin to the nuclear periphery due to the chromatin-lamina interactions in Eq. (6) induces a preferential nucleation of LADs. A schematic representation of heterochromatin compaction along the nuclear periphery resulting in LAD growth is shown in Fig.2e. As with the interior heterochromatin droplet, phase-separation drives the heterochromatin compaction (left({phi }_{h}={phi }_{h}^{max }right)) within the LADs, while the chromatin immediately outside corresponds to the euchromatin energy minimal well (left({phi }_{h}=0right)). Far away from the peripheral LAD nucleation sites, the chromatin composition remains undisturbed at the average composition of (left({bar{phi }}_{h},{bar{phi }}_{e}right)). The variation of chromatin composition with distance from nuclear periphery is shown in Fig.2e (blue line). Like in the case of the interior heterochromatin droplets, the heterochromatin composition gradient driven diffusive influx is balanced by the epigenetic and transcriptional regulated heterochromatin outflux, which determines the rescaled steady-state thickness of the LADs (refer to the Supplementary Section S7, Supplementary Eq. (S34)),

$${widetilde{x}}_{t}^{{ss}}=frac{{widetilde{Gamma }}_{{me}}left(1-{bar{phi }}_{n}right)}{{phi }_{h}^{max }left(1+{widetilde{Gamma }}_{{me}}+kappa {widetilde{Gamma }}_{a}right),}-frac{delta {widetilde{Gamma }}_{a}}{2}$$

(4)

As with the interior domains, we observe that the LADs become thicker with increase in methylation, while they become thinner with increasing acetylation or chromatin extrusion rates. A quantitative dependence of steady state LAD thickness on transcription rate based on Eq. (4) is plotted in Fig.2g (red dashed line). Our theory predicts an increase in the sizes of LADs along the nuclear periphery upon inhibition of transcription. While the theoretical analysis helps develop a fundamental biophysical understanding of the role of energetics and kinetics in chromatin phase separation, a nucleus-wide chromatin organization and its dynamic evolution can only be obtained numerically.

Next, we use the in-silico model to make testable quantitative predictions of the meso-scale chromatin organization in the nucleus. We also report the in-vivo nuclear chromatin reorganization upon transcription inhibition using complimentary STORM19 and ChromSTEM on nuclei from multiple cell lines. The choice of the parameters for rates of acetylation ({widetilde{Gamma }}_{{ac}}), methylation ({widetilde{Gamma }}_{{me}}), and the strength of chromatin-lamina interactions ({widetilde{V}}_{L}), were held constant for all the following simulations, and the choice of the level of spatial noise is discussed in the Supplementary Section S8. We calibrate the active chromatin supercoiling-driven loop extrusion rate ({Gamma }_{a}) to obtain an in-silico change in the interior domain sizes quantitatively comparable to that observed upon transcriptional inhibition. The calibrated model is then used to predict the change in LAD thickness due to inhibition of transcription, which upon comparison with experimental images serves to validate the model. A schematic for the workflow utilized to calibrate and cross-validate the model predictions in the interior and along periphery of the nucleus is shown in Supplementary Fig. (S14) (Supplementary Section S8).

ChromSTEM was used to obtain super-resolution images in terms of statistical descriptions of chromatin packing domains for BJ fibroblasts. ChromSTEM allows the quantification of 3D chromatin conformation with high resolution22. ChromSTEM mass density tomograms were collected for BJ fibroblasts treated with Actinomycin D (ActD) (Fig.3a, center) and compared to DMSO treated mock controls (Fig.3a, left) to evaluate the average size and density of chromatin packing domains. We have previously demonstrated that chromatin forms spatially welldefined higherorder packing domains and that, within these domains, chromatin exhibits a polymeric power-law scaling behavior with radially decreasing mass density moving outwards from the center of the domain23. As the ChromSTEM intensity in the reconstructed tomogram is proportional to the chromatin mass density, we estimated the size of the domains based on where the chromatin mass scaling and the radial chromatin density deviate from their predicted behavior (discussed in Supplementary Section S1.11). Based on the statistical analysis of individual packing domains, in a single tomograph shown in Fig.3a, we observed 71 domains in DMSO and 48 domains in the ActD-treated nucleus. Of the identified domains, the average domain radius ((pm) S.E) of BJ cells treated with DMSO and ActD was estimated to be 103.5 (pm) 4.73nm and 129.7 (pm) 6.78nm, respectively (Fig.3a, right panel), representing a 20.2% increase in size. Overall, fewer domain centers, and larger chromatin packing domains were experimentally observed upon ActD treatment compared to the control.

a ChromSTEM tomogram reconstructions for DMSO (left panel) and ActD treated (center panel) BJ fibroblasts. The domains radii for BJ cells treated ActD (right panel, n=48 domains) show 1.25 times (unpaired two tail t-test, p=0.002) increase compared to control (n=71 domains). b Representative live-cell PWS images (1-hour ActD treatment). Scale bars=5m. Box plots compare the domain sizes between DMSO control and ActD treated cells. Sample size HCT116: n=63 nuclei (control), 65 (ActD), p=0.05; A549: n=102 (control), 84 (ActD), p=1e7; U2OS: n=116 (control), 75 (ActD), p=1e12; n=103 (control), 150 (ActD), p=0.04. c Heatmap density of DNA super-resolution images in DMSO control (left panel, n=19 nuclei) and ActD (right panel, n=20 nuclei) treated HeLa nuclei. All scale bars 3m. d Loss of chromatin loop extrusion due to absence of RNAPII results in increased heterochromatin domain size (in red, nucleosomes not shown for clarity). e Numerical prediction of chromatin organization in DMSO control and ActD treated nucleus. f Zoomed in views of DMSO and ActD treated nuclei localized to the nucleus interior (top panels) and the periphery (bottom panels). Red and blue boxes shown in c are zoomed into. All scale bars 1m. g Left: Simulations predict domains in ActD nuclei are on average 1.63 times larger than in DMSO nuclei (n=127 (DMSO), 77 (ActD) unpaired two tail t-test, p=0) while LADs are 1.37 times thicker (n=38 (DMSO), 15 (ActD); unpaired two tail t-test, p=0). Right: Domain radii observed experimentally in ActD treated nuclei (n=3584 loci, 20 nuclei) are 1.61 times (unpaired two tail t-test, p=0) larger than in DMSO nuclei (n=5830 loci,19 nuclei), while LADs are 1.3 times thicker (n=1082 loci (DMSO), 1015 loci (ActD), unpaired two tail t-test, p=0.0006). All boxplots show the mean (cross), median (horizontal line), upper and bottom quartiles (box outlines) and the maximum and minimum non-outlier data points (whiskers). All source data are provided as a source data file.

In addition to evaluating domain properties using ChromSTEM, we utilized live-cell partial wave spectroscopy (PWS) imaging to observe the change in chromatin organization after transcription inhibition in various cell lines (Fig.3b). The PWS images demonstrate a significant reduction in average chromatin packing scaling upon ActD treatment in live cells across four different cell types. Next, the size of the domains is quantitatively approximated via polymer scaling relationships discussed in Supplementary Section S1.1322,24. The quantification of the domain sizes (boxplots in Fig.3b) shows that, for all cell types studied, packing domains are larger for upon transcription inhibition with ActD treatment in agreement with the ChromSTEM results on BJ fibroblasts.

Additionally, we have previously used STORM imaging to observe the nucleus wide changes in chromatin organization caused by transcription abrogation in HeLa nuclei after ActD treatment19. Heatmaps of chromatin density obtained via Voronoi tessellation-based color-coding of STORM images (see19 for analysis) are shown in Fig.3c. The zoomed in images of heatmaps of the chromatin cluster density (Fig.3f) clearly show the increasing heterochromatin domain sizes when RNAPII activity is inhibited, in agreement with our theoretical and numerical predictions (Fig.2d, e). Importantly, we see that the changes in chromatin organization occur not only in the interior domains of the nucleus but also along its periphery (Fig.3f, g).

Altogether these complementary imaging techniques establish that nucleus wide increase in sizes of compacted chromatin domains occurs upon the loss of transcription in a wide range of cell lines.

The chromatin cluster density maps obtained from STORM imaging were further analyzed to quantify the sizes of heterochromatin domains after DMSO and ActD treatment. A density-based threshold was used to isolate the high-density heterochromatin regions, which were then clustered via a density based spatial clustering algorithm (see Supplementary Section S1.8) and further sub-classified into LADs and interior domains depending on the distance from nuclear periphery (Supplementary Section S1.9). The quantitatively extracted distribution of interior heterochromatin domain radii for DMSO and ActD treated nuclei shows that their mean radius after transcription inhibition was nearly 1.61 times that in DMSO controls (Fig.3g).

Indeed, our model (Eq 3-4, Fig.2d, g) predicts that loss of transcription results in increased heterochromatin domain size. This is because under control conditions, extrusion of heterochromatin phase into euchromatin occurs. We assume, based on previous experimental findings19, that the presence of RNAPII activity drives the supercoiling of the DNA loop, thereby extruding it from the heterochromatin phase into the euchromatin phase at the phase boundaries (Fig.3c, left panel). However, when RNAPII is inhibited with ActD treatment (Fig.3c, right panel), the absence of this driving force for supercoiling-mediated loop extrusion keeps more DNA in the heterochromatin phase thereby increasing the domain sizes. The in-silico chromatin distribution predicted under control (left panel) and transcription inhibited (({Gamma }_{a}=0), right panel) conditions is shown in Fig.3e. The phase separated heterochromatin domains (left({phi }_{h}={phi }_{h}^{max }right)) are shown in red in a loosely compacted euchromatin background (blue, ({phi }_{h}=0)). We quantify the change in the sizes of the heterochromatin domains predicted by the model as the active extrusion rate ({Gamma }_{a}) is parametrically varied. The value of ({Gamma }_{a}) under control conditions is chosen (Supplementary TableS2) such that the change in the interior domain sizes with respect to transcription inhibition (with ({Gamma }_{a}=0)) is quantitatively the same as observed experimentally.

Next, we quantitatively validate the choice of ({Gamma }_{a}) under control conditions by comparing the predicted change in LAD thickness against that quantified from the STORM images. Our theoretical predictions (Eq. (4)) show that the reduction in transcription increases the thickness of the LADs reflecting the behavior predicted in the interior of the nucleus (Fig.2d, g). Our simulations of chromatin distribution in the nucleus (Fig.3e) show that inhibition of transcription (({Gamma }_{a}=0)) results in thicker LADs. Of note, the chromatin-lamina interaction strength (left({V}_{L}right)) stays unchanged between the two simulations. Yet, we see a higher association of chromatin with the periphery. Upon quantitative comparison (Fig.3g, left panel) we see that the LADs grow approximately 1.37 times thicker upon loss of transcription.

To validate this prediction, we compare the predicted change in LAD thickness with that quantified from in-vivo STORM imaging. (Fig.3g, refer to Supplementary Sections S1.8 and S1.9 for procedure). The quantified comparison of LAD thickness between DMSO and ActD nuclei (Fig.3g) shows nearly 1.3 times increase upon ActD treatment, in close quantitative agreement with the model prediction. Overall, with both model predictions and cellular observations, our results suggest that impairment of transcription plays a significant role in determining the size scaling of the interior heterochromatin domains and LADs.

We next enquire how, in addition to altering the size of the compacted domains, abrogation of transcription changes the extent of DNA packing. For this we analyzed the chromatin distribution in HeLa nuclei under DMSO and ActD treatments from STORM images previously generated19. Under control conditions the distribution of DNA is qualitatively more homogenous while ActD treated nuclei exhibit more isolated distinct domains of compacted chromatin surrounded by region of very low chromatin density (Fig.4a). For quantification, we plot the chromatin intensity along a horizontal line chosen to run across two heterochromatin domains with euchromatin between them (see zoomed images in Fig.4b, blue and red horizontal line). The chromatin intensity, plotted in Fig.4c (in blue) shows that even in the euchromatin region, the DNA presence is substantial. On the other hand, chromatin intensity across a horizontal line chosen across a heterochromatin domain in ActD nucleus (Fig.4b, c; in red) shows a much steeper gradient outside the domain.

a Grayscale heatmap density rendering of super-resolution images of DNA in control (DMSO, left panel, n=19 nuclei) and actinomycin D (ActD, right panel, n=20 nuclei) treated HeLa nuclei. All scale bars 3m. b Zoomed in views of DMSO and ActD treated nuclei. Boxes shown in a are zoomed into. All scale bars - 1m. c Along the blue (DMSO) and red (ActD) line segments, we plot the chromatin heatmap intensity (corresponding to the total DNA content) for the DMSO-treated control nucleus (in blue) and ActD-treated nucleus (in red). The DMSO-treated nucleus shows a wider distribution of small heterochromatin domains, while the ActD treated nucleus shows a greater compaction with isolated large heterochromatin domains. d Numerical prediction of distribution of total DNA (in grayscale) in a nucleus with (DMSO) and without (ActD) transcription mediated chromatin extrusion. e Distribution of total DNA content along the blue (red) line in d under DMSO (ActD) treatment. The black dashed line shows the level of total DNA predicted in the euchromatin phase of DMSO and ActD treated nuclei.

The increased presence of DNA in the euchromatic phase in presence of transcription as observed experimentally is captured by the simulations. The in-silico distribution of DNA (measured as the sum of volume fractions of the chromatin phases, ({phi }_{e}+{phi }_{h})) in a nuclear region far from LADs is plotted in Fig.4d for control and transcription inhibited in-silico nuclei. We see that the euchromatic phase (outside white circles) is darker when transcription is inhibited, indicating the presence of much lesser DNA than in control euchromatin. A quantification of the total DNA along cut-lines chosen in the control and ActD in-silico nuclei confirm the observations (Fig.4e).

Since the lack of transcription inhibits supercoiling-mediated chromatin loop extrusion from heterochromatin into euchromatin, we see a reduced density of DNA in the euchromatin phase of the nucleus under ActD conditions. Further, due to the lack of chromatin extrusion out of the heterochromatin domains when transcription is inhibited, we also observe that they are larger in size. Thus, transcription, via chromatin loop extrusion, results in removal of DNA from compacted heterochromatin region by converting it into active euchromatin form.

Taken together, our results suggest that transcription not only affects the scaling of the lengths (radius or thickness) of the heterochromatin domains, but also significantly changes the relative amounts of DNA in the euchromatin and heterochromatin phases.

We have established that change in transcription activity affects the global chromatin organization of the nucleus via altered supercoiling mediated loop extrusion. In turn, chromatin loop extrusion is initiated by the loading of cohesin onto DNA via a balance between cohesin loaders such as NIPBL and cohesin unloaders like WAPL (Fig.1c2,12,25,26). If the chromatin loop extrusion is responsible for the global chromatin reorganization, altering the cohesin loading/unloading balance must also result in chromatin reorganization. Thus, next, we study the chromatin arrangement in WAPL-deficient (WAPL) nuclei marked by increased levels of loaded cohesin.

In vivo, WAPL depletion causes an accumulation of large amounts of cohesin on chromatin27. This results in a much more homogenous distribution of DNA, which was previously termed blending due to excessive extrusion of chromatin loops, as shown schematically in Fig.5a19. In our mathematical model, WAPL deficiency is simulated as an increase in the rate of chromatin extrusion (({Gamma }_{a})). Based on the theoretical size scaling of the interior heterochromatin domains and LADs, as seen from Eq. (3) and Fig.2g, our model predicts that increase in ({Gamma }_{a}) would result in a decrease in the radius of the steady state heterochromatin domains (Fig.5b).

a Schematic representation of chromatin loop extrusion. WAPL-depletion results in increased cohesin loading and excessive transcription-driven chromatin loop extrusion. Note that nucleosomes, despite being present, are not shown to improve clarity. b Numerical prediction of distribution of heterochromatin domains in the interior and the LADs along the periphery (all domains in red) in a nucleus without (Cas9) and with (WAPL) cohesin unloading disruption. c Heatmap density of DNA super-resolution images in d control (Cas9, left panel) and WAPL knock-out (WAPL) treated HeLa nuclei. All scale bars - 3m. d Left: Zoomed in views of Cas9 and WAPL treated nuclei focusing on the interior heterochromatin domains. White solid boxes shown in c are zoomed into. All scale bars - 1m. Right: Zoomed in views of Cas9 and WAPL treated nuclei along the nuclear periphery. White dashed boxes shown in c are zoomed into. All scale bars - 1m. e Quantification of heterochromatin domain radius in the interior of Cas9- and WAPL - treated nuclei. (n=2386 loci in 6 nuclei for Cas9-treatment and 2416 loci in 7 nuclei for WAPL treatment). WAPL treated nuclei exhibit a significantly lower ((sim) 0.86 times) mean heterochromatin radius (unpaired two-tailed t-test, p=6e10). Quantification of LAD thickness along the periphery of Cas9- and WAPL - treated nuclei. (n=219 loci in 6 nuclei for Cas9-treatment and 169 loci in 7 nuclei for WAPL treatment). WAPL treated nuclei exhibit a significantly lower ((sim) 0.43 times) mean LAD thickness (unpaired two-tailed t-test, p=1e13). f Boxplot in left panel shows the distribution of domain radii predicted numerically. WAPL nuclei have a mean domain radius 0.8 times that of Cas9-treated nuclei (unpaired two-tailed t-test, p=0). Boxplot in right panels shows the distribution of LAD thicknesses predicted numerically. WAPL nuclei have a mean LAD thickness 0.82 times that of Cas9-treated nuclei. All boxplots show the mean (cross), median (horizontal line), upper and bottom quartiles (box outlines) and the maximum and minimum non-outlier data points (whiskers) of the plotted distribution. All source data are provided as a source data file.

STORM images of HeLa nuclei without (labeled Cas9) and with WAPL-deficiency previously revealed genome-wide changes in the chromatin organization induced by excessive loading of cohesin (Fig.5c, d)19. A visual comparison between representative zoomed-in regions (white boxes in Fig.5c) demonstrates the reduction of heterochromatin domain sizes in the interior of the nuclei in WAPL nuclei (Fig.5d). Using clustering analysis (refer Supplementary Section S1.8 and S1.9), we quantify the altered chromatin domain sizes in control and WAPL HeLa cell nuclei. We observe that WAPL nuclei with increased chromatin blending have heterochromatin domains with a mean radius approximately 15% smaller than control nuclei (Fig.5e).

In-silico, we parametrically vary the active chromatin extrusion rate ({Gamma }_{a}) above the control level (SupplementaryTable S2, determined forcontrol treatment). The value of ({Gamma }_{a}) for WAPL nuclei is chosen (Supplementary TableS2) such that the decrease in the size of interior heterochromatin domains reduces by 15% (Fig.5f) to agree with the experimental observation (Fig.5e).

As discussed previously (Fig.2g), the model predicts that the effects of chromatin extrusion observed in the interior domains of the nucleus are replicated along the nuclear periphery. Simulation of nuclear chromatin organization (Fig.5b) reveals that by changing only the rate of chromatin extrusion ({Gamma }_{a}), keeping all other parameters including chromatin-lamina interaction potential ({V}_{L}) constant, we see a reduction in the association of chromatin with the lamina. Specifically, a 2.5-fold increase in ({Gamma }_{a}) calibrated to occur due to WAPL-deficiency predicts a 51.2% decrease in the average LAD thickness, as shown in Fig.5f.

The predicted change in LAD thickness is consistent with previous experimental observations and was further quantitatively validated by measuring the thickness of LADs in STORM images of control and WAPL nuclei (Fig.5e)19. A reduction in the sizes of domains, as seen in the nucleus interior, can also be observed at the nuclear periphery, as shown in a representative zoomed in region (white dashed boxes in Fig.5c) in Fig.5d. The mean thickness of the LADs at the nuclear periphery is approximately 20% smaller for WAPL nuclei (Fig.5h) as compared to the control-treated nuclei.

Together, these results confirm that the meso-scale spatial chromatin organization is strongly regulated by the chromatin loop formation, and this effect can be modulated not only by the transcription activity, but also by altering the extent of loading or unloading of cohesin rings on the DNA. These results provide further evidence for the link between transcriptional regulation and nucleus-wide chromatin distribution via transcription-driven supercoiling mediated chromatin loop extrusion.

Since we have established, via both quantitative analysis of experimental data and simulations, that extrusion of chromatin loops is governed by both cohesin loading/unloading balance and RNAPII mediated transcription, a question of their tandem role emerges.

To simulate the individual effects of cohesin loading and transcriptional activity, we decompose the overall active chromatin extrusion rate into its distinct constitutive steps. The individual steps involved in the process of supercoiling mediated chromatin loop extrusion from heterochromatin into euchromatin (as discussed previously in Section Introduction) are shown in Fig.6a. As a first step, a balance between the loading of cohesin via NIPBL/MAU225 on chromatin occurring at a rate ({Gamma }_{l}) and its unloading via by WAPL/PDS52,12,26 occurring at a rate ({Gamma }_{{ul}}) results in the association of cohesin rings with chromatin at an overall rate ({Gamma }_{{coh}}={Gamma }_{l}-{Gamma }_{{ul}}). In other words, ({Gamma }_{{coh}}) denotes the overall rate of cohesin loading on DNA. The entrapment of DNA by cohesin is followed by the extrusion of supercoiled loops of chromatin via DNA supercoiling by the RNAPII mediated transcription, at a rate denoted by ({Gamma }_{{tr}}). Thus, as shown in Fig.6a, by assuming a first-order reaction kinetics for both steps, the overall rate of active chromatin extrusion ({Gamma }_{a}) at the interface of heterochromatin and euchromatin is proposed to be multiplicatively decomposed as,

$$begin{array}{c}{Gamma }_{a}={Gamma }_{{tr}}{Gamma }_{{coh}}={Gamma }_{{tr}}left({Gamma }_{l}-{Gamma }_{{ul}}right)end{array}$$

(5)

a Schematic showing the associative sub-steps of chromatin extrusion incorporating cohesin loading v/s unloading balance and active transcriptional work done by RNAPII. The rate of active extrusion of chromatin loops (left({Gamma }_{a}right)) is determined by both sub-steps. Note that nucleosomes, despite being present, are not represented in this schematic to better display the chromatin loops. b Numerical prediction of distribution of heterochromatin domains in the interior and the LADs along the periphery (all domains in red) in a nucleus in control (Cas9-DMSO treatment, top-left panel), transcription inhibited (Cas9-ActD, top right), WAPL knock-out treated (WAPL-DMSO, bottom left) and simultaneous WAPL knock-out along with transcription inhibition treated (WAPL-ActD, bottom right). c Heatmap density rendering of super-resolution images of DNA in control (Cas9-DMSO treatment, left panel), transcription inhibited (Cas9-ActD, center left), WAPL knock-out treated (WAPL-DMSO, center right) and simultaneous WAPL knock-out along with transcription inhibition treated (WAPL-ActD) HeLa nuclei. All scale bars 3m. d Quantification of heterochromatin domain radius in the interior (plain colored boxes) as well as the LAD thickness along the nuclear periphery (hatched boxes) of Cas9-DMSO (3328 loci in 13 nuclei), Cas9-ActD (4042 loci in 11 nuclei), WAPL-DMSO (1548 loci in 10 nulcei) and WAPL-ActD (1926 loci in 11 nuclei) treated nuclei. As previously, ActD treated nuclei exhibited a significantly increased domain size (unpaired two-tailed t-test, p=0) while WAPL treated nuclei exhibit a significantly lower mean heterochromatin radius (unpaired tw-tailed t-test, p=0). However, the differences between Cas9-ActD treated and WAPL-ActD treated nuclei was insignificant (unpaired two-tailed t-test, p (sim) 0.9). All boxplots show the mean (cross), median (horizontal line), upper and bottom quartiles (box outlines) and the maximum and minimum non-outlier data points (whiskers) of the plotted distribution. All source data are provided as a source data file.

In addition to the extrusion of loops via RNAPII mediated DNA supercoiling activity12,13,19,28,29,30, in vitro experiments proposed that cohesin once transiently loaded onto DNA, could independently drive the formation of loops via its ATPase machinery9,11,31,32,33. Cell based experiments demonstrated that in WAPL cells, clusters of cohesin in WAPL cells assemble together into vermicelli-like structures and these structures disappear upon transcription inhibition, but not upon partial loss of cohesin19. These results, taken together, present strong evidence for the important role of transcription in powering cohesin mediated loop extrusion. While the relative role of cohesins motor activity and transcription in loop extrusion inside cells remains to be determined, here we focus on the latter given the previous in vivo experimental findings. We indeed show that a kinetic model captured by Eq. (5) sufficiently explains the effect of extrusion of the specific chromatin loops extending from transcriptionally silenced heterochromatin into genetically active euchromatin on determining the meso-scale chromatin domain sizes.

The chromatin organization is simulated in a nucleus under control and transcription inhibition treatments for nuclei with and without WAPL deficiency. The chromatin organization in a control nucleus (labeled Cas9-DMSO), simulated via parameters listed in Supplementary TableS1 is shown in Fig.6b, top-left panel. The individual inhibition of transcriptional activity without affecting the cohesin loading (Cas9-ActD) results in a chromatin organization with increased heterochromatin domains sizes and LAD thickness, as shown in Fig.6b, top-right panel. On the other hand, the simulation of chromatin distribution in nucleus with depleted cohesin unloading, without disturbing the transcriptional activity, (WAPL-DMSO) is shown in Fig.6b, bottom-left panel. Finally, the chromatin distribution predicted in a WAPL nucleus with inhibited transcription (WAPL-DMSO-treatment) is shown in Fig.6b, bottom-right panel. As shown in Fig. 3e and Fig. 3g, ActD (mathematically, ({Gamma }_{{tr}}=0) in Eq. (5)) results in larger heterochromatin domains and thicker LADs, while WAPL nuclei (increased cohesin loading; mathematically, ({Gamma }_{{ul}}/{Gamma }_{l}) increases in Eq. (5)) show the opposite effect with smaller heterochromatin domains and LADs. For a WAPL nuclei in which transcription is inhibited (WAPL ActD; mathematically, ({Gamma }_{{tr}}=0) and ({Gamma }_{{ul}}/{Gamma }_{l}) increases in Eq. (3)), the model predicts that inhibition of transcription returns the chromatin organization to the control (Cas9-ActD) levels. Transcription inhibition thus blocks the reduction in chromatin domain sizes induced due to WAPL deficiency due to lack of impetus for chromatin supercoiling.

To quantitatively validate the model predictions, we investigate the in-vivo chromatin organization under individual and tandem changes in transcription and cohesin unloading by re-analyzing previously reported super-resolution images shown as heatmap density plots in Fig.6c19. Visual inspection of this data agrees with the model predictions that transcriptional inhibition counteracts the chromatin blending observed in DMSO treated WAPL nuclei, which was also previously reported19. We thus focused on extracting the radius of heterochromatin domains and LAD thickness to further validate the model results quantitatively (Fig.6d). Cas9 ActD treated nuclei show an increased heterochromatin domain radius compared to control while WAPL nuclei show a significant reduction in domain radius and LAD thickness (Fig.6d). However, WAPL ActD treated nuclei show no significant difference in comparison to Cas9 ActD treated nuclei (Fig.6d), in quantitative agreement with the numerical predictions.

These results further confirm that the effect of transcription on global chromatin distribution occurs via supercoiling mediated chromatin loop extrusion, especially at the interface of heterochromatin and euchromatin phases. Furthermore, these results also present a significant validation of the mathematical phase-field model of chromatin organization in the nucleus.

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Identification of DERBPs during EMT in breast cancer cells

By differential expression analysis of breast cancer cells at different stages of EMT, we identified a large number of DEGs among the comparison groups (Supplementary Data S1). We found that the number of DEGs decreased first and then increased during the transformation process, and there were fewer DEGs among the comparison groups in the intermediate state (Supplementary Fig. S2A).

We combined all identified DEGs and intersected them with known human RBP genes and found that 504 RBP genes were differentially expressed during EMT in breast cancer cells (Fig.1A)4. WGCNA was used to analyse the coexpression relationships among DERBPs (Supplementary Data S2). We found that according to the expression of RBPs in different EMT stages, these genes could be divided into different modules, and the expression of genes in each module was relatively similar at different stages (Supplementary Fig. S2B). Using the WGCNA process, we calculated the correlation between the genes of each module and the EMT state and found that the MEgreen, MEbrown and MEturquoise modules were significantly correlated with the EMT state (Fig.1B). Among them, the MEgreen module gene was highly expressed in the intermediate state, the MEbrown module gene was highly expressed in the E state cells, and the MEturquoise module gene was highly expressed in the M state cells (Fig.1C).

Identification of EMT-related RBPs in a breast cancer cell line. (A) Venn diagram showing the overlap of DEGs and RBP genes. (B) The correlation between DERBPs in different modules and EMT state. Module-trait associations were computed by a LME model with all factors on the x axis used as covariates. All Pearson s correlation value and P values are displayed. (C) Heatmap of module eigengenes sorted by average linkage hierarchical clustering. FPKM values were log2-transformed and then median-centred by each gene (color figure online). (DF) Heatmap showing the expression profile of DERBPs of green, brown and turquoise module. FPKM values were log2-transformed and then median-centred by each gene (color figure online). (G) The top 5 most enriched GO terms were illustrated for DERBP genesin the three modules.The colour scale showing the row-scaled significance (log10 corrected P value) of the terms.

RBPs in the MEgreen, MEbrown and MEturquoise modules were further extracted, and a heatmap of expression was drawn. Most of the RBPs in the MEgreen module were highly expressed in the intermediate state (Fig.1D). Some of the RBPs in the MEbrown module gene were highly expressed in E state cells, while other RBPs were expressed at extremely low levels in this state (Fig.1E). Most RBPs of MEturquoise module genes were highly expressed in M3 state cells, while a small portion of RBPs were expressed at extremely low levels in M3 state cells (Fig.1F). These results suggested that the expression level of RBPs might affect the conversion process of EMT in breast cancer.

The genes of MEgreen, MEbrown and MEturquoise were extracted for GO pathway analysis. The results showed that pathways enriched in MEbrown genes mainly included the innate immune response, immune system processes, mRNA processing (Supplementary Fig. S2C). The pathways enriched in MEgreen genes mainly included spermatogenesis, cell differentiation, RNA splicing, mRNA processing. (Supplementary Fig. S2D). The pathways of enriched in MEturquoise genes included mRNA processing and RNA splicing (Supplementary Fig. S2E). We further extracted the common GO functional pathways enriched in MEgreen, MEbrown and MEturquoise genes. The results showed that the MEgreen gene had the highest degree of enrichment in RNA splicing and spermatogenesis pathways, the MEturquoise gene had the highest degree of enrichment in mRNA processing, and the MEbrown gene had the highest degree of enrichment in innate immune pathways (Fig.1G).

According to the above results, high expression of RBPs in breast cancer cells in the E state might regulate the expression of immune-related genes in cancer cells to achieve immune escape. Breast cancer cells in intermediate state overexpressed RBPs related to splicing regulation and promoted EM progression. Breast cancer cells with M status highly expressed RBPs related to mRNA processing and realised the transformation of the M phenotype.

Based on the transcriptome data of 18 breast cancer cell samples at different EMT stages, AS events were analysed according to the use of splicing sites using the SUVA pipeline. Five types of AS events, such as alternative 5' splice site, were identified (Supplementary Fig. S3A).

The SUVA pipeline was used to compare the pSAR used in the same splicing event between the two groups of samples. We identified a large number of AS events, such as those involving alternative 5' splice sites and alternative 3' splice sites, between different comparison groups (Fig.2A). In addition, by matching the splicing events detected by SUVA to classical splicing events, 10 kinds of splicing events, including a large number of events involving alternative 3 splice sites, were found (Supplementary Fig. S3B). According to the pSAR used by each differential splicing event, the median pSAR value of the differential AS event was calculated. We found that most of the differential AS events had pSAR values greater than 50% (Fig.2B). Principal component analysis was performed based on pSAR values of differential splicing events with pSAR50% in each sample. The results showed that breast cancer cells at the E, EM1, EM2, EM3, M1 and M2 stages were clustered together. This suggested that these differential AS events can be used to distinguish breast cancer cells at different EMT stages (Fig.2C).

Identification of EMT-related AS in a breast cancer cell line. (A) Bar plot showing number of RAS detected by SUVA in each group. (B) Bar plot showing RAS with different pSAR. RAS with pSAR50% were labeled. (C) Principal component analysis based on RAS with pSAR50%. The ellipse for each group was the confidence ellipse. (D) Heatmap showing the splicing ratio of RAS (pSAR50%). Splicing ratio were log2-transformed and then median-centred by each gene (color figure online). (E) Bar plot exhibited the most enriched GO biological process results of the RAS with pSAR50%.

A heatmap was drawn with pSAR values of differential splicing events with pSAR50%. The pSAR values of some splicing events in breast cancer cells at the E, EM and M2 stages were higher than those at other stages (Fig.2D).

To identify the potential functions of these differential AS events, we extracted the genes responsible for these differential AS events and performed GO and KEGG analyses. GO analysis showed that these genes were enriched in pathways including cellular response to DNA damage stimulus, cell division, cell cycle, positive regulation of GTPase activity, protein transport, tRNA methylation (Fig.2E). KEGG analysis showed that these genes were enriched in pathways including adherens junction, Epstein-Barr virus infection, fatty acid biosynthesis, ferroptosis, yersinia infection (Supplementary Fig. S3C).

Given that RBPs can regulate the AS of some genes during EMT in breast cancer, we extracted differential splicing events with pSAR50% and RBPs in MEgreen, MEbrown, and MEturquoise modules associated with EMT. By using the expression levels of these RBPs and the pSAR of differential AS events to establish a coexpression relationship, we obtained the AS events potentially regulated by RBPs related to EMT. The genes involved in these differential splicing events were extracted for GO function analysis. We found that these genes were significantly mainly enriched in cell adhesion, the integrin-mediated signalling pathway, lipid transport, positive regulation of GTPase activity (Fig.3A).

DERBPs potentially regulated AS associated with cell adhesion in a breast cancer cell line (A) The most enriched GO biological process results of the coexpressed RAS (pSAR50%) potentially regulated by DERBPs. Cutoffs of P value0.01 and Pearson coefficient0.9 or0.9 were applied to identify the coexpression pairs. (B) Heatmap showing the splicing ratio of RAS in cell adhesion pathway. Splicing ratio were log2-transformed and then median-centred by each gene (color figure online). (C) Regulatory networks for differential AS events and coexpressed RBPs on genes in the cell adhesion pathway. (D) The reads distribution and splicing ratio of clualt3p26826 ITGA6. The expression levels of PCBP3 in breast cancer cells at different EMT stages were showed in the right part.

In view of the important role of cell adhesion in EMT and cancer metastasis29, we further extracted differential AS events corresponding to genes enriched in cell adhesion pathways. According to their pSAR values, a heatmap was drawn. The pSAR of some differential splicing events was higher in the E and EM stages, while the pSAR of other differential splicing events was higher in the M stage (Fig.3B).

We constructed regulatory networks for differential AS events and coexpressed RBPs on genes in the cell adhesion pathway and found that 88 RBPs may regulate 37 differential splicing events on 19 cell adhesion pathway genes (Fig.3C). RBM47, PCBP3, FRG1, SRP72 and other RBPs might regulate AS of ITGA6, ADGRE5, TNC and other genes and affect the EMT process of breast cancer cells (Fig.3D and Supplementary Fig. S4).

We further downloaded the sequencing data of breast cancer patients and related clinical information from the TCGA database and extracted the expression levels of the above 88 RBPs with regulatory effects. In the RBPs-related analysis, 1216 breast cancer patients were screened (Supplementary Table S1). The median follow-up was 905days (interquartile range 4621694days), with 200 deaths. We constructed a risk model based on the expression levels of these RBPs and found that ADAT2, C2orf15, SRP72, PAICS, RBMS3, APOBEC3G, NOA1, and ACO1 could be used for risk assessment in terms of breast cancer prognosis (Fig.4AC). Patients predicted to be at high risk using this model had a significantly worse prognosis (Fig.4D). We found significant differences in the expression levels of all 8 RBPs in breast cancer tissues without metastasis compared with normal breast tissues. Perhaps due to the small number of metastatic samples, the expression levels of the 8 RBPs in breast cancer tissues with vs. without metastasis were not significantly different (Fig.4E). Further analysis showed that the expression levels of 8 RBPs in breast cancer tissues were significantly correlated with the prognosis of patients (Fig.4F).

EMT-related RBPs were significantly correlated with the prognosis of breast cancer patients. (A) The result of LASSO regression analysis. (B) LASSO coefficient profiles of the candidate RBPs by tenfold cross-validation. (C) Prognostic value of the candidate RBPs in breast cancer. The HR and P values were calculated using the univariate Cox regression analysis. (D) Comparison of overall survival according to the risk score calculated from candidate RBPs. (E) The boxplot showing the FPKM of candidate RBPs in Tumour, Metastatic and Normal samples. *0.05;**0.01;***0.001. (F) Relationship between expression level of candidate RBPs and prognosis of breast cancer.

In the AS-related analysis, 90 breast cancer patients were screened (Supplementary Table S2). The median follow-up was 1268days (interquartile range 7742129days), with 26 deaths. We used SUVA to identify differential AS events between breast cancer tissue and normal tissue in TCGA and obtained pSAR values of 37 differential splicing events related to 19 cell adhesion pathway genes. Risk analysis based on pSAR values of splicing events showed that splicing events occurring on TNC and COL6A3 could be used to evaluate breast cancer prognosis (Fig.5AC). The analysis found that patients with high-risk differential splicing events had a poor prognosis (Fig.5D). We found that there were significant differences in the pSAR values of these two splicing events in breast cancer tissue without metastasis compared with normal breast tissue (Fig.5G). Further analysis showed that the pSAR values of these two differential splicing events in breast cancer tissues were significantly correlated with the prognosis of patients (Fig.5EF).

EMT-related AS were significantly correlated with the prognosis of breast cancer patients. (A) The result of LASSO regression analysis. (B) LASSO coefficient profiles of the candidate AS by tenfold cross-validation. (C) Prognostic value of the candidate AS in the breast cancer. The HR and P values were calculated using the univariate Cox regression analysis. (D) Comparison of overall survival according to the risk score calculated from candidate AS. (E,F) Relationship between the pSAR of candidate AS and prognosis of breast cancer. (G) The boxplot showing the splicing ratio of clualt5p25729 COL6A3 and clualt3p46274 TNC in Tumour and Normal samples. *0.05;**0.01;***0.001.

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Inocras and IMBdx Announce Strategic Partnership to Transform Cancer Patient Care in the U.S.

Inocras and IMBdx have announced a partnership aimed at enhancing care for cancer patients in the U.S. Through this partnership, Inocras, a leader in whole genome sequencing diagnostics, and IMBdx, an innovator in liquid biopsy-based diagnostics for cancer, will be able to provide broader offerings for cancer patients and their providers.

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L to R: Jehee Suh, CEO of Inocras, Tae-You Kim, CEO of IMBdx (Photo: Business Wire)

Inocras specializes in cancer profiling using whole genome sequencing (WGS), capturing over 99% of an individual's genomic makeup. This allows for a broader analysis of mutations and complex variations, allowing for deeper insights into a patient's condition. IMBdx has developed a comprehensive portfolio of liquid biopsies for the cancer diagnosis and treatment cycle. The company was recently listed on the KOSDAQ in South Korea.

The partnership will focus on Inocras providing IMBdxs liquid biopsy-based cancer diagnostics in the U.S. market, creating alternatives for patients and their providers when genetic testing from tissue biopsy is not feasible. This joint initiative introduces a new model of patient care, leveraging advanced two technologies in genetic testing - whole genome data and liquid biopsy.

"Our collaboration with IMBdx marks a significant step forward in our mission to transform healthcare for cancer and rare disease patients," said Jehee Suh, CEO of Inocras. "We think of patients at the core and are trying to create more options for patients and providers. By combining our strengths, we're not just delivering advanced diagnostic services, we're contributing to creating a pathway to a more effective cancer care journey.

About Inocras Inc:

Inocras Inc. (formerly Genome Insight, Inc) is a pioneering provider of whole genome sequencing and analytics for cancer and rare diseases. The company is dedicated to unlocking the potential of genomic data to enable precision health for everyone. For more information about Inocras and our services, please visit Inocras.com.

About IMBdx:

IMBdx is at the forefront of liquid biopsy technology, developing advanced diagnostic tools that capture and analyze circulating biomarkers to detect and monitor diseases more effectively and non-invasively.

Sohee Cho / media@inocras.com

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Inocras and IMBdx Announce Strategic Partnership to Transform Cancer Patient Care in the U.S. - Morningstar