Daily Archives: September 2, 2021

By Michael Marshall

Human brains have been shaped by DNA that evolves quickly

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Many of the fastest-evolving sections of the human genome are involved in brain development. These rapidly changing segments of DNA may have played key roles in the evolution of the human brain and in our cognitive abilities.

Chris Walsh at Boston Childrens Hospital in Massachusetts and his colleagues studied sections of the human genome dubbed human accelerated regions (HARs). These stretches of DNA are virtually identical in many other mammals that have been studied, suggesting they have important functions but they differ in humans, implying our evolution has changed them.

Previous studies have identified 3171 possible HARs, but Walsh says it is unlikely that they are all important. Probably hundreds of them are, but probably not thousands, he says. His team set out to identify HARs that have played important roles in the evolution of our brains.

The researchers placed copies of each HAR, as well as their chimpanzee equivalents, into developing brain cells from mice and humans. In each cell line, they tracked how much each gene in the genome was expressed. This allowed them to determine whether each HAR enhanced the activity of genes, compared with the equivalent sequence from a chimp.

Using this and other methods, the team identified 210 HARs that significantly enhanced gene activity in the neural cells. These HARs probably affect human brain development.

The researchers then zeroed in on a gene called PPP1R17, which is expressed in some of the cells of the developing brain and regulated by several HARs, so it therefore behaves differently in humans than in other mammals. They compared the expression of PPP1R17 in the developing brains of mice, ferrets, rhesus macaques and humans. In the macaques and humans, the gene was expressed in the cerebral cortex, but it wasnt in the mice and ferrets.

This gives an example of how dynamic these enhancers are over the course of evolution, says Walsh.

It isnt clear why PPP1R17 came to be activated differently in humans, but it may be related to our unusually large brains. Big brains need lots of cells, each of which is likely to contain harmful mutations that need to be fixed. These repairs take time, and PPP1R17 is known to make cells take longer to grow and divide.

Journal reference: Neuron, DOI: 10.1016/j.neuron.2021.08.005

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Rapidly evolving bits of DNA helped develop the human brain - New Scientist

Study on yeast cells reveals how gene affects speed of damage caused by UV radiation

WSU researchers discovered a gene involved in repairing genetic pathways that can lead to cancerous conditions, neurological defects and cause aging.

The gene named ELOF1 was discovered 20 years ago in humans. It was solely recognized for its role in transcribing DNA. The WSU research team is the first to recognize its role in repairing damaged DNA from UV radUnaiation, said Kathir Selvam, postdoctoral researcher with the WSU School of Molecular Biosciences.

Selvam said he and his team cannot test ELOF1 because it is only found in humans, so they are studying its genetic counterpart, Elf1, which can be found in yeast cells. By exposing the cells to UV light, which can cause cancerous conditions or premature aging, the researchers are testing Elf1s role in DNA repair.

In the yeast cell trials, Elf1 is knocked out of the yeast. The cells, which no longer have Elf1, are exposed to UV light in a controlled environment for about one week. When Elf1 is reintroduced to the cell, it is repaired in about one day.

To analyze the process, John Wyrick, WSU School of Molecular Biosciences associate professor, said a genetic sequencing method was developed. The sequence maps where the damage, which can be randomly distributed, forms across a genome.

Genome maps are then compared to analyze the repairing properties of the Elf1 gene, Selvam said.

So far, results show that cells die more readily when Elf1 is removed following UV irradiation, Wyrick said.

Because Elf1 and ELOF1 are counterparts, Elf1s effect in yeast cells DNA repair pathway would be similar to ELOF1s effect in human DNA repair, Selvam said.

Wyrick said he and Selvams group are collaborating with researchers at other universities. This includes the Erasmus University Medical Center in the Netherlands, which focuses on ELOF1s effect on human cells.

Wyrick said the research began in summer 2019 and is centered around studying how DNA is damaged, resulting in possible mutations in genes. The researchs goal was to study a specific type of DNA damage resulting from exposure to UV light.

Nucleotide excision repair is a critical repair pathway that helps repair cell damage, Wyrick said. When there are genetic defects in a DNA pathway, it significantly increases the likelihood an individual will have cancer.

NER repairs damage to DNA lesions caused by UV exposure, Wyrick said. Normally, NER goes along the pathway, searching for distorted DNA lesions and tries to repair them.

Its like finding a needle in a haystack, he said.

Postdoctoral student Kathir Selvam, left, stands with Professor John Wyrick, right.

Typically, DNA is transcribed into RNA by a protein called RNA polymerase. But when the DNA is damaged, the RNA polymerase stalls and the damage has to be repaired.

Failure in this repair can cause a number of human diseases, including Cockayne syndrome a rapidly aging disease that can cause neurological defects and prevent individuals from surviving past their teen years and make an individual more susceptible to cancerous conditions, Selvam said.

Essentially its like a car going along and if a cow gets in your way, then you stop the car, Wyrick said.

If something goes wrong when DNA is transcribed into RNA, a NER subpathway is initiated to repair the damage so transcription can continue.

The cow then gets moved out of the way and the car keeps driving, Wyrick said.

Wyrick said ELOF1 likely plays a role in the pathway because it binds to the RNA polymerase and travels with it, helping to repair and bypass the damage.

Basically, we want to understand what genes are playing a role in that [repair] pathway, and what role they play, Wyrick said.

ELOF1 is not exclusive to repairing damage from UV exposure. It is equally essential in repair pathways found in human genetic diseases or mutations, Wyrick said.

In previous research, Wyrick said mouse embryos were not able to develop without the presence of the ELOF1 gene, indicating it is likely needed for survival.

The research is just focusing on understanding the mechanisms, understanding how the damages are bad, Selvam said. And long term, understanding how mechanisms can, of course, lead to improving treatment.

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Researchers redefine gene involved with DNA repair The Daily Evergreen - The Daily Evergreen

Can we recreate dinosaurs from their DNA? A paleontologist answers the question. Image via Deb Dowd/ Unsplash.

By William Ausich, Ohio State University

Would it really be possible to get the DNA of dinosaurs and then recreate them? Lucie R., age 5, Atlanta, Georgia

As a paleontologist thats a scientist who studies ancient life Im asked this question all the time. After all, the scientists in Jurassic Park (and later, Jurassic World) used DNA to recreate dozens of dinosaurs: Triceratops, Velociraptor and T. rex. And if you saw any of those movies, you had to wonder: Could real scientists do that today?

DNA which stands for deoxyribonucleic acid is something in every cell of every organism that ever lived on Earth including dinosaurs. Think of DNA as molecules that carry the genetic code, a set of instructions that helps bodies and minds grow and thrive.

Your DNA is different from everyone elses. It determines many of the characteristics that define you, like the color of your eyes or whether your hair is straight or curly.

DNA is much easier to find in the soft parts of an animal their organs, blood vessels, nerves, muscle and fat. But a dinosaurs soft parts are long gone. They either decomposed or were eaten by another dinosaur.

Dinosaur fossils are all thats left of those prehistoric animals. Immersed for tens of millions of years in ancient mud, minerals and water, the fossils come from the dinosaurs so-called hard parts its bones, teeth and skull.

We find dinosaur fossils in the ground, in riverbeds and lakes, and on the sides of cliffs and mountains. Every now and then, someone finds one in their backyard. Often, theyre quite near the surface, and usually, theyre embedded in sedimentary rock.

With enough fossils, scientists can build a dinosaur skeleton: what you see when you go to the museum.

But scientists have a big problem when trying to find DNA in dinosaur fossils. DNA molecules eventually decay. Recent studies show DNA deteriorates and ultimately disintegrates after about 7 million years. That sounds like a long time, but the last dinosaur died at the end of the Cretaceous Period. Thats more than 65 million years ago.

Dig up a fossil today, and any dino-DNA within would have long since fallen apart. That means, as far as scientists know, and even using the best technology available today, its not possible to make a dinosaur from its DNA.

Although its too late to find dino-DNA, scientists recently found something almost as intriguing. They discovered DNA fragments in the fossils of Neanderthals and other ancient mammals, such as woolly mammoths.

Now that makes sense. Those fragments are less than 2 million years old, well before all of the DNA would decay.

Just for fun, lets imagine that somehow, sometime in the future, researchers came up with fragments of dinosaur DNA. With only fragments, scientists still could not make a complete dinosaur. Instead, they would have to combine the fragments with the DNA of a modern-day animal to create a living organism.

That creature, however, could not be called an actual dinosaur. Rather, it would be a hybrid, a blend of dinosaur and, most likely, a bird or reptile. Think thats a good idea? After all, the scientists in the Jurassic movies tried that. And you know what happened there.

William Ausich, Professor Emeritus of Paleontology, Ohio State University

This article is republished from The Conversation under a Creative Commons license. Read the original article.

Bottom line: We cant recreate dinosaurs from their DNA because the DNA no longer exists. DNA disintegrates in about 7 million years, and dinosaurs lived 65 million years ago.

Members of the EarthSky community - including scientists, as well as science and nature writers from across the globe - weigh in on what's important to them. Photo by Robert Spurlock.

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Can we recreate dinosaurs from their DNA? - EarthSky

ABOVE: Artist rendering of the male found deceased on Aug. 1, 1978 at Poole Knobs Recreation area in La Vergne.

Relatives of missing man James Sanders waited 43 years before learning last week their brothers remains were found in 1981 in Immokalee, Fla., based on DNA samples submitted by Rutherford County Sheriffs detectives.

32-Year-old Sandersof Portland, Tenn., was lastseen Jan. 1, 1978 at a bus stop in Tennessee en route to North Carolina where he planned to work at an aunts tobacco farm. Sanders never arrived at his aunts farm and was never heard from again.

Brother Eddie Sanders contacted Rutherford County Sheriffs Detective Sgt. Dan Goodwin and Steve Kohler in 2014, who were trying to find the identity of a man whose burned body was found Aug. 1, 1978 at Poole Knobs Recreation area in La Vergne.

Sanders wondered if the man found in La Vergne might be his brother. He, his sister and his brothers children gave DNA samples to the detectives who submitted the samples in January 2015 to the North Texas University DNA lab for testing.

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Lab employees notified the detectives six months later the man found in La Vergne was not Sanders (above is a photo of James Sanders as he appeared prior to the last time he was seenon Jan. 1, 1978).

The DNA samples submitted by the Sanders family remained at the lab and now those samples have a match.

Sheriff Kevin Rambosk of Collier County, Florida, announced Friday the lab matched bone from the body found in Immokalee, Florida to Sanders DNA submitted by Rutherford County detectives.

This important development was made possible thanks to DNA technology and the dedication of everyone involved in this investigation, said Rambosk.

Without the combined efforts of multiple agencies, Mr. Sanders would still be unidentified after 40 years, Rambosk said. Now that our victim has a name, the homicide investigation can continue to be actively worked.

The case for the identity of the missing man found in La Vergne is being investigated by Detective Richard Brinkley of the Rutherford County Sheriffs Office.

Brinkley is still trying to identify the suspects who killed the unidentified man while focusing on the identity of the victim.

An autopsy report described the unidentified man as being in his late 30s who was 5-feet-10 tall. He had long, brown hair with a receding hairline and a reddish brown and gray beard. He had a scar on his stomach, no teeth but an upper denture and a quarter-sized mole near his waistline.

Detectives worked with Dr. Lee Meadows Jantz from the state Forensic Anthropology Center who said the mans DNA was submitted to the FBIs Combined DNA Index System to search for a match. No results have been found.

A profile of the unidentified man was added to the National Missing and Unidentified Persons System.

People may view the mans profile athttps://www.namus.gov/UnidentifiedPersons/Case#/1585?nav.

The sheriffs office is now partnering with Othram, a private DNA lab who recovers human DNA to solve murder cases with hopes for an identification. The team is actively working on matches.

Were pursuing the same thing that solved the Florida case, DNA evidence, Brinkley said.

People who may have information on the man may contact Brinkley atrbrinkley@rcsotn.orgor at 615-904-3045.

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DNA from Rutherford County Leads to Identification of Body Found in Florida 40-Years-Ago - Wgnsradio

Significance

During the aging process, senescent cells secrete inflammatory factors, causing various age-related pathologies. Thus, controlling the senescence-associated secretory phenotype (SASP) can tremendously benefit human health. Although SASP seems to be induced by the alteration of chromosomal organization, its underlying mechanism remains unclear. Here, it has been revealed that noncoding RNA (ncRNA) transcribed from pericentromeric repetitive elements impairs the DNA binding of CCCTC-binding factor, resulting in the alteration of chromosomal accessibility and the activation of SASP-like inflammatory genes. Notably, the ncRNA was transferred into surrounding cells via small extracellular vesicles, acting as a tumorigenic SASP factor. Our study highlights a novel mechanism regulating chromatin interaction and inflammatory gene expression in senescence and cancer.

Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and noncell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.

Cellular senescence is a state of essentially irreversible cell cycle arrest induced by several stressors under various physiological and pathological conditions (1). Senescent cells that accumulate in vivo over the course of aging communicate with surrounding tissues through the production of proinflammatory proteins, termed the senescence-associated secretory phenotype (SASP), which are thought to promote multiple age-related diseases, including some cancers, such as breast and colon cancer (27). Therefore, elucidating the regulatory mechanism of the SASP is essential for developing new preventive and therapeutic strategies against age-related cancer.

Recent studies have reported that abnormal nuclear morphologies, observed as micronuclei or nuclear buds, induce SASP gene expression via the activation of the DNA-sensing pathway during cellular senescence (812). In addition, cellular senescence causes a dramatic alteration of chromatin organization, characterized by an increase in short-range chromatin contacts and genome-wide shrinkage of chromosome arms (13). Chromatin organization and global gene expression are coordinately maintained by the CCCTC-binding factor (CTCF), a zinc-finger (ZF) nucleic acidbinding protein, and the cohesin complex; together, these factors orchestrate higher-order chromatin conformation through the formation of intrachromosomal and interchromosomal loops (1416). Given that the nuclear localization and RNA-binding capacity of CTCF dynamically change due to cellular stress (17) and the alteration of CTCF distribution and/or followed by chromatin reorganization occur during cellular senescence (13, 18), postulating that CTCF distribution is associated with SASP gene expression in senescent cells is reasonable. However, the molecular mechanism underlying the connection between CTCF regulation and its association with SASP gene expression remains elusive.

In this study, we demonstrate that noncoding RNA (ncRNA) transcribed from pericentromeric repetitive satellite sequences changes the distribution of CTCF binding on the genome, thereby inducing SASP-like inflammatory gene expression via the functional impairment of CTCF in senescent cells. Furthermore, pericentromeric satellite RNA provokes tumorigenesis in a cell-autonomous or noncell-autonomous manner via a pathway involving exosomes, a type of small extracellular vesicle (EV). Our findings reveal a mechanism of CTCF regulation by pericentromeric satellite RNA during cellular senescence, which may contribute to the risk of tumorigenesis.

To elucidate the molecular mechanism underlying the alteration of chromatin organization and gene expression during cellular senescence, we first compared genome-wide chromatin accessibility between X-rayinduced senescent and proliferating IMR-90 cells, which are normal human diploid fibroblasts. Assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis revealed that the peak intensities in 16,325 regions were dramatically altered (false discovery rate [FDR] < 0.05) during cellular senescence (Fig. 1 A and B), and a high incidence of distal intergenic regions and introns was identified (SI Appendix, Fig. S1A). From ATAC-seq analysis, 14,356 peaks were identified as higher chromatin accessibility (red) in X-rayinduced senescent cells versus proliferating IMR-90 cells; 1,969 peaks were identified as lower chromatin accessibility (blue; Fig. 1B). The 16,325 ATAC-seq peaks that showed differential chromatin accessibility values in X-rayinduced senescent cells compared to those in proliferating cells (Fig. 1B) were annotated to 652 transcripts using databases, including GRCh37/hg19 (coding genes and some noncoding regions) and RepeatMasker (repetitive elements). Next, we focused on these 652 transcripts and reanalyzed their expression level using published RNA-sequencing (RNA-seq) data of proliferating and X-rayinduced senescent IMR-90 cells (GSE130727; Fig. 1C) (19). Thus, loci containing pericentromeric repetitive sequences called human satellite II (hSATII), which are epigenetically silenced in normal somatic cells, were highly accessible (yellow; Fig. 1B), and hSATII ncRNA expression was markedly up-regulated in X-rayinduced senescent IMR-90 cells compared to proliferating cells (log10 fold change = 2.8) among the transcripts showing an FDR < 1010 (Fig. 1C). When we integrated our ATAC-seq data with published RNA-seq data of proliferating and X-rayinduced senescent IMR-90 cells (GSE130727) (19), a comparative analysis of senescent and proliferating cells represented both higher chromatin accessibility and increased transcription at hSATII loci in senescent cells versus proliferating cells (Fig. 1D). In accordance with previous studies of senescent cells and many types of cancer (2023), we detected hSATII RNA expression by RT-qPCR and Northern blot analysis in H-RasV12 and serial passageinduced senescent cells (SI Appendix, Fig. S1 BF). To interpret the biological effects of hSATII RNA expression, we overexpressed hSATII RNA in SVts8 cells, a conditionally immortalized human fibroblast cell line suitable for transfection analysis (24). The ectopic expression of hSATII RNA, but not centromeric human satellite alpha RNA (hSAT), induced SASP-like inflammatory gene expression, which was shown as the enrichment of signatures related to the inflammatory response and SASP by gene set enrichment analysis and altered the chromatin accessibility of the loci of SASP genes (Fig. 1 EG and SI Appendix, Fig. S2 AD) (25). Importantly, the knockdown of hSATII RNA diminished the expression of SASP genes in X-rayinduced senescent SVts8 cells (Fig. 1H) or X-ray and serial passageinduced senescent IMR-90 cells (SI Appendix, Fig. S2 E and F). These data suggest that hSATII RNA regulates SASP-like inflammatory gene expression by altering chromatin accessibility during cellular senescence.

Pericentromeric hSATII RNA regulates SASP factor gene expression during cellular senescence. (AC) Screening of unique transcripts showing increased chromatin accessibility and active transcription during X-rayinduced senescence in IMR-90 cells. (A) A scheme of the screening steps. (B) Volcano plot of ATAC-seq signals showing fold change (FC) (x-axis) and FDR (y-axis) of chromatin accessibility between proliferating and X-rayinduced senescent IMR-90 cells. Red peaks show significantly increased chromatin accessibility in X-rayinduced senescent cells. Blue peaks showing significantly increased chromatin accessibility in proliferating cells. Black peaks show no significant changes. Yellow peaks containing hSATII loci show significantly increased chromatin accessibility. (C) Volcano plot of RNA-seq data (GSE130727) showing FC (x-axis) and FDR (y-axis) concerning 652 transcripts involved in an increased chromatin accessibility region between proliferating and X-rayinduced senescent IMR-90 cells from ATAC-seq analysis in B. The 47 transcripts showing FDR < 1010 are shown as red (up-regulated) or blue (down-regulated) dots. (D) Peaks of uniquely mapped reads by ATAC-seq and RNA-seq (GSE130727) in hSATII loci in proliferating or X-rayinduced senescent IMR-90 cells. Two biological replicates are shown. (EG) RNA-seq analysis of hSAT or hSATII-overexpressed and X-rayinduced senescent SVts8 cells. (E) Heatmap regarding SASP-related gene expression in hSAT or hSATII-overexpressed and X-rayinduced senescent SVts8 cells. (F) Scatterplot showing FC in hSAT (x-axis) or hSATII (y-axis) RNA-overexpressed SVts8 cells compared to empty vectorexpressed cells. Red dots indicate genes up-regulated (FC > 10) in vicinity of specific chromatin accessible peaks in hSATII RNAoverexpressed cells. (G) Gene set enrichment analysis of signatures associated with senescence (Upper) and inflammatory response (Lower) in hSAT or hSATII RNAoverexpressed SVts8 cells. NES, normalized enrichment score. (H) The effect of hSATII RNA knockdown on hSATII RNA and SASP gene expression in proliferating or X-rayinduced senescent SVts8 cells by RT-qPCR. The relative expression is shown as the FC from control small-interfering RNAtreated proliferating cells. Each bar represents mean SD of three biological replicates. ***P < 0.001 by one-way ANOVA, followed by the Tukeys multiple comparisons post hoc test.

To understand how hSATII RNA promotes SASP-like inflammatory gene expression, we attempted to identify hSATII RNAbinding proteins. Several studies have reported the association of centromeric hSAT RNA with specific proteins (26); however, thus far, none have reported such an association for pericentromeric hSATII RNA. RNA pull-down and mass spectrometry analysis identified 280 hSATII RNAbinding proteins (Fig. 2A and SI Appendix, Fig. S3A and Table S1). Among these proteins, we identified 26 chromatin-binding proteins by Gene Ontology (GO) analysis (GO: 0003682) and focused on CTCF because of both its high intensity score (unique peptides) and its relevance to chromatin organization (15, 16, 27) (Fig. 2A). Unlike hSAT RNA, hSATII RNA bound to CTCF, whereas both ncRNAs bound to lamin B1 (Fig. 2B) (26). Because CTCF binding to genomic DNA is important for the maintenance of genomic integrity and CTCF is an RNA-binding protein (2831), we performed RNA immunoprecipitation (RIP) analysis, demonstrating that the ZF DNA- and RNA-binding domains of CTCF are important for their binding to not only an exogenous hSATII RNA in human embryonic kidney (HEK)-293T cells (Fig. 2 C and D and SI Appendix, Fig. S3 B and C) but also an endogenous hSATII RNA in X-rayinduced senescent IMR-90 cells (SI Appendix, Fig. S3D). Of the 11 ZF domains of CTCF, the binding of ZF1 or ZF10 of CTCF to RNA is important for CTCF to form chromatin loops and regulate gene expression (31); however, we found that hSATII RNA bound to neither ZF1 nor ZF10 (SI Appendix, Fig. S3 B and C). Further analysis revealed that ZF3-ZF6 of CTCF-, known as DNA-binding domain (32), deficient mutant (CTCF ZF3-6) could not bind to hSATII RNA (Fig. 2 C and D), indicating that ZF3-ZF6 of CTCF is important for binding to hSATII RNA. Note that CTCF also binds to RNA through ZF domains; therefore, ZF3-6 might be an unfolding protein and not function appropriately. Further analysis will be needed to determine the interaction between CTCF and hSATII RNA. Importantly, the up-regulation of SASP-like inflammatory gene expression caused by hSATII RNA was canceled in the presence of excessive CTCF in SVts8 cells (Fig. 2E and SI Appendix, Fig. S3E). In contrast, CTCF depletion by RNA interference resulted in SASP-like inflammatory gene expression in proliferating cells (Fig. 2F and SI Appendix, Fig. S3F). Based on these results, we considered it likely that SASP-like inflammatory gene expression induced by hSATII RNA depends on the functional impairment of CTCF. Unexpectedly, we found that hSATII RNA expression was also up-regulated by CTCF depletion (Fig. 2F). Moreover, CTCF expression decreased during cellular senescence (SI Appendix, Fig. S3 G and H) (33). Together, these findings imply that CTCF regulates hSATII RNA expression during cellular senescence.

Pericentromeric hSATII RNA binds to CTCF. (A) GO analysis of 280 hSATII RNAbinding proteins (Left). Among these proteins, 26 genes were categorized as chromatin-binding (GO: 0003682), and the top 10 ranked genes and unique peptides are listed (Right). (B) RNA pull-down assay using SVts8 cell lysate followed by Western blotting confirmed hSATII RNA but not hSAT RNA bound to CTCF. (C) Western blot analysis of FLAG-tagged CTCF (WT: wild type) or CTCF ZF (deletion of ZF domain) in HEK-293T cells. (D) RIP followed by qPCR confirmed the binding of FLAG-tagged CTCF WT, but not CTCF ZF1-11 or ZF3-6, to hSATII RNA in HEK-293T cells. (E) RT-qPCR analysis of SASP-like inflammatory genes in hSATII RNAoverexpressed SVts8 cells with excess CTCF. The relative expression shows the value normalized from empty vectorexpressed cells. (F) RT-qPCR analysis of SASP-like inflammatory genes in CTCF-depleted SVts8 cells. The relative expression shows the value normalized from small-interfering control (siControl)treated cells. Each bar represents mean SD of three technical replicates repeated in two independent experiments (D, E, and F). *P < 0.05, **P < 0.01, ***P < 0.001, or N.S. (not significant) by one-way ANOVA, followed by the Tukeys (D and E) or Dunnetts (F) multiple comparisons post hoc test.

Furthermore, we investigated the expression of mouse major satellite (MajSAT) RNA, which is located at the pericentromeric locus of chromosomes, as well as human hSATII RNA. In mouse embryonic fibroblasts (MEFs), DNA damage induced by doxorubicin increased MajSAT RNA expression along with some canonical markers of cellular senescence (SI Appendix, Fig. S3I). As expected, the induction of MajSAT RNA was negatively correlated with the expression of CTCF (SI Appendix, Fig. S3I), and CTCF bound to pericentromeric MajSAT RNA but not mouse centromeric minor satellite (MinSAT) RNA, resulting in the up-regulation of SASP-like inflammatory genes (SI Appendix, Fig. S3 J and K). Taken together, these findings indicate that CTCF is crucial for the regulation of both pericentromeric satellite RNA and the expression of SASP-like inflammatory gene during cellular senescence (SI Appendix, Fig. S3M).

Because the ZF3-ZF6 DNA binding domain of CTCF was relevant to its binding to hSATII RNA (Fig. 2D), we hypothesized that hSATII RNA changes the DNA-binding capacity of CTCF via direct binding to its ZF domains. As expected, the ectopic expression of hSATII RNA altered the distribution of CTCF at its binding sites (Fig. 3 A and B). Remarkably, both chromatin immunoprecipitation (ChIP)-qPCR and electrophoretic mobility shift assay (EMSA) revealed that hSATII RNA inhibited the DNA-binding capacity of CTCF to an imprinting control region (ICR) positioned between IGF2 and H19, a well-known representative CTCF binding site, in a dose-dependent manner (Fig. 3 C and D) (15). In accordance with this data, pericentromeric MajSAT RNA bound to CTCF and diminished the binding of CTCF to ICR (SI Appendix, Fig. S3L). These notions raised the possibility that pericentromeric satellite RNA could change chromatin interaction as the binding of CTCF to DNA is important to maintain genomic integrity. To validate this assumption, we performed a chromosome conformation capture (3C) assay of the SASP genes in the vicinity of the CXCL10/CXCL11 locus because a robust interaction was noted in proliferating fibroblasts and various cell lines (SI Appendix, Fig. S4 AF) (34). We discovered that the ectopic expression of hSATII RNA significantly weakened interactions in the T2 and T22 regions, as revealed by 3C-qPCR assay, and increased chromatin accessibility within these loci, as revealed by ATAC-seq analysis, followed by the up-regulation of SASP-like inflammatory gene expression, as also observed in X-rayinduced senescent cells (Fig. 3 E and F). Together, these data indicate that the up-regulation of hSATII RNA in senescent cells causes a conformational change of chromatin structure in some SASP gene loci. Chromatin organization and global gene expression are coordinately regulated by CTCF during a variety of physiological and pathological events, such as embryonic development and carcinogenesis (27). However, the molecular mechanism underlying the connection between CTCF regulation and its association with SASP gene expression during cellular senescence has not been elucidated. Our findings demonstrated that pericentromeric satellite RNA influences chromatin interaction by interfering with CTCF function, resulting in changes in SASP-like inflammatory gene expression (SI Appendix, Fig. S4G).

Pericentromeric hSATII RNA changes chromosomal interaction via CTCF disturbance. (A and B) Venn diagram showing overlap of CTCF binding sites from ChIP-seq analysis. (B) Enrichment of peaks from ChIP-seq analysis whose signals on peak summit 2 kb region are shown as profile plot (Left) and heatmaps split into two clusters using the k-means algorithm (Right) over sets of genomic regions in SVts8 cells. Wilcoxon rank-sum test P values are shown. (C) ChIP-qPCR for CTCF binding to an ICR positioned between IGF2 and H19. (D) EMSA showing the effect of hSAT or hSATII RNA on CTCF binding to ICR. (E and F) RNA-seq, CTCF ChIP-seq, and ATAC-seq profiles of SVts8 cells in representative loci of the SASP factor genes, CXCL10 and CXCL11 (E), and chromatin conformation by 3C-qPCR assay (F). The interaction of a constant primer (C) with each target primer (T) is shown. Each bar represents mean SD of three technical replicates repeated in two independent experiments (C and F). *P < 0.05, ***P < 0.001, or N.S. (not significant) by one-way ANOVA, followed by the Tukeys multiple comparisons post hoc test (C) or unpaired two-sided t test (F).

Human and murine satellite RNAs have the potential to induce chromosomal instability (CIN), leading to tumorigenesis (26, 35, 36). Hence, we explored the possibility that the loss of CTCF could contribute to satellite RNAinduced CIN. In accordance with previous reports, we confirmed that the ectopic expression of hSATII RNA provoked multipolarity and chromosomal bridge formation (SI Appendix, Fig. S5 AC), which are typical characteristics of CIN (37). Furthermore, hSATII RNAoverexpressing cells exhibited obvious phenotypes of tumor cells, such as an abnormal chromosomal number and anchorage-independent growth (SI Appendix, Fig. S5 D and E). Notably, we found that excessive CTCF expression abolished CIN induced by hSATII RNA (SI Appendix, Fig. S5 F and G), a finding suggesting that CTCF plays a role in hSATII RNAinduced CIN, which is a risk factor for tumor development. Similarly, the ectopic expression of MajSAT RNA in MEFs also provoked multipolarity and chromosomal bridge formation in mitosis (SI Appendix, Fig. S6 AC), causing the formation of transformed foci and polyploidy transition (SI Appendix, Fig. S6 D and E). Surprisingly, these cells exhibited the ability to form tumors in immunodeficient mice, although control cells did not (SI Appendix, Fig. S6F). Collectively, we concluded that pericentromeric satellite RNA may promote susceptibility to carcinogenesis.

Furthermore, to gain insight into the biological significance of our findings, we focused on the function of hSATII RNA in the tumor microenvironment. We and others recently reported that small EVs secreted from cancer cells and/or senescent stromal cells dynamically contribute to tumor incidence and progression in a noncell-autonomous manner in the tumor microenvironment (3841). Intriguingly, the amounts of hSATII RNA, but not those of hSAT, were higher in small EVs derived from senescent cells than in those derived from proliferating cells (Fig. 4A). From an analysis of recently published RNA-seq data (42), we discovered that hSATII RNA could be detected in exosomes, a type of EV, secreted from cells of different human colon cancer cell lines (SI Appendix, Fig. S7A). Based on these observations, we speculated that hSATII RNA derived from senescent stromal cells would be transferred into surrounding cells through small EVs and function as a SASP-like inflammatory factor. Supporting this hypothesis, small EVs derived from senescent cells promoted anchorage-independent growth and CIN in normal cells (SI Appendix, Fig. S7 B and C). To assess the involvement of hSATII RNA in these phenotypes, we used EXOsomal transfer into cells (EXOtic) synthetic biology (43) to establish a designer exosome that contains hSATII RNA and found that these designer exosomes have tumorigenic activity similar to those of small EVs derived from senescent cells (SI Appendix, Fig. S7 DF). Importantly, these designer exosomes promoted SASP-like inflammatory gene expression (Fig. 4B). Together, these findings show that pericentromeric hSATII RNA in small EVs secreted from senescent cells promote SASP-like inflammatory gene expression and CIN in neighboring cells in the tumor microenvironment.

Pericentromeric hSATII RNA promotes tumor development in a cell-autonomous and noncell-autonomous manner. (A) RT-qPCR analysis of hSATII and hSAT RNA in the same number of small EVs derived from RPE-1/hTERT cells. Each value represents three biological replicates. *P < 0.05 or ***P < 0.001 by one-way ANOVA followed by the Dunnetts multiple comparisons post hoc test. (B) An effect of the designer exosome produced by the EXOtic devices on SASP-like inflammatory gene expression in SVts8 cells was evaluated by RT-qPCR. Each value was normalized from EXOtic-Nluc-treated cells. (CE) Representative and magnified (100) images (C) or quantified data (D and E) of RNA-ISH with hSATII RNA probe in colon cancer specimens. Black and red arrows indicate normal epithelial and tumor cells, respectively. Black and red arrowheads indicate fibroblasts and cancer-associated fibroblasts, respectively. (Scale bar, 200 m.) In the boxplot, the bottom and top hinges indicate the first and third quartile, respectively. The horizontal lines into the boxes indicate the median. The upper and lower whiskers define the highest and lowest value within 1.5 times of the interquartile range, respectively. n = 20 for each sample. **P < 0.01 or ***P < 0.001 by the Wilcoxon rank-sum test. Statistical analysis was performed using all samples and included outliers. (F) Schematic representation of this study. The up-regulation of pericentromeric satellite RNA during cellular senescence or aging provokes the expression of aberrant SASP-like inflammatory gene by interfering with the function of CTCF. In the tumor microenvironment, inflammatory proteins and hSATII RNA in small EVs are secreted from senescent stromal cells into surrounding tissue, where they act as SASP factors, thereby increasing the risk of carcinogenesis.

Finally, we checked the expression of pericentromeric ncRNA in the tumor microenvironment. Because the expression of murine satellite RNA is higher in malignant organoids derived from colon cancer (Apc716 Trp53R270H/R270H) than in nonmalignant organoids (Apc716) and accompanied by the reduction of CTCF and the up-regulation of SASP-like inflammatory genes (44) (SI Appendix, Fig. S7G), we evaluated the expression of hSATII RNA in surgical resection specimens from patients with primary colon carcinoma. We found that colon cancer cells expressing hSATII RNA were highly abundant in specimens compared with normal epithelial cells by RNA in situ hybridization (RNA-ISH) analysis (Fig. 4 C and D). Strikingly, we also observed that the population of hSATII RNApositive cells was significantly higher among cancer-associated fibroblasts than among fibroblasts in normal stromal tissues (Fig. 4 C and E). In summary, our findings suggest that senescent stromal cells expressing hSATII RNA support tumor development in a noncell-autonomous manner in the tumor microenvironment via the secretion of SASP-like inflammatory factors and small EVs containing hSATII RNA (Fig. 4F).

Cellular senescence causes a dramatic alteration of chromatin organization (13, 18); however, its effect on gene expression and implication for senescent cells are not fully understood. We identified ncRNA derived from pericentromeric repetitive elements as a novel inducer of SASP-like inflammatory gene expression that acts by altering chromatin interaction. Importantly, pericentromeric satellite RNA is up-regulated during cellular senescence and aging in vivo (Fig. 1 AD and SI Appendix, Fig. S1 BF), which decreases CTCF binding to genomic DNA and alters both chromatin interaction and transcription in SASP-like inflammatory gene loci, thereby increasing the risk of tumor development (Fig. 4F). To verify the physiological role of pericentromeric satellite RNA, we confirmed that the expression level of ectopic ncRNA was equivalent to that found in senescent cells. Furthermore, the knockdown of endogenous pericentromeric satellite RNA diminished the expression of SASP-like inflammatory genes in senescent cells despite having no effect on the induction of senescence, clearly indicating that endogenous pericentromeric satellite RNA plays a role in the expression of SASP-like inflammatory genes.

CTCF and the cohesin complex are essential for stabilizing chromatin organization, which has divergent effects on gene regulation, embryonic development, and tumorigenesis (27), through their binding ability to specific sequences in genomic DNA (15, 16, 45). Recently, some groups have reported that CTCF shows a high affinity for specific RNA and depends on binding with RNA to form chromatin loops and genome organization in mouse embryonic stem cells (30, 31). Another group also has shown that the interaction of CTCF with long ncRNA, such as Tsix and Xite, mediates long-range chromosomal interactions, inducing homologous X chromosome pairing in mouse embryonic stem cells (29). In contrast, in senescent cells (in pathological conditions), we have revealed a novel molecular mechanism in which pericentromeric satellite RNA regulates chromatin interaction and gene expression by CTCF disturbance. Because pericentromeric satellite RNA expression is at an extremely low level in normal cells (SI Appendix, Fig. S1 BF) (20), we considered that it is insufficient for the RNA to disturb CTCF function in physiological conditions but not in senescent and tumor cells that aberrantly express pericentromeric satellite RNA. In the previous study, Zirkel et al. revealed that, upon senescence entry, the high-mobility group B protein (HMGB2) nuclear depletion provokes the alteration of CTCF distribution and CTCF spatial clustering (18). Moreover, Lehman et al. showed that stressors, such as acute oxidative stress, cause CTCF reduction from nuclear speckles and changes in CTCF RNA interaction (17). These reports support our findings that pericentromeric satellite RNA up-regulated during cellular senescence directly binds to CTCF and disturbs CTCF function.

Furthermore, our observations also showed that CTCF maintains pericentromeric satellite RNA expression at extremely low levels in normal cells by directly binding the pericentromeric hSATII locus (46); however, CTCF expression significantly decreased during cellular senescence (SI Appendix, Fig. S3 G and H). Therefore, once pericentromeric satellite RNA is predominantly expressed by CTCF depression, satellite RNA alters chromatin interaction and induces CIN and SASP-like inflammatory gene expression via CTCF disturbance (SI Appendix, Fig. S3M). Previous studies have revealed that CTCF binds to pericentromeric/centromeric regions and recruits the centromeric protein CENP-E to these regions in mitotic chromosomes (46, 47). In addition to these findings, we have identified a regulatory machinery involving CTCF that controls pericentromeric satellite RNA expression.

In patients with gastrointestinal cancer, driver mutations were detected in the ZF domains of CTCF that provoke CIN and aberrant gene expression (48). Although CTCF-knockout mouse embryos die at early implantation stages (49), CTCF haploinsufficient (Ctcf+/) mice are markedly susceptible to cancer, and transformed foci are observed in Ctcf+/ mousederived MEFs (35). These reports strongly support our conclusion that the disturbance of CTCF function caused by pericentromeric satellite RNA results in aberrant gene expression, CIN, and tumorigenesis. Centromeric satellite RNA (hSAT and MinSAT RNA) is associated with CIN in some cell lines (26), but we believe that there must be another mechanism, as these centromeric satellite RNAs neither bind to CTCF nor contribute to SASP-like inflammatory gene expression in normal fibroblasts (Figs. 1 E and F and 2B and SI Appendix, Figs. S2 A and B and S3K).

We conclude that pericentromeric satellite RNA plays a prominent role in tumorigenesis by cell-autonomous and noncell-autonomous pathways in vivo: 1) DNA damage caused by various oncogenic stresses induces cellular senescence of normal epithelial cells, thereby acting as a tumor-suppressor mechanism. In these senescent cells in benign tumors, pericentromeric satellite RNA is up-regulated and leads to SASP-like inflammatory induction (1, 2, 4, 50). If these senescent cells override their cell cycle arrest, pericentromeric satellite RNA may contribute to transformation from a benign to a malignant tumor through CIN (51). 2) Pericentromeric satellite RNA could be transferred into cancer cells via small EVs from senescent stromal cells, provoking CIN and SASP-like inflammatory gene expression, resulting in tumor progression. Our data suggest that secreted pericentromeric satellite RNA also functions as a tumorigenic SASP factor via small EVs in the tumor microenvironment. Furthermore, the down-regulation of CTCF expression with age may trigger the up-regulation of pericentromeric satellite RNA expression and diminish CTCF function via a positive-feedback loop, subsequently promoting SASP-related inflammation and tumorigenesis during aging (Fig. 4F). Our findings clearly indicate that pericentromeric satellite RNA represents a therapeutic target for age-related pathologies.

A full description of the following methods is described in the SI Appendix, Supplementary Information Methods: cell culture, plasmid construction, RNA interference, RT-PCR, RT-qPCR, Northern blot, RNA pull-down assay, Western blotting, mass spectrometric analysis, ChIP followed by ChIP sequencing (ChIP-seq), 3C-qPCR, ATAC-seq, RIP, EMSA, immunofluorescence imaging, karyotype analysis, focus formation assay, anchorage-independent soft agar colony formation assay, RNA-seq, extraction and application of exosome-like EVs, RNA-ISH, organoid culture experiments, in vivo allograft assays, bioinformatical analysis, and statistical analysis.

TIG-3 cells (11, 38, 52) and IMR-90 cells were obtained from the Japanese Cancer Research Resources Bank and American Type Culture Collection, respectively. TIG-3 cells, IMR-90, and IMR-90/ER:H-RasV12 cells (52) were cultured in Dulbeccos Modified Eagles (DME) medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Sigma-Aldrich) at physiological oxygen conditions (92% N2, 5% CO2, and 3% O2) at 37 C. RPE-1/hTERT cells (39) and HEK-293T cells (52) were cultured in DME medium (Nacalai Tesque) supplemented with 10% FBS and penicillin/streptomycin (Sigma-Aldrich) in a 5% CO2 incubator at 37 C. SVts8 cells (24) were cultured in DME medium (Nacalai Tesque) supplemented with 10% FBS and penicillin/streptomycin (Sigma-Aldrich) in a 5% CO2 incubator at 34 C. MEFs were generated from CD-1 mice as previously described (53) and then cultured in DME medium (Nacalai Tesque) supplemented with 10% FBS and penicillin/streptomycin (Sigma-Aldrich) at physiological oxygen conditions (92% N2, 5% CO2, and 3% O2) at 37 C. All cell lines used were negative for mycoplasma.

Cell pellets were lysed in lysis buffer (0.1 M TrisHCl pH 7.5, 10% glycerol, and 1% sodium dodecyl sulfate [SDS]), boiled for 5 min, and then centrifuged for 10 min at 15,000 rpm. All protein concentrations were determined by BCA Protein Assay Reagent (Pierce). Each cell lysate was electrophoresed by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking with 5% skim milk (Megumilk) or 5% bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% Tween 20 (TBST), the membrane was treated with primary antibodies to p16 (IBL, #11104, 1:250 dilution), lamin B1 (Abcam, #ab16048, 1:1,000 dilution), GAPDH (Proteintech, #60004-1-lg, 1:10,000 dilution), vinculin (Sigma-Aldrich, #V9131, 1:1,000), CTCF (Cell Signaling Technology, #3418, 1:1,000 dilution), DDDK-tag (MBL, #M185-3L, 1:5,000), and ras (Oncogene, #OP41, 1:1,000 dilution) overnight at 4 C in blocking buffer. Membranes were then washed three times in TBST and incubated with an enhanced chemiluminescence (ECL) anti-mouse IgG, horseradish peroxidaselinked whole antibody (GE Healthcare, NA931V) or ECL anti-rabbit IgG, horseradish peroxidaselinked whole antibody (GE Healthcare, NA934V) for 1 h at room temperature. After washing the membrane three times in TBST, the signal was resolved with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and imaged on a FUSION imaging system (Vilber Lourmat).

hSATII RNA was detected on formalin-fixed paraffin-embedded (FFPE) sections in primary colon cancer specimens using an Advanced Cell Diagnostics (ACD) RNAscope 2.5 HD Reagent Kit-BROWN (ACD, #322300) and the RNAscope Target Probe Hs-HSATII (ACD, #504071) according to the manufacturers instructions. For each sample (n = 10), two images (100) of normal mucosa, submucosa, and tumor were randomly selected. The areas of hSATII RNA positivity and total cells were analyzed using the ImageJ software (https://imagej.nih.gov/ij/docs/faqs.html). The hSATII RNApositive area per field (percent) of each type of cell was calculated as the proportion of the total positive area to the total area of cells.

The FFPE sections in primary colon cancer specimens were collected from patients who provided informed consent for genetic and cell biological analyses. All methods were performed in accordance with protocols approved by the Institutional Review Board (approval number: 2013-1090) of the Japanese Foundation for Cancer Research (JFCR).

MEF/Vector or MEF/MajSAT RNA (5 106 cells) in Hanks Balanced Salt Solution (Gibco/Thermo Fisher Scientific) were subcutaneously injected with an equal volume of Matrigel (BD Pharmingen) into 4- or 5-wk-old female BALB/c-nu/nu mice (Charles River Laboratories). After 20 or 30 d of cell injection, the mice were euthanized and tumor weight was measured. All animal procedures were performed using protocols approved by the JFCR Animal Care and Use Committee in accordance with the relevant guidelines and regulations (approval number: 1804-05).

Parametric statistical analyses were performed using the unpaired two-tailed Students t test (Fig. 3F and SI Appendix, Figs. S1C, S3 G, I, J, and L, S5 BE, S6 BE, and S7 EG) or one-way ANOVA, followed by the Dunnetts (Figs. 2F and 4A and SI Appendix, Figs. S2A and S7C) or Tukeys (Figs. 1H, 2 D and E, and 3C and SI Appendix, Figs. S2 E and F, S3 C and D, and S5G) multiple comparisons post hoc test using the R software for statistical computing (64-bit version 3.6.1). Nonparametric statistical analyses were performed using the Wilcoxon rank-sum test (Figs. 3B and 4 D and E) or the KruskalWallis H test (one-way ANOVA on ranks) followed by the Steels multiple comparisons post hoc test (SI Appendix, Fig. S6F) using the R software for statistical computing. P < 0.05 was considered statistically significant. All experiments, except for mass spectrometric analysis, were repeated at least twice.

The sequence and processing data have been deposited in the DNA Data Bank of Japan with the accession numbers DRA009771 for RNA-seq, DRA010750 for ChIP-seq, and DRA010749 for ATAC-seq. All other data supporting the findings of this study are available within the article and SI Appendix.

We thank K. Nagasaka for valuable suggestions; T. Yamamoto and N. Saitoh for technical assistance for chromatin conformation analysis; K. Matsumoto for technical assistance for northern blot; S. Kuraku, C. Obuse, S. Adachi, and T. Natsume for mass spectrometry; N. Tanaka for bioinformatics analysis; G. Hannon and D. Beach for providing the MaRX vector; R. Asaka, K. Baba, and K. Takeuchi for technical advice for in situ experiments; H. Siomi for technical assistance for the RNAprotein interaction study; and members of the A.T. laboratory for helpful discussions during the preparation of this manuscript. This work was supported in part by the Japan Science and Technology Agency (JST)-Precursory Research for Embryonic Science and Technology (PRESTO) under grant number JPMJPR17H7; JST-Moonshot R and D under grant number JPMJPS2022; the Japan Agency of Medical Research and DevelopmentAdvanced Research and Development Programs for Medical Innovation (PRIME) under Grant 19gm6110023h0001; the Japan Society for the Promotion of Science (JSPS) under Grants 19H03507, 18K15254, and 20K16344; the Princess Takamatsu Cancer Research Fund; the Mitsubishi Foundation; and the Takeda Science Foundation. This research was also supported by the Research Fellowships for Young Scientists from JSPS under Grant 19J00796.

Author contributions: K.M. and A.T. designed research; K.M., Y.I., S.H., M. Nishio, T.M.L., R.O., R.F., L.J., H.Z., T.S., and A.T. performed research; L.Y., T.N., R.M., R.K., M. Nakayama, M.O., S.N., H. Seimiya, T.H., and H. Saya contributed new reagents/analytic tools; K.M., K.U., S.T., K.S., E.H., and A.T. analyzed data; and K.M. and A.T. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2025647118/-/DCSupplemental.

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Pericentromeric noncoding RNA changes DNA binding of CTCF and inflammatory gene expression in senescence and cancer - pnas.org

Illustration. Credit: Yuval Robichek, Weizmann Institute of Science

Proteins communicating through the DNA molecule constitute a newly discovered genetic switch.

Proteins can communicate through DNA, conducting a long-distance dialogue that serves as a kind of genetic switch, according to Weizmann Institute of Science researchers. They found that the binding of proteins to one site of a DNA molecule can physically affect another binding site at a distant location, and that this peer effect activates certain genes. This effect had previously been observed in artificial systems, but the Weizmann study is the first to show it takes place in the DNA of living organisms.

A team headed by Dr. Hagen Hofmann of the Chemical and Structural Biology Department made this discovery while studying a peculiar phenomenon in the soil bacteria Bacillus subtilis. A small minority of these bacteria demonstrate a unique skill: an ability to enrich their genomes by taking up bacterial gene segments scattered in the soil around them. This ability depends on a protein called ComK, a transcription factor, which binds to the DNA to activate the genes that make the scavenging possible. However, it was unknown how exactly this activation works.

(l-r) Dr. Nadav Elad, Dr. Haim Rozenberg, Dr. Gabriel Rosenblum, Jakub Jungwirth and Dr. Hagen Hofmann. Twisting a rope from one end. Credit: Weizmann Institute of Science

Staff Scientist Dr. Gabriel Rosenblum led this study, in which the researchers explored the bacterial DNA using advanced biophysical tools single-molecule FRET and cryogenic electron microscopy. In particular, they focused on the two sites on the DNA molecule to which ComK proteins bind.

They found that when two ComK molecules bind to one of the sites, it sets off a signal that facilitates the binding of two additional ComK molecules at the second site. The signal can travel between the sites because physical changes triggered by the original proteins binding create tension that is transmitted along the DNA, something like twisting a rope from one end. Once all four molecules are bound to the DNA, a threshold is passed, switching on the bacteriums gene scavenging ability.

We were surprised to discover that DNA, in addition to containing the genetic code, acts like a communication cable, transmitting information over a relatively long distance from one protein binding site to another, Rosenblum says.

A 3D reconstruction from single particles of bacterial DNA (gray) and ComK proteins (red), imaged by cryogenic electron microscopy, viewed from the front (left) and at a 90 degrees rotation. ComK molecules bound to two sites communicate through the DNA segment between them. Credit: Weizmann Institute of Science

By manipulating the bacterial DNA and monitoring the effects of these manipulations, the scientists clarified the details of the long-distance communication within the DNA. They found that for communication or cooperation between two sites to occur, these sites must be located at a particular distance from one another, and they must face the same direction on the DNA helix. Any deviation from these two conditions for example, increasing the distance weakened the communication. The sequence of genetic letters running between the two sites was found to have little effect on this communication, whereas a break in the DNA interrupted it completely, providing further evidence that this communication occurs through a physical connection.

Knowing these details may help design molecular switches of desired strengths for a variety of applications. The latter may include genetically engineering bacteria to clean up environmental pollution or synthesizing enzymes to be used as drugs.

Long-distance communication within a DNA molecule is a new type of regulatory mechanism one that opens up previously unavailable methods for designing the genetic circuits of the future, Hofmann says.

Reference: Allostery through DNA drives phenotype switching by Gabriel Rosenblum, Nadav Elad, Haim Rozenberg, Felix Wiggers, Jakub Jungwirth and Hagen Hofmann, 20 May 2021, Nature Communications.DOI: 10.1038/s41467-021-23148-2

The research team included Dr. Nadav Elad of Weizmanns Chemical Research Support Department; Dr. Haim Rozenberg and Dr. Felix Wiggers of the Chemical and Structural Biology Department; and Jakub Jungwirth of the Chemical and Biological Physics Department.

Dr. Hagen Hofmann is the incumbent of the Corinne S. Koshland Career Development Chair in Perpetuity.

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Calling Through the DNA Wire: A Newly Discovered Genetic Switch - SciTechDaily

We found a handful of new Jurassic World items today. Our finds include a Mr. DNA reversible bucket hat, a Dinosaur Observation Committee crossbody bag, a Camp Cretaceous lunch bag, and a pin set. Lets dig in to the new merchandise.

Both sides of this bucket hat are bright and colorful.

The blue side has the Jurassic World logo, surrounded by DNA strands and Mr. DNA himself.

Universal Studios text and a mosquito are embroidered on the other side.

Reversing the hat reveals the yellow side.

The yellow side is all about Mr. DNA.

A Universal Studios tag is stitched to one side.

The Jurassic World logo hangs from the bag.

Two zippered pockets are on the opposite side.

A Dinosaur Observation Committee badge is attached in the corner. The artwork has been used on other Jurassic World merchandise.

Inside, we find a Jurassic World tag and an additional zippered pocket.

This lunch box is perfect for day trips to Camp Cretaceous.

We dont think anyone will steal our lunch with this dino protecting it. Three unique patches round out the design.

The opposite side has additional artwork and a Universal Studios text logo. A single mesh pouch is provided to hold a bottle.

The zipper pulls are dinosaur teeth, complete with the Jurassic World logo!

This Jurassic World pin set includes three separate pins.

The largest pin is a blue Jurassic World sign pin. It is surrounded by DNA.

The Mr. DNA pin is the most colorful in the set.

An atom rounds out the set. We found all four items in Jurassic Outfitters. The reversible hat and lunch bag are also available at Dinostore.

For more Universal Studios news from around the world, follow Universal Parks News Today on Twitter, Facebook, and Instagram.

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PHOTOS: New Jurassic World Pin Set, Crossbody, Lunch Bag, and Mr. DNA Reversible Bucket Hat Available at Universal's Islands of Adventure - wdwnt.com

DUBLIN, Sept. 1, 2021 /PRNewswire/ -- The "DNA Vaccines: Technologies and Global Markets 2021-2026" report has been added to ResearchAndMarkets.com's offering.

Research and Markets Logo

The global market for DNA vaccines should grow from $3.1 billion in 2021 to $11.5 billion by 2026 with a compound annual growth rate (CAGR) of 30.1% for the period of 2021-2026.

DNA vaccines market for research tools should grow from $2.2 billion in 2021 to $8.7 billion by 2026 with a compound annual growth rate (CAGR) of 32.3% for the period of 2021-2026.

DNA vaccines market for clinical vaccines should grow from $924 million in 2021 to $2.8 billion by 2026 with a compound annual growth rate (CAGR) of 24.5% for the period of 2021-2026.

Report Scope

The study's scope includes DNA vaccine products that already are commercialized or likely to be in the next five years. Both human and animal health markets are studied. DNA vaccine delivery technologies are also included. DNA vaccine candidates in clinical trials are examined by indication, and future market growth from 2021 through 2026 is forecast.

The role that DNA vaccines play in the overall vaccine industry is examined, as well as how the vaccine industry structure and dynamics are changing.

We examine DNA synthesis, biotechnology and pharmaceuticals firms, strategic industry alliances, and the role of gene delivery and synthesis technologies. The major markets for DNA vaccines, including infectious diseases, cancers, animal health, allergies and biodefense, are analyzed, and the main companies in these fields are highlighted.

The Report Includes

14 data tables and 55 additional tables

An overview of the global market for DNA vaccines and related technologies

Analyses of global market trends, with data from 2019, 2020, estimates for 2021, and projections of compound annual growth rates (CAGRs) through 2026

Coverage of delivery and synthesis technologies, the forces driving market growth, product formats, and market applications for these products

Identification of new opportunities, challenges, and technological changes within the industry and highlights of the market potential for DNA vaccines by delivery technology, format, function and region

Coverage of life cycle status and commercial status of DNA vaccine technologies and brief description of the Human Immune System

Details about vaccines, their evolution, and types including DNA vaccines and cancer DNA vaccines, their function, scope and clinical trials, and information on changing vaccine paradigm, antigen discovery, plasmid design and manufacture and delivery technologies

Detailed analysis of the current market trends, market forecast, and discussion of technological, and regulatory elements that are affecting the future marketplace

Information about major technologies for the formulation of DNA vaccines and assessment of their relation to biotechnology, gene therapy, DNA delivery, pharmaceuticals, and biodefense companies

Comprehensive profiles of leading companies in the field as well as updates to alliance, merger, and acquisition activities, including Astellas Pharma Inc., Bristol-Myers Squibb Co., Merck & Co., Novartis AG, Pfizer Inc. and Sanofi-Aventisa

Vaccines, once a stable though unexciting sector of the pharmaceutical industry, are becoming an attractive growth opportunity. DNA vaccines are an emerging vaccine platform within this changing paradigm. DNA vaccines target a wide range of traditional pharmaceutical markets, such as cancers and allergies, as well as infectious diseases. The vaccine industry has proved that it can generate products with nontraditional applications and blockbuster potential. Products such as that from ViroCyt, now part of Sartorius Stedim Biotech, are leading the field of rapid virus quantification. DNA vaccines are poised to generate significant future market potential.

Story continues

New biotechnologies and nanotechnologies are driving DNA vaccine development. Particularly key to DNA vaccines reaching their potential are emerging delivery technologies such as electroporation (EP), innovative vaccine formats such as DNA prime-adenovector boost, and novel molecular adjuvant technologies. These technologies are providing the means for achieving the higher efficacy in humans that is required for the commercialization of DNA vaccines.

DNA vaccines have already made significant progress to date. There are three approved DNA vaccines for animal health applications and nearly 100 clinical trials underway in humans for a wide range of diseases. There is a deep pipeline of preclinical projects. A small but strategic market segment is commercial today, consisting of research tools and animal health applications.

The high growth is the result of a low starting base and a forecast introduction of several DNA vaccines late in the period. While research tools and animal health clinical applications currently dominate the market, human clinical DNA vaccines will make up the vast majority of this market through 2026.

Key Topics Covered:

Chapter 1 Introduction

Chapter 2 Summary

Chapter 3 Market Overview

Introduction

DNA Vaccine Technologies Covered in This Report

Global Market for DNA Vaccines

Forces Driving DNA Vaccine Market Growth

Life Cycle Status of DNA Vaccine Technologies

Commercial Status of DNA Vaccine Technologies

DNA Vaccine Industry

Chapter 4 DNA Vaccine Technologies

Introduction

Human Immune System

Vaccines

Evolution of Vaccines

Types of Vaccines

DNA Vaccines

Changing Vaccine Paradigm

Function and Scope of DNA Vaccines

Cancer DNA Vaccines

DNA Vaccine Technology Value Chain

Antigen Discovery

Plasmid Design

Plasmid Manufacture

Delivery Technologies

Uncomplexed pDNA

Electroporation

Liposomes

Gold Particles

Nanoparticles

Bacterial Ghosts

Bacteriophages

Viruses

Targeting Technologies

Adjuvant Technologies

DNA Vaccine Technology Needs

Chapter 5 DNA Vaccine Applications

Research Tool Applications

Clinical Application

Overview to DNA Vaccine Clinical Trials Process

Summary of DNA Vaccine Clinical Trials

Chapter 6 DNA Vaccine Industry

Vaccine Industry Structural Shifts

Industry Structure

DNA Vaccine Industry Competitors

DNA Vaccine Commercial Value Chain

DNA Vaccine Competitor Strategic Positioning

Research & Development

Chapter 7 DNA Vaccine Markets

Drivers of Growth

DNA Vaccine Markets

Market Forecasts

DNA Vaccine Research Tool Markets

DNA Vaccine Clinical Markets

DNA Vaccine Market by Region

Research Tools for DNA Vaccine Market by Region

Clinical DNA Vaccine Market by Region

Chapter 8 Impact of COVID-19

Vaccines, Treatments and Diagnostics

Vaccines

Therapeutics

Economic Impact of COVID-19

Chapter 9 Company Profiles

Astellas Pharma Inc.

Astrazeneca Plc

Bristol-Myers Squibb Co.

Gilead Sciences Inc.

Johnson & Johnson

Merck & Co.

Novartis Ag

Pfizer Inc.

Sanofi-Aventis

For more information about this report visit https://www.researchandmarkets.com/r/o8dxo1

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The Worldwide DNA Vaccines Industry Should Reach $11.5 Billion by 2026 - Yahoo Finance

After the dire misery of last year, vaccines have emerged as mankinds main weapon against the COVID-19pandemic. Governments all over the world are striving to get as many people vaccinated as fast as possible.

India has administered around 65 crore doses, with around 15 crore fully vaccinated individuals and 50 crore vaccinated with a single shot. If you're one of the vaccinated, theres a lot of information out there to give an idea of what exactly happens when the COVID-19 vaccine is injected into the body.

Here we talk about what is injected via a vaccine, what happens inside the body when it responds to the jab, why many people get some common side-effects and finally, why is there a need with several COVID-19 vaccines to get a second shot.

What happens when vaccine serum is injected into the body?

By definition, vaccine is a process of acquired immunity to fight off a future infection. Vaccines contain an agent resembling the virus, bacteria or parasite causing a disease, which in the case of COVID-19 is the SARS-COV-2 virus. It is either a weakened or dead microorganism, or its toxins or a surface protein. This agent carries the virus genetical material, which can then be read by the body to formulate the immune response.

When the vaccine is injected, the agent goes into the cells of our tissues. It captures the attention of certain 'dendritic' cells, which have a specific function to monitor intruders that may have entered the body. These patrolling cells come in contact with this new never-before-seen agent and alarm the body against it.

The dendritic cells do this by reading the genetic instructions about the virus carried into the body by the vaccine agent. The information is then replicated for the immune system to read and react.The cells travel to a lymph node to identify the right cells in the body and then activates them against the virus.

Why does the vaccine give side-effects to some people?

When it comes to vaccines, most side-effects literally mean that your immune system is responding as it is supposed to. With COVID-19 vaccines, common side effects range from soreness or swelling in the arm where the vaccine was injected, fatigue, headache, fever, chills, muscular pain and nausea.

Vaccines trick the immune system in believing that an actual pathogen has entered the body as it cannot tell the difference between the actual virus and a vaccine agent. The white blood cells rush to the spot to break down the virus and antibodies then attack the debris spread around from the breakdown. This makes the spot of injection akin to a tiny battlefield.

Fatigue and soreness after receiving the vaccine shot is due to cytokines and chemokines. These substances are directing more of the immune cells from other parts of the body to the infected site. This also causes inflammation and temporary swelling in lymph nodes in the armpit area.

Why the need for a second shot?

Several of the COVID-19 vaccines, including the COVAXIN and Covishield being administered in India, need a second booster shot after some time. This is because the first shot creates neutralizing antibodies in the body that block the SARS-COV-2 virus from making one sick but this formation of protective antibodies can be short-lived. Thus, a second dose is needed in most cases to help the body generate a more robust and long-term response against illness by locking the memory of the virus.

The second shot helps the body form long-term memory-cells in addition to short-term protective antibodies. This is also why several people see much stronger side-effects after the second shot, because the body now has a stronger, faster and better-equipped immune response against the virus.

The fear of side effects might tempt many to forego the second shot, but it should be remembered that the potential effects of a COVID-19 infection could be much worse and even fatal.

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DNA Explainer: Received your COVID-19 vaccine? Know what's going on inside your body - DNA India

If in space, no one can hear you scream, then what happens when DNA silently breaks in microgravity? Spoiler alert: it heals itself.

This is what a CRISPR experiment that won the Genes in Space 6 contest SYFY WIRE was at the recent launchof the Genes in Space 8 experiment to the ISS found out. DNA damage or breakage can mean the potential for degenerative diseases such as cancer, but the fact that it can also self-repair (and in microgravity!) has enormous implications for medical treatments above and below Earths atmosphere.

Genes in Space picks the brains of teen scientists in grades 7 through 12 to come up with a DNA analysis experiment that can be carried out in the ISS U.S. National Lab, which has really become more like a molecular laboratory in space. Aarthi Vijayakumar, Michelle Sung, Rebecca Li, and David Li were the winners who wanted to know if broken DNA could heal in zero-G.

Along with NASA microbiologists Sarah Rommel and Sarah Wallace, the students recently published a study in PLOS ONE.

We have the capabilities to extract, amplify, purify, prepare sequencing libraries, and sequence nucleic acids onboard the ISS, Wallace told SYFY WIRE. While we have come a long way, there is still a lot that could advance the work further.

You cant just pack up an experiment and launch it to the ISS as is. Wallace and Rommel, in collaboration with biologist Emily Gleason of miniPCR and the Genes in Space Program, needed to create custom kits that took both the conditions in space and safety of the crew into account. Everything in those kits must undergo a tough toxicological assessment to make sure the astronauts are not being exposed to anything dangerous almost 250 miles from the ground. If something happens, astronauts cant just wash their hands or roll the window down.

The ISS, as Wallace sees it, is a semi-closed system. Anything that is brought in from outside could possibly be a hazard, which is why scientists much make sure that whatever is on a payload will not endanger astronauts or mess with life support systems. Procedures also need to be streamlined. There is not that much crew time to go around, since the crew have multiple experiments to manage and also take care of other tasks, such as going on spacewalks to resolve maintenance issues. Everything in a kit needs to be packaged for one convenient use.

The end result is a custom kit that has everything the astronauts need to perform the experiment we have planned, said Wallace. We use a lot of things from commercial kits, but we also use reagents we make in our labs, so it really is similar to what molecular labs on Earth are doing.

There is a huge advantage to going through with the entire experiment in space rather than just breaking the DNA on Earth and sending it up to the ISS to see whether it can be repaired. Not everything used in a lab on terra firma can transition to microgravity. At least CRISPR (which is really an acronym for Clustered Regularly Insterspaced Short Palindromic Repeats) does. It replicates an immune response in which bacteria copy DNA sequences to RNA, which sends a protein to cut the DNA. CRISPR can break DNA in a specific place without random damage.

"It has been hypothesized that DNA is more likely to be repaired by the error prone method in microgravity than it is on Earth, but there hasn't been a good way to study this before now." Gleason also told SYFY WIRE. "The students wanted to know ifDNA is repaired differently in microgravity andproposed using the CRISPR/Cas9 system to damage DNA so that we could study how it was repaired in space."

Astronauts created breaks in both strands of the double helix and waited to see what would happen. Our bodies do repair DNA on their own, but would these genes, taken from yeast, be salvageable in space? Replicating these processes in space was previously prevented by technical and safety risks that have now been worked out. After the DNA had repaired itself, it was sequenced to make sure that all its components had gone back to where they belonged. The methods used on the ISS arent limited to space, something Wallace is excited about.

I believe that there is massive potential for this type of portable method in the diagnostic space, she said. There are many potential applications of these methods and technology, ranging from environmental monitoring, pathogen identification, revealing antimicrobial resistance, and so much more.

Wallace and her team are currently working on a collaboration that will optimize an ISS method to see what kinds of microbes are crawling around in hospital rooms, since antibiotic resistance is an issue that (especially in this pandemic era) threatens healthcare. The miniPCR tech that is being used on the ISS has already been used in COVID-19 diagnostics, andGleason also looks forward to updates that will be made to the ISS lab and Genes in Space.

"There's so much that can be done with the tools and procedures already on the ISS," she said. "Genes in space has plans to add flurorescence visulaization and cell-free protein synthesis technology next year."

This goes to show that studies done in microgravity this can go from Earth to space and back again.

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CRISPR just proved genes can self-heal in space for the first time ever - SYFY WIRE