Daily Archives: July 17, 2022

Ultima CEO Gilad Almogy, Ph.D./courtesy of Ultima Genomics

The information stored within the confines of the human genome is some of the most important data we can use in the diagnosis of disease, prevention efforts and therapeutics. Despite the fact that the technology to conduct whole genome sequencing (WGS) has been around for decades, financial barriers have stood in the way not just for patients and doctors looking for information, but for researchers as well.

Leveraging over $600 million in funding and five years of hard work, Newark, California-based Ultima Genomics has designed a way to surmount that financial barrier lowering the cost of a human genome from the realm of $1,000 to just $100.

Ultima has achieved this through the use of its sequencing architecture which replaces the traditional flow cell, a channel that contains all of the surfaces where chemistry and imaging occur during sequencing, with a silicon wafer. The technology serves the same function but at a lower cost with larger surface area, allowing for billions of reads. The process is easier to scale, amounting in large volumes of genetic data, and avoids costly and complicated fluidics.

The company also touts novel scalable chemistry, which combines the speed, efficiency and read lengths of natural nucleotides with the accuracy and scalability of endpoint detection. Add machine learning at the genome scale that can deliver accurate results and youve got yourself a cost-effective and useful human genome ready for interpretation.

Why it Matters

The importance of cost goes beyond simply enabling access to a larger quantity of existing genomic solutions. It also enables qualitatively different experiments to be envisioned and executed, not just once, but routinely, Ultima CEO Gilad Almogy, Ph.D. said in an interview with BioSpace. This can enable scientists to ask new questions they previously couldnt answer, and it can change the way genomic information is incorporated into the broader healthcare system.

The $100 genome stands to make genomics research that was once thought of as impossible, possible. In 2020, an article celebrating the 20th anniversary of Nature Reviews Genetics discussed the future of genetics and genomics research. In the piece, Stacey Gabriel, senior director of the genomics platform at the Broad Institute, commented that the real promise of genome sequencing lies in true population-scale sequencing at the scale of tens of millions of individuals that would enable the comprehensive, unbiased study of the human genome and the variations found within it.

Genomic research has provided physicians with a wealth of knowledge about genes that can increase a persons risk of developing a certain condition, such as the BRCA2 gene which is linked to an increased risk of developing breast and ovarian cancer. However, without the ability to conduct large-scale studies, simply understanding the role that one gene or a handful of genetic mutations plays in developing disease is often not enough information to elucidate the genome's full impact. With scalable and cost-effective WGS, it will become much easier for researchers to understand the parties within our genome that contribute to the manifestation of disease, which could ultimately lead to targeted therapeutics.

Genomic Data can Inform Treatment

Gabriel stated that she believes WGS should become a part of the electronic health record. There are plenty of good reasons to collect and include genomic data as it relates to health and disease. Beyond using this data to understand the risk someone is at for a certain condition, genomic information can help direct treatment. For example, some cancer therapies specifically target genetic mutations or alterations that have occurred in the tumor microenvironment. If patients and physicians have access to more affordable genomic testing, they can use the information to choose a targeted therapy that will work best for them.

We envision a future where in nearly every interaction patients have with the healthcare system, their genomic information will be sequenced to reveal not just their inherited DNA, but also what changes in their bodies are encoded into circulating DNA, RNA, methylation and proteomics, Almogy said.

Early Application

The $100 genome is already proving its worth. Researchers from Stanford University utilized the low-cost genomic sequencing to investigate the trajectories in precancerous polyps to early colorectal adenocarcinoma. The paper, not yet peer-reviewed but published on bioRxiv, demonstrated the technologys ability to observe changes in DNA methylation that occur early in the malignant transformation process, providing clues as to what happens at a molecular level when a polyp turns cancerous. This type of research could one day translate into clinical use, where physicians could use genomic sequencing to detect DNA changes in cells that might signal the danger of an impending malignant tumor.

Low-cost genomics helps therapeutic development in a couple of fundamental ways, Almogy said. Firstly, many companies seek to understand the genomic drivers of disease by sequencing populations and looking for associations between variants and disease. This type of work inherently requires large numbers to be useful, and the $100 genome certainly enables larger studies in a wider variety of populations. Second, low-cost genomics enables large-scale experiments to reveal the function of many genes.

Ultima isnt prepared to stop at $100 though. As evidenced by its recent collaboration with Exact Sciences, the goal is to continue driving the price down. The companies entered into a long-term supply agreement in June aimed at lowering the cost of sequencing and improving patient access to genomics-based testing. As part of the alliance, Ultima and Exact will develop one or more of Exacts advanced cancer diagnostic tests that will be developed using Ultimas technology. Earlier in June, Ultima paired with Olink Holding AB to combine the latter's Explore assay with its sequencing system to enable larger-scale projects.

Were currently in early access mode, so were focused on optimizing the platform with our initial customers before making it available for broad commercial launch next year, Almogy explained. "Beyond that, we continue to develop improvements in the architecture, because for us the $100 genome is only the beginning and were committed to continuously [driving] down the cost of genomic information.

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Here's how the $100 Human Genome will Change Medicine - BioSpace

Like most geneticists, Ryan Doan learned in school that the vast majority of the genome is useless so-called junk DNA that doesnt code for proteins. But in 2014, while doing postdoctoral research, Doan started to rethink that belief. He was bothered by the fact that autism genetics research, which has largely focused on the coding genome, hasnt made the progress many had hoped for especially in providing autistic people with genetic information that informs potential treatments.

Were not finding as much as we wouldve thought, says Doan, assistant professor of pediatrics at Boston Childrens Hospital in Massachusetts. The next best place is trying to branch out into the noncoding regions.

Now, Doans autism research is primarily focused on the largely unexplored 99 percent of the genome that lies beyond the protein-coding exome. According to his unpublished work, at least 3 percent of autistic people have noncoding mutations that contribute to their condition.

The future seems bright, but the noncoding space will be difficult for quite a while. Ivan Iossifov

Aided by new databases and cheaper whole-genome sequencing, many autism genetics researchers are, like Doan, taking tentative steps into the wide-open noncoding space. Their results so far are mixed, and the challenges remain large. Whole-genome sequencing still costs two or three times as much as exome sequencing, which limits sample size, and the effects of noncoding mutations are likely to be more subtle than those of their coding counterparts, Doan and other scientists say. But many say they hope that probing noncoding DNA will unearth genetic causes of autism for more people and reveal new details about the conditions biology.

The future seems bright, but the noncoding space will be difficult for quite a while, says Ivan Iossifov, associate professor of genetics at Cold Spring Harbor Laboratory in New York. For now, everyone is simply taking baby steps, he says very expensive baby steps.

Researchers had no way to navigate the noncoding genomes 3 billion base pairs until the launch of the Encyclopedia of DNA Elements (ENCODE) in 2003. A little more than a decade later, its spinoff, psychENCODE, started to map gene regulatory elements within that vast uncharted space in the human brain and other tissues work that is still underway.

Those maps made it possible for researchers to begin devising targeted strategies to explore the links, if any, between noncoding mutations and autism. It might be tempting to search the entire noncoding space to ensure that important mutations connected to autism arent missed especially given how little is known about the DNA there. But starting with stretches of DNA with known functions, such as the promoters and enhancers that help regulate a genes expression, stands to increase the likelihood that any discovered mutations will be meaningful.

Some people are very agnostic to location, says Santhosh Girirajan, associate professor of genomics at Pennsylvania State University. And some are looking at some star in some galaxy somewhere.

Promoters the focus of Doans study are located next to the genes they regulate. Enhancers, which may be farther away, carry more mutations in autistic people than in their non-autistic siblings, according to a 2021 analysis. In autistic people, genes linked to autism also tend to have an overabundance of transposons sections of noncoding DNA that can jump randomly around the genome and disrupt other genes another study found.

Iossifov is surveying yet another source of noncoding DNA: stretches located within genes called introns. About 6 percent of autistic people have an intron mutation that likely contributes to their condition, according to his 2021 analysis of these sections in nearly 2,000 autistic children and their non-autistic siblings. To bolster the finding, his team is studying gene expression levels, reasoning that if a gene with an intron mutation has atypical expression in autistic people, its likely that mutation is involved in the condition.

Early results look promising, Iossifov says. Expression abnormalities in a gene are rare enough that they can be used as this very useful filter for pointing at de novo noncoding mutations which might be contributory.

For researchers who are exploring the entire noncoding space, machine learning is proving to be a useful tool. A 2018 analysis of whole genomes from nearly 2,000 families with one autistic and one non-autistic child, for example, initially turned up no relevant noncoding mutations compared with controls. But using a machine-learning tool that identifies multiple types of noncoding variants revealed an excess of mutations in promoter regions among the autistic participants.

Similarly, only a neural network trained on functional genomics data could spot differences between autistic children and their non-autistic siblings across some 200,000 noncoding variants in another 2021 study. More noncoding mutations occurred near autism-linked genes in children with autism than in those without. Overall, though, noncoding mutations occurred equally closely to the nearest gene in both autistic and non-autistic people, highlighting the challenge of identifying these causal mutations, the investigators wrote.

Noncoding and coding mutations may contribute to autism in similar proportions: they are found in about 4.3 and 5.4 percent of autistic children, respectively, according to a 2019 analysis that used machine learning to estimate an individual mutations likelihood of contributing to the condition.

Yet a third strategy involves looking at the whole noncoding space but limiting the analysis to a cohort thats more likely to have rare mutations. A February study of 22 families with high rates of inter-family marriage, for example, found likely disease-causing variants in promoters and enhancers for five autism-linked genes. The team is now using CRISPR to study the variants functions in cells, as well as repeating the work in a new cohort of African children with autism.

Eventually, all of this information in aggregate will be able to tell us about the molecular mechanisms underlying autism, says lead investigator Maria Chahrour, assistant professor of genetics and neuroscience at the University of Texas Southwestern Medical Center in Dallas.

Even when a noncoding mutation contributes to autism, its individual effect is small, the results so far suggest. That means noncoding mutations probably arent acting on their own to cause autism, Girirajan says. Rather, several may act together or in tandem with a coding mutation.

How noncoding mutations affect the genome might also be far more subtle and difficult to nail down than for coding mutations. A given mutation may matter in only one cell type or at a specific point in development, for example. Parsing this kind of complexity, while enormously challenging, could help to explain autisms heterogeneity, Girirajan says. Autism subtypes might reflect not just mutations in a specific gene, but how a genes expression varies across time.

Eventually, all of this information in aggregate will be able to tell us about the molecular mechanisms underlying autism. Maria Chahrour

Its so complex. We are living in a naive land where everything is genes, Girirajan says. What we are not thinking about is gene regulation at different stages of development and tissues. Oh gosh.

To move forward, Girirajan and others say, the field needs to build up whole-genome databases in a big way: At present, autism researchers have access to the exomes of around 50,000 autistic people, and even that has been barely enough to find results in the much simpler coding space, Doan says.

For the noncoding space, you cut your samples 5-fold, but increase complexity 50-fold, he says. You have a huge power problem and thats just something we have to deal with for a while.

Geneticists also need to refine the maps that autism researchers are using to find their way. The ENCODE project, for one, is working to release data on the time periods and cell types in which promoters, enhancers and other regulatory elements influence genes.

Still, results from other fields are encouraging: Other neuropsychiatric conditions are now linked to many mutations in the noncoding region. Of 22 regions implicated in schizophrenia in one large study, for example, 13 are located in noncoding regions within or between genes.

In autism, this is still behind, Iossifov says, but adds that it is only a matter of time before similar findings emerge. Theres no doubt.

Cite this article: https://doi.org/10.53053/WHLV1876

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The final frontier: Autism geneticists take on the noncoding genome | Spectrum - Spectrum

With its multiple variants such as Delta and Omicron, the COVID-19 pandemic highlighted the need for genomic surveillance, to monitor virus evolution and its implications on transmission dynamics and response measures like vaccines. Sequencing informationprovides crucial decision-making information during epidemics and pandemics. On 8-9 June 2022, WHOs Eastern Mediterranean Regional Office convened a meeting in Egypt with partner organizations and countries to discuss the framework for integratedrespiratory pathogen surveillance including the role of genomic surveillance.The regional laboratory focal point set the scene:

Currently, 19 out of the 22 countries in the Eastern Mediterranean Region have genomic sequencing capabilities. A regional network has been established to enable all countries to have access to sequencing, and to strengthen their capacities coherentlyand collaboratively to be able to detect, investigate and respond to COVID-19 and other emerging and re-emerging infectious diseases with epidemic and pandemic potential.

- Dr Amal Barakat, Regional Laboratory Focal Point, WHO

Some highlights from stories shared by countries in the meeting:

Following the significant increase in molecular diagnostic capacity for SARS-CoV-2 in the country enabling up to 250,000 tests per day, the National Influenza Centre at the Ministry of Health (MOH) recognized early that the need for SARS-CoV-2 sequencingwas also increasing. To address this, Morocco set up a national consortium of four laboratories two public and two private to cover different geographic regions in the country.

The Consortium enables us to address genomic surveillance needs by bringing in the capacities and capabilities of the private sector. This was a major achievement and presents an opportunity for us as we think about the next generation of publichealth surveillance.

- Professor Hisham Ouzmil, National Influenza Centre, Morocco

The MOH Central Public Health Laboratory (CPHL) serves as WHOs regional reference laboratory for COVID-19. The CPHL linked with national and local academic partners to strengthen workforce capacities, increase national genomic surveillance coverage,and develop algorithms for selecting cases for sequencing so that virological trends associated with different sub-populations such as travelers, severely ill patients and cases from different geographic regions could be well understood.

Genomics have helped us to better understand the epidemiology of COVID-19 in Oman. Linking genomic data to epidemiological and clinical data, and analyzing trends from other countries maximizes the utility and power of genomics. We are happy towork with other countries, share our experiences and strengthen collaborations as we learn lessons for future pandemic preparedness.

- Dr Hanan Alkindi, Central Public Health Laboratory, Oman

A massive effort was undertaken to expand genomic surveillance so that the viral phylo-dynamics could be understood in all geographic regions of the country and to look at patterns among severe cases, travel-related cases, post-vaccination cases and re-infections.

More than 60,000 SARS-CoV-2 samples have been sequenced from around the country. We have the opportunity to use the capacity established for various public health threats and are ready for future emergencies.

- Dr Ahmed Albarraq, Public Health Authority, Saudi Arabia

Outputs from the meeting and reflections from countries on the role of genomics during the COVID-19 pandemic and future emergencies will enable the Region to plan effectively and focus attention on the future of integrated respiratory pathogen surveillanceinclusive of genomic surveillance. The regional operational framework for integrated surveillance is being finalized and will be available later this year including the opportunities for genomic surveillance in the context of the recently launchedGlobal Genomic Surveillance Strategy for Pathogens with Pandemic and Epidemic Potential 20222032.

The 10-year Global strategy will enable countries in the Eastern Mediterranean Region, as well as other regions, to capitalize on the gains made and to solidify the role of genomics in future public health practice. Click here to learn more about the Global Genomic Surveillance Strategy for Pathogens with Pandemic and Epidemic Potential 20222032.

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The Eastern Mediterranean Region reflects on genomic sequencing and its future within integrated surveillance of respiratory viruses - World Health...

Contributed Commentary by Per Hammer, Cytiva

July 15, 2022 | Historically, the heavily regulated biopharma industry has been slow to adopt new technologies. However, a shift toward automation is vital to ensure that next-generation solutionssuch as cell and gene therapiesare produced at scale.

Less than one in five senior pharma executives strongly believe that frontier technologies, such as artificial intelligence, are widely adopted to support automation and increase the speed of specific processes. With cell therapies approved by global regulatory bodies, it is time to accelerate smart technologies and cell and gene therapy manufacturing.

Todays cell therapy treatments are often made on a small-scale, include manual preparation steps, and are produced for a clinical trial setting. Researchers spend days processing cellular material, monitoring its growth during the expansion phase, and preparing for re-administration to the patient. This process is demonstrated in administering autologous treatments so that every patient receives a unique living drug.

Though the current process is complex, it offers inspiring outcomes. For example, on April 1, 2022, the Food and Drug Administration (FDA) approved Kite Pharmas Yescarta, a chimeric antigen receptor (CAR) T-cell therapy for adult patients with large B-cell lymphoma. This kind of cancer is usually resistant to initial treatment and relapses within one year. With FDA approval, Yescarta (axicabtagene ciloleucel) is now the second-line treatment, a first for an autologous CAR T-cell therapy.

Cell Therapy Enters Mainstream

The exceptional results emerging from cell therapy clinical trials suggest we are entering a new phase of medical treatmentone where we can expect far more from our healthcare interventions than we ever imagined. Following the regulatory approval of autologous CAR T-cell therapies, the global cancer treatment landscape is changing, and the future is bright.

The success of COVID-19 vaccines signaled the arrival of the genomic medicines ageone where we hope to see cell and gene therapies deliver long-term remission and even cures for patients with some of the most complex diseases. According to the Alliance for Regenerative Medicine 2021 Annual Report, nearly 60% of the ongoing regenerative medicine clinical trials studied prevalent diseases by the end of the calendar year. But to get these powerful treatments to those who need them, we must have an automated manufacturing infrastructure that can generate cell therapies to meet increased demand in the coming years.

Saving Time Through Automation

Time is of the utmost importance, as biopharma manufacturing involves patient cells that have limited viability. Manual approaches to cell therapy production are time-consuming, and tasks such as checking cells at regular intervals during expansion are laborious. Another time-draining factor is the workflow and cleaning routines involved in maintaining a safe lab environment.

Automated solutions reduce or remove many of these challenges. After setting up a process, an operator can focus on other things while critical parameters such as temperature, pH level, gas transfer, and flow rates are monitored and controlled without human intervention.

Reducing Risks for Better Results

Manual cell processing solutions are complex, with many checkpoints across isolation, expansion, harvesting, and preservation stages. Unfortunately, each of these steps increases the risk potential. Despite the research teams expertise, there is still a chance that materials could be inadvertently contaminated during numerous open stages.

Additionally, limited process control can lead to difficulties in achieving high reproducibility. An automated modular solution minimizes these risks by bringing multiple steps within a closed, highly regulated, and controlled system.

Improving Manufacturing Efficiency

Changing a manufacturing process requires multiple manual routines and adjustments that must be checked and documented. However, documentation and protocols are less helpful when a customized process is used because they only apply to that specific setup.

Standardization would effectively improve manufacturing efficiency. This approach would ensure that what is learned in one project can be referenced in future work, with data and documentation applicable across different technology applications. A modular chain of connected systems allows for process variation with instruments running in customized configurations. Additionally, having control of an individual instrument leads to the straightforward use of built-in software and sensors.

Automated Manufacturing: The Way Forward for Cell and Gene Therapy

By using automated manufacturing to minimize human interaction, time, and resource requirements, it is possible to increase production speed and lower some risks and costs associated with commercialization.

The industry is ever-changing and adjusting its complex, yet exciting challenges will take some time. However, automation can create a significant advantage over competitors, providing the tools needed to produce cell therapies with the highest levels of safety and efficacy for patients.

Per Hammer has two decades of experience in the biopharma industry, mainly supporting customers in academics through process development and manufacturing. Per joined Cytiva in 2001, taking on several distinct roles in the company. Most recently, he progressed from Product Manager Leader for the Bioprocess Automation and Digital Team to Senior Global Product Manager for the Cell & Gene Therapy Automation and Digital Solutions. He can be reached at per.hammer@cytiva.com.

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Automating the Genomic Medicines of the Future - Bio-IT World

A genetic setup to investigate boundary function in vivo

We previously demonstrated that a 150-kilobase (kb) region, the EP boundary, is sufficient to segregate the regulatory activities of the Epha4 and Pax3 TADs10 (Extended Data Figs. 1 and 2). The DelB background carries a large deletion that removes this boundary region, and the Epha4 gene, resulting in the ectopic interaction between the Epha4 limb enhancers and the Pax3 gene. This causes Pax3 misexpression and the shortening of fingers (brachydactyly) in mice and in human patients. In contrast, the DelBs background carries a similar deletion but not affecting the EP boundary, which maintains the Epha4 and Pax3 TADs and confines the Epha4 enhancers within their own regulatory domain (Fig. 1a and Extended Data Fig. 1).

a, cHi-C maps from E11.5 distal limbs from DelBs mutants at 10-kb resolution. Data were mapped on a custom genome containing the DelBs deletion (n=1 with an internal control comparing 6 different experiments; Methods). The red rectangle marks the EP boundary region. Insets represent a magnification (5-kb resolution) of the centromeric (left) and telomeric (right) loops highlighted by brackets on the map. Cen, Centromeric; Tel, Telomeric. Arrowheads represent reverse- (light blue) and forward- (orange) oriented CBSs. Below, Lac-Z staining (left) and WISH (right) of E11.5 mouse forelimbs show activation pattern of Epha4 enhancers and Pax3 expression, respectively. b, CTCF ChIPseq track from E11.5 mouse distal limbs. Schematic shows CBS orientation. c, Insulation score values. The gray dot represents the local minima of the insulation score at the EP boundary. BS, boundary score. d, Relationship between BS and the number of CBSs (data from ref. 26). The boxes in the boxplots indicate the median and the first and third quartiles (Q1 and Q3). Whiskers extend to the last observation within 1.5 times the interquartile range below and above Q1 and Q3, respectively. The rest of the observations, including maxima and minima, are shown as outliers. N=8,127 insulation minima found in mESC Hi-C matrices. e, WISH shows Pax3 expression in E11.5 forelimbs from CBS mutants. Note Pax3 misexpression on the distal anterior region in R1, F1 and F2 mutants (white arrowheads). Scale bar, 250m. f, Pax3 qPCR analysis in E11.5 limb buds from CBS mutants. Bars represent the mean and white dots represent individual replicates. Values were normalized against DelBs mutant (Ct) (two-sided t-test *P0.05; NS, nonsignificant; P values from left to right: DelBs versus R1: 0.02; DelBs versus R2: 0.11; DelBs versus F1: 0.02; DelBs versus R3: 0.23; DelBs versus F2: 0.02; DelBs versus R4: 0.73). Cen, Centromeric; Tel, Telomeric.

To characterize the EP boundary in vivo, we performed CTCF ChIPseq on developing limbs. This analysis revealed the presence of six clustered CBSs at the EP boundary region (Fig. 1a,b and Extended Data Fig. 2), a profile that is conserved across tissues25,26. CTCF motif analyses confirmed the divergent orientation of these sites, a signature of TAD boundaries, with four CBSs in reverse (R) and two in forward orientation (F). Other features associated with boundaries, such as active transcription or housekeeping genes, were not found in the region27 (Extended Data Fig. 3). cHi-C data from DelBs stage E11.5 distal limbs28 revealed chromatin loops connecting the two forward-oriented CBSs (F1 and F2) with the telomeric boundary of the Pax3 TAD, and the centromeric boundary of the Epha4 TAD with the reverse-oriented CBSs R1, R2 and R3 (Fig. 1a,b). However, the close genomic distances between R2 and F1 and between R3 and F2 preclude the unambiguous assignment of loops to specific sites. RAD21 (cohesin subunit) ChIPseq experiments in E11.5 distal limbs revealed that R1, F1 and F2, as well as R2 and R3 to a lesser degree, are bound by cohesin (Extended Data Fig. 3), an essential component for the formation of chromatin loops21,29,30. These results delineate the EP element as a prototypical boundary region with insulating properties likely encoded and controlled by CBSs.

Boundary regions are predominantly composed of CBS clusters31, suggesting that the number of sites might be relevant for their function. We explored this by calculating boundary scores32 on available Hi-C maps26, and categorizing boundaries according to CBS number. We observe that boundary scores increase monotonically with CBS number, reaching a stabilization at ten CBSs (Fig. 1d). According to this distribution, the EP boundary falls within a range where its function might be sensitive to alterations on CBS number. To test this, we employed a mouse homozygous embryonic stem cell (mESC) line for the DelBs background28, which we edited to generate individual homozygous deletions for each of the six CBSs of the EP boundary region (Supplementary Fig. 1). ChIPseq experiments revealed that the disruption of the binding motif was sufficient to abolish CTCF recruitment (Supplementary Fig. 2). Subsequently, we employed tetraploid complementation assays to generate mutant embryos and measure the functional consequences of these deletions in vivo33,34.

Whole-mount in situ hybridization (WISH) on E11.5 mutant embryos revealed that the insulation function of the EP boundary can be sensitive to individual CBS perturbations (Fig. 1e). However, this effect was restricted to CBSs displaying prominent RAD21 binding (R1, F1 and F2) (Extended Data Fig. 3). The altered boundary function was evidenced by Pax3 misexpression on a reduced area of the anterior limb, while the expression domains in other tissues remained unaltered (Supplementary Fig. 3). The disruption of the other CBSs (R2, R3 and R4) did not alter Pax3 expression, demonstrating that the EP boundary can also preserve its function despite a reduction in CBS number.

To quantify Pax3 misexpression, we performed quantitative PCR (qPCR) in E11.5 forelimbs. Similarly, we observed a modest, but significant, upregulation in R1, F1 and F2 mutants (Fig. 1f). Importantly, the functionality of individual CBSs is not strictly correlated with CTCF occupancy as the deletion of R3, displaying the highest levels of CTCF binding among the cluster (Fig. 1b and Extended Data Fig. 3), does not result in measurable transcriptional changes (Fig. 1f). Thus, while CBS number influences insulation, the characteristics of individual sites are major determinants of boundary function.

To explore CBS cooperation, we retargeted our R1 mESC line to generate double knockout mutants with different (R1+F2) or identical CBS orientations (F1 and F2 in F-all) (Fig. 2a). WISH revealed an expanded Pax3 misexpression towards the posterior region of the limb, demonstrating that the EP boundary is compromised in both mutants. Next, we determined the nature of CBS cooperation by qPCR. These experiments revealed that, in both mutants, Pax3 misexpression exceeded the summed expression levels from the corresponding individual deletions (Fig. 2b). These negative epistatic effects indicate that CBSs are partially redundant, compensating for the absence of each other.

a, WISH shows Pax3 expression in E11.5 forelimbs from CBS mutants. Arrowheads represent reverse- (light blue) and forward- (orange) oriented CBSs. Crosses indicate deleted CBSs. Note increased Pax3 misexpression towards the posterior regions of the limb. Scale bar, 250m. b, Pax3 qPCR analysis in E11.5 limb buds from CBS mutants. Bars represent the mean and white dots represent individual replicates. Values were normalized against DelBs mutant (Ct) (**t-test **P0.01; R1+F2 versus F-all: 0.008). c, cHi-C maps from E11.5 mutant distal limbs at 10-kb resolution (top). Data were mapped on a custom genome containing the DelBs deletion (n=1 with an internal control comparing 6 different experiments; Methods). Insets represent a magnification (5-kb resolution) of the centromeric (left) and telomeric (right) loops highlighted by brackets on the map. Gained or lost chromatin loops are represented by full or empty dots, respectively. Subtraction maps (bottom) showing gain (red) or loss (blue) of interactions in mutants compared with DelBs. d, Insulation score values. Lines represent indicated mutants. Dots represent the local minima of the insulation score at the EP boundary for each mutant. e, Virtual 4C profiles for the genomic region displayed in c (viewpoint in Pax3). The light-gray rectangle highlights the Epha4 enhancer region. Note increased interactions between the Pax3 promoter and the Epha4 enhancer in R1+F2 and F-all (purple and orange) compared with DelBs mutants (gray).

To gain insights on the mechanisms of CBS cooperation, we generated cHi-C maps of the EP locus from E11.5 distal limbs (Fig. 2c and Supplementary Fig. 4). Maps from R1+F2 embryos denoted a clear partition between the EphaA4 and Pax3 TADs, analogous to DelBs control mutants (Fig. 2c). However, subtraction maps revealed decreased intra-TAD interactions for the Epha4 and Pax3 TADs, and a concomitant increase in inter-TAD interactions. In addition, we observed the appearance of a loop connecting the outer boundaries of the Epha4 and Pax3 TADs (meta-TAD loop; Extended Data Fig. 4)35. Accordingly, the boundary score of the EP boundary in R1+F2 mutants was decreased, reflecting a weakened structural insulation (Fig. 2d). Virtual Circular Chromosome Conformation Capture (4C) profiles revealed increased chromatin interactions between the Pax3 promoter and the Epha4 limb enhancers (Fig. 2e), consistent with the upregulation of Pax3. In addition, two of the chromatin loops that connect the EP boundary and the telomeric boundary were abolished, due to the deletion of the F2 anchor and the associated loss of RAD21 (Fig. 2c and Extended Data Figs. 4 and 5). Consequently, the adjacent chromatin loop exhibited a compensatory effect, with increased interactions mediated by the F1 anchor, consistent with higher RAD21 occupancy (Extended Data Figs. 4 and 5). At the centromeric site, the deletion of R1 causes the relocation of the loop anchor towards an adjacent region containing a reverse-oriented (R2) and the only remaining forward CBS (F1). While the loop extrusion model would predict a stabilization at a reverse CBS15,16, the short genomic distance between R2 and F1 precludes an unambiguous assignment of the loop anchor. We also observed increased contacts at R3 and R4, suggesting that these sites are functionally redundant.

Then, we examined cHi-C maps from F-all mutants, which display a more pronounced Pax3 misexpression (Fig. 2b). Interaction maps revealed a partial fusion of the Epha4 and Pax3 domains (Fig. 2c), accompanied by a notable decrease of the boundary score (Fig. 2d). Virtual 4C profiles confirmed increased interactions between Pax3 and the Epha4 enhancers in F-all compared with R1+F2 mutants, in agreement with the more pronounced Pax3 upregulation (Fig. 2e). The deletion of all CBSs with forward orientation abolishes the chromatin loops connecting with the telomeric Pax3 boundary (Fig. 2c and Extended Data Fig. 4). Towards the centromeric side, R1 maintains RAD21 binding and its chromatin loop with the centromeric Epha4 boundary (Extended Data Figs. 4 and 5). However, other chromatin loops are still discernible and anchored by the R3 and R4 sites, confirming that these sites perform distinct yet partially overlapping functions. These results demonstrate that CBSs can cooperate but also partially compensate for the absence of each other, conferring functional robustness to boundaries.

Chromatin loops are predominantly anchored by CBS pairs with convergent motif orientation14,36. Intriguingly, we observed that the combined F1 and F2 deletion (F-all) not only disrupts the loops in the expected orientation (telomeric), but also impacts the centromeric one, as observed in the subtraction maps (Fig. 2c). This effect is noticeable at the R2/F1 site, which was associated with a centromeric chromatin loop in the DelBs background (Fig. 1a). This demonstrates that the main loop anchor point was not the R2 but the F1 site (Extended Data Fig. 4), suggesting that this CBS can form loops in a nonconvergent orientation. Such mechanism is described by the loop extrusion model, which predicts that loops could create steric impediments that might prevent additional cohesin complexes from sliding through anchor sites15,16. This effect would stabilize these additional cohesin complexes, resulting in the establishment of simultaneous and paired nonconvergent and convergent loops (Fig. 3a).

a, Schematic of a convergent loop that indirectly generates a nonconvergent loop in the opposite direction. b, Percentage of loop anchors establishing bidirectional loops (n=12,635 loops from mESCs from ref. 26). Anchor categories: convergent-only (only CBSs oriented in the same direction as their anchored loops, n=7,769), nonconvergent (anchor loops in a direction for which they lack a directional CBS, n=960) and no-CTCF (no CBS, n=3,906). c, Loop strengths in pairs of convergent/nonconvergent loops classified into Non-conv.-associated (nonconvergent loop sharing the nonconvergent anchor with a convergent loop in the opposite direction, n=322) and Conv.-associated (convergent loop sharing one anchor with a nonconvergent loop in the opposite direction, n=496). Boxplots defined as in Fig. 1c. Two-sided BenjaminiHochberg-corrected MannWhitney U-test P=6.2106. d, Aggregated loop signal for categories in c. Arrows represent CBS orientation. e, Pax3 WISH in E11.5 forelimbs from CBS mutants. Arrowheads represent reverse- (blue) and forward- (orange) oriented CBSs. Crosses indicate deleted CBSs. Note the positive correlation between expanded Pax3 misexpression and increased number of deleted CBSs. Scale bar, 250m. f, Pax3 qPCR analysis in E11.5 limbs from CBS mutants. Bars represent mean and dots individual replicates. Values were normalized against DelBs mutant (Ct). Note the positive correlation of Pax3 misexpression with the increase in deleted CBSs (Pearson correlation significantly>0; ***P0,001). g, cHi-C maps from E11.5 mutant distal limbs at 10-kb resolution (top). Data were mapped on a custom genome containing the DelBs deletion (n=1 with an internal control comparing 6 different experiments; Methods). Insets represent a magnification (5-kb resolution) of the centromeric (left) and telomeric (right) loops highlighted by brackets on the map. Gained or lost chromatin loops are represented by full or empty dots. Subtraction maps (bottom) showing gain (red) or loss (blue) of interactions in mutants compared with DelBs. h, Insulation score values. Dots represent the local minima of the insulation score at the EP boundary for each mutant. i, Virtual 4C profiles for the region in g (viewpoint in Pax3). The gray rectangle highlights Epha4 enhancers. Note increased interactions between the Pax3 promoter and the Epha4 enhancers in R-all compared with DelBs.

We searched for further biological indications of this mechanism by analyzing ultra-high-resolution Hi-C datasets26. First, we identified loop anchors and classified them according to the orientation of their CBS motif and associated loops. Loop anchors were split into convergent-only (only CBSs oriented in the same direction as their anchored loops), nonconvergent (anchor loops in a direction for which they lack a directional CBS) and no-CTCF (no CBS). While most loop anchors belong to the convergent-only category14,36, 7.6% of them were classified as nonconvergent. Then, we explored whether these nonconvergent loops could be explained by the nonconvergent anchor simultaneously establishing a convergent loop in the opposite direction (Fig. 3a). We calculated the frequency of anchors involved in bidirectional loops for each category and discovered that, while only 5% of convergent-only or no-CTCF anchors participate in bidirectional loops, this percentage increases significantly up to 45% for nonconvergent anchors (Fig. 3b; chi-squared test, P<10225). To gain further insights into the mechanisms that establish convergent/nonconvergent loop pairs, we calculated the strength of each corresponding paired loop22. We observed that the convergent loops linked to a nonconvergent loop are significantly stronger than their nonconvergent counterparts (Fig. 3c,d; MannWhitney U-test, P=6106). Next, we explore if convergent loops paired to nonconvergent loops are particularly strong in comparison with other types of convergent loops. This analysis revealed that the strength of these convergent loops is similar to other unpaired convergent loops across the genome (Extended Data Fig. 6; single-sided convergent category). However, paired convergent/nonconvergent loops appear to be mechanistically different from unpaired loops, as they are more often associated with TAD corners (Extended Data Fig. 6c; chi-squared test, P<3.5106) and therefore connect anchor points that are located farther away in the linear genome (Extended Data Fig. 6d; MannWhitney U-test, P<4.8108). A comparison against pairs of convergent/convergent loops, which are similarly associated with TAD corners (Extended Data Fig. 6b; category double-sided convergent), revealed that the convergent loops in convergent/nonconvergent pairs are on average stronger (MannWhitney U-test, P=7105). This type of convergent/nonconvergent loops can be observed at relevant developmental loci, such as the Osr1, Ebf1 and Has2 loci (Extended Data Fig. 7). Overall, our analyses suggest that a considerable number of nonconvergent loops could be mechanistically explained by the presence of a stronger and convergent chromatin loop in the opposite orientation and anchored by the same CBS.

To validate these findings in vivo, we sequentially retargeted our R1 mESCs to create a mutant that only retains the forward F1 and F2 sites, which have strong functionality (Fig. 2a,b). During the process, we obtained intermediate mutants with double (R1+R3) and triple CBS deletion (R1+R3+R4), as well as the intended quadruple knockout lacking all reverse CBSs (R-all). WISH revealed an expanded Pax3 expression pattern towards the posterior limb region, an effect that increases with the number of deleted CBSs (Fig. 3e). Expression analyses by qPCR confirmed a significant increasing trend in Pax3 misexpression levels across mutants (Fig. 3f; Pearson correlation>0, P2107). These results demonstrate again that R2, R3 and R4 are functionally redundant sites, despite the absence of measurable effects upon individual deletions (Fig. 1b). However, we noted that Pax3 levels were only moderately increased (threefold) compared with the expression in mutants retaining only-reverse CBSs (ninefold, F-all). Importantly, R-all mutants retain two intact CBSs in the forward orientation, while up to four CBSs are still present in F-all mutants, suggesting that these two forward CBSs (F1 and F2) grant most of the insulator activity of the EP boundary. These experiments indicate that the functional characteristics of specific CBSs can outweigh other predictive parameters of boundary function such as the total number of sites.

As expected, cHi-C maps from R-all mutant limbs revealed a clear partition between the Epha4 and Pax3 TADs (Fig. 3g), consistent with the reduced Pax3 misexpression. Boundary scores at the EP boundary were also only moderately reduced (Fig. 3h), in comparison with the broader effects of the F-all mutant (Fig. 2d). Accordingly, intra-TAD interactions modestly decreased while inter-TAD interactions increased, as also observed in virtual 4C profiles (Fig. 3i). Despite the multiple deletions, the telomeric chromatin loops remained unaffected and anchored by the F1 and F2 sites, both occupied by RAD21 (Fig. 3g and Extended Data Figs. 4 and 5). However, we noticed the persistence of centromeric chromatin loops anchored by the F1 and F2 sites, despite their nonconvergent forward orientation. A higher contact intensity is observed at F1, which would be the first CBS encountered by cohesin complexes sliding from the centromeric side (Extended Data Figs. 4 and 5).

Finally, we investigated if the formation of nonconvergent loops might be associated with the accumulation of cohesin complexes over a limited number of CBSs. We generated a mutant that only retains the R3 CBS (R3-only), which is prominently bound by CTCF (Fig. 1b). We hypothesized that, in the absence of others, this CBS may accumulate the cohesin and form a nonconvergent loop. However, although R3 was the only site able to stall cohesin in this background (Extended Data Fig. 4), cHi-C maps revealed a single convergent loop towards the centromeric side (Extended Data Fig. 8). This loop displays a weak insulator function, denoted by a decreased boundary score, an Epha4 and Pax3 TAD fusion and prominent Pax3 misexpression. Therefore, our results in transgenic mice support our findings at the genome-wide level (Fig. 3ac), demonstrating that specific CBSs can create chromatin loops independently of their motif orientation, seemingly through loop interference.

Previous studies identified divergent CBS clusters as a signature of TAD boundaries, suggesting a role on insulation13,31. While our analysis on mutants with reverse-only CBS orientation (F-all) showed a severe impairment of boundary function (Fig. 2c), this was not the case for R-all mutants, which retain CBSs only in the forward orientation (Fig. 3f). Indeed, the levels of Pax3 misexpression evidenced that insulation is more preserved in R-all than in R1+F2 mutants, which still conserve a divergent CBS signature (Fig. 2c).

This prompted us to explore the relation between CBS composition at boundaries and insulation strength. We examined available Hi-C datasets, classifying boundary regions according to different parameters of CBS composition (that is, number and orientation) and calculating boundary scores (Fig. 4a). Our analysis revealed that, for the same CBS number, boundaries with divergent signatures generally display more insulation than their nondivergent counterparts. However, up to 6% of nondivergent boundaries display scores above 1.0, a value associated with robust functional insulation (Fig. 1c). Manual inspection at specific loci showed that nondivergent boundaries with strong boundary scores present clear TAD partition and no evidence of coregulation for genes located at either side (Extended Data Fig. 9). These results suggest that a divergent signature is not strictly required to form strong functional boundaries.

a, Relation between BSs and the number of CBSs for divergent and nondivergent boundaries in mESC Hi-C data26. Boxplots defined as in Fig. 1c. b, WISH shows Pax3 expression in E11.5 forelimbs from CBS mutants. Arrowheads represent reverse- (light blue) and forward- (orange) oriented CBSs. Crosses indicate deleted CBSs. Light-gray rectangle marks inverted region. Note similar Pax3 misexpression pattern between F-all-Inv and F-all mutants. Scale bar, 500m. c, Pax3 qPCR analysis in E11.5 limb buds from CBS mutants. Bars represent the mean and white dots represent individual replicates. Values were normalized against DelBs mutant (Ct) (two-sided t-test P value). d, cHi-C maps from E11.5 mutant distal limbs at 10-kb resolution (top). Data mapped on custom genome containing the DelBs deletion and the inverted EP boundary (n=1 with an internal control comparing 6 different experiments; Methods). Insets represent a magnification (5-kb resolution) of the centromeric (left) and telomeric (right) loops highlighted by brackets on the map. Gained or lost chromatin loops are represented by full or empty dots, respectively. Subtraction maps (bottom) showing gain (red) or loss (blue) of interactions in mutants compared with DelBs. e, Insulation score values. Lines represent mutants. Dots represent the local minima of the insulation score at the EP boundary for each mutant. f, Virtual 4C profiles for the genomic region displayed in d (viewpoint in Pax3). Light-gray rectangle highlights Epha4 enhancer region. Note similar interaction profile between F-all-Inv (yellow) and F-all mutants (orange).

Next, we explored if the genomic contexts might explain the prominent insulation differences between only-reverse (F-all) or only-forward (R-all) mutants. To evaluate this, we generated a mutant with a homozygous inversion of the boundary region, on the F-all background (F-all-Inv) (Fig. 4b and Supplementary Fig. 5).

WISH and qPCR experiments showed that Pax3 expression is almost indistinguishable from the F-all mutants, both spatially and at the quantitative level (Fig. 4b,c). Moreover, cHi-C maps from F-all-Inv mutants revealed a similar fusion of the Epha4 and Pax3 TADs (Fig. 4d). However, subtraction maps showed a redirection of chromatin loops, which now interact mainly with the telomeric Pax3 boundary instead of the centromeric Epha4 boundary. These ectopic loops are mainly anchored by the R1 site, which preserves its marked functionality. Despite these local differences, boundary scores and virtual 4C profiles remained comparable between F-all-Inv and F-all mutants (Fig. 4e,f). These results suggest that the orientation of entire boundary regions, as well as the differences in the surrounding genomic context, play a minor role in insulator function.

To determine to what extent CTCF binding contributes to the EP boundary function, we generated a sextuple knockout with all CBSs deleted (ALL). WISH revealed a further expansion of Pax3 misexpression, covering the distal limb entirely. This expanded expression mirrors that of DelB mutants, in which the entire boundary region is deleted (Fig. 5a). Expression analyses revealed that Pax3 misexpression in ALL mutants exceeds the combined sum of expression from R-all and F-all mutants (Fig. 5b), again indicating the cooperative and redundant CBS action. Intriguingly, Pax3 misexpression in the R3-only background was comparable to ALL, suggesting that a functionally weak CBS is not sufficient to hinder enhancerpromoter communication (Extended Data Fig. 8). Nevertheless, ALL mutants only reach 65% of the Pax3 misexpression observed in DelB mutants (Fig. 5b), which may be attributed to the 150-kb inter-CBS region that differentiates both mutants.

a, WISH shows Pax3 expression in E11.5 forelimbs from CBS mutants. Arrowheads represent reverse- (light blue) and forward- (orange) oriented CBSs. Crosses indicate deleted CBSs and the gray rectangle represents the deleted region. Note the similarities in expression pattern between mutants. Scale bar, 250m. b, Pax3 qPCR analysis in E11.5 limb buds from CBS mutants. Bars represent the mean and white dots represent individual replicates. Values were normalized against DelBs mutants (Ct) (*two-sided t-test P0.05, ALL versus DelB: 0.03). c, cHi-C maps from E11.5 mutant distal limbs at 10-kb resolution (top). Data mapped on custom genome containing the DelBs deletion (n=1 with an internal control comparing 6 different experiments; Methods). Insets represent a magnification (5-kb resolution) of the centromeric (left) and telomeric (right) loops highlighted by brackets on the map. Gained or lost chromatin loops represented by full or empty dots, respectively. Subtraction maps (bottom) showing gain (red) or loss (blue) of interactions in mutants compared with DelBs (left) and DelB (right). d, Insulation score values. Lines represent mutants. Dots represent the local minima of the insulation score at the EP boundary for each mutant. e, Virtual 4C profiles for the genomic region displayed in c (viewpoint in Pax3). Light-gray rectangle highlights Epha4 enhancer region.

To investigate the reduced Pax3 misexpression in ALL, compared with DelB mutants, we performed cHi-C experiments (Fig. 5c). These experiments revealed a prominent Epha4 and Pax3 TAD fusion, with increased intensity of the meta-TAD loop (Extended Data Fig. 4). This results from the severe disruption of the EP boundary, denoted by a reduced boundary score (Fig. 5d) and the complete absence of RAD21 binding or anchored loops (Extended Data Figs. 4 and 5). In fact, the interaction profile at the EP boundary is not different from other internal locations of the Epha4 TAD (Fig. 5c). Of note, higher insulation is observed in R3-only compared with ALL, despite the comparable Pax3 misexpression between both genetic backgrounds (Extended Data Fig. 8). However, virtual 4C profiles from ALL and R3-only mutants confirmed a similar interaction between Epha4 enhancers and Pax3 (Fig. 5e and Extended Data Fig. 8). These enhancergene interactions were reduced in comparison with DelB, in which Pax3 misexpression is more prominent (Fig. 5e and Extended Data Fig. 8). ChIPseq datasets for epigenetic marks did not reveal additional regions with regulatory potential within the 150-kb region (Extended Data Fig. 3), indicating that the enhanced Pax3 misexpression in DelB mutants is unlikely caused by the deletion of regulatory elements. Taken together, these results suggest that enhancerpromoter distances might influence gene expression levels.

PAX3 misexpression during limb development can cause shortening of thumb and index finger (brachydactyly), in human patients and mouse models10. Therefore, our mutant collection provides an opportunity to study how boundary insulation strengths translate into developmental phenotypes.

We obtained mutant E17.5 fetuses and performed skeletal stainings, measuring relative digit length as a proxy for the phenotype (Fig. 6a,b). First, we analyzed R1 mutants, which displayed moderate Pax3 misexpression in the anterior distal limb (Fig. 1f). Finger length ratios revealed that R1 limbs develop normally, demonstrating that the detrimental effects of Pax3 misexpression can be partially buffered.

a, Skeletal staining of forelimbs from E17.5 mutant and control fetuses. White arrowheads indicate reduced index finger lengths. Black bracket shows the region of the finger measured for the quantification. Finger length correlates negatively with increased Pax3 misexpression. Scale bar, 500m. b, Index lengths relative to ring finger lengths in E17.5 mouse forelimbs. Bars represent the mean and white dots represent individual replicates. Values were normalized on control (CTRL) animals (two-sided t-test **P0.01; two-sided t-test ***P0.001; R1+F2 versus CTRL: 0.007; F-all versus CTRL: 0.0002). c, Correlation between the number of remaining CBSs at the EP boundary and the levels of Pax3 expression in the different mutants described in this study. Pearson regression lines are shown together with R2 values, both for the whole collection of mutants (black) and discarding combined CBS deletions involving CBSs with forward orientation (turquoise). d, Correlation and R2 between BSs and the brachydactyly phenotype penetrance measured as the index to ring finger length ratio for controls, R1+F2 and F-all mutants. The color of the dots represents the level of Pax3 limb misexpression as measured by qPCR. e, Model for boundary insulation as a quantitative modulator of gene expression and developmental phenotypes. Left, a strong boundary (B) efficiently insulates gene A from the enhancers located in the adjacent TAD (E). The boundary shows a cluster of CBSs with different orientations represented with arrowheads. The colored arrow represents a CBS with prominent contribution to boundary function. Middle, the absence of specific CBSs results in a weakened boundary, moderate gene misexpression (limb, indicated in yellow) and mild phenotypes (reduced digits, indicated in red and pointed out by white arrowhead). Right, the absence of the boundary causes a fusion of TADs, strong gene misexpression and strong phenotypes.

In contrast, R1+F2 mutants displayed a moderate reduction of index digit length (Fig. 6a,b), consistent with their increased Pax3 misexpression (Fig. 2b). This demonstrates that weakened boundaries can be permissive to functional interactions between TADs, resulting in altered transcriptional patterns and phenotypes. Importantly, the phenotypes of R1+F2 mutants occur despite an observable partition between Epha4 and Pax3 TADs and across a boundary region displaying high boundary scores (Fig. 2c,d; boundary score=0.8). Analyses on ultra-high-resolution Hi-C datasets26 revealed that many boundary scores fall within the ranges described in our mutant collection (Extended Data Fig. 10). Of note, 40% of boundaries display scores lower than 0.8. According to our observations, those boundaries could be permeable for functional interactions across domains.

Finally, we analyzed the F-all mutants, in which the Epha4 and Pax3 TADs appear largely fused (Fig. 2c). This disruption of TAD organization led to a prominent reduction of digit length (Fig. 6a,b), consistent with the higher Pax3 misexpression (Fig. 2b). Overall, these results illustrate how boundary insulation strength can modulate gene expression and developmental phenotypes, by allowing permissive functional interactions between TADs.

Continued here:
In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation - Nature.com

Every week, we update this list with new meetings, awards, scholarships and events to help you advance your career.If youd like us to feature something that youre offering to the bioscience community, email us with the subject line For calendar. ASBMB members offerings take priority, and we do not promote products/services. Learn how to advertise in ASBMB Today.

This in-person meeting will be held Sept. 29 through Oct. 2 in Snowbird, Utah. Sessionswill cover recent advances and new technologies in RNA polymerase II regulation, including the contributions of non-coding RNAs, enhancers and promoters, chromatin structure and post-translational modifications, molecular condensates, and other factors that regulate gene expression. Patrick Cramer of the Max Planck Institute will present the keynote address on the structure and function of transcription regulatory complexes. The deadline for abstracts for talks is now July 21. The early registration deadline ($50 in savings) is Aug. 1. The deadline for poster presentation abstracts is Aug. 18. The regular registration deadline is Aug. 28.Learn more.

The American Society for Investigative Pathology, American Society for Matrix Biology and the histochemical Society have teamed up for a series of webinars about science careers. The next one will be at noon Eastern on July 27 titled "Career Options in Science Industry vs. Academia." It will have four panelists from Genentech, FENIX Group, GE Healthcare and the University of Saskatchewan. Learn more and register.

The National Institutes of Health Office of Research on Women's Health has a free quarterly lecture series titled "Diverse Voices: Intersectionality and the Health of Women." The July 28 event will include presentations from Heather Shattuck-Heidorn of the University of Southern Maine and Stephaun Wallace of the Fred Hutchinson Cancer Research Center. Register.

Most meetings on epigenetics and chromatin focus on transcription, while most meetings on genome integrity include little attention to epigenetics and chromatin. This conference in Seattle will bridge this gap to link researchers who are interested in epigenetic regulations and chromatin with those who are interested in genome integrity. The oral and poster abstract deadline and early registration deadline is Aug. 2. The regular registration deadline is Aug. 29.Learn more.

This five-day conference will be held Aug. 1418 in person in Cambridge, Massachusetts, and online. It will be an international forum for discussion of the remarkable advances in cell and human protein biology revealed by ever-more-innovative and powerful mass spectrometric technologies. The conference will juxtapose sessions about methodological advances with sessions about the roles those advances play in solving problems and seizing opportunities to understand the composition, dynamics and function of cellular machinery in numerous biological contexts. In addition to celebrating these successes, the organizers also intend to articulate urgent, unmet needs and unsolved problems that will drive the field in the future. The registration deadline was July 1, but you have until July 12 to register to participate virtually.Learn more.

For Discover BMB, the ASBMB's annual meeting in March in Seattle, we're seeking two types of proposals:

The American Physiological Society is hosting a free webinar that will cover polycystic ovary syndrome, an endocrine disorder associated with modestly elevated androgens, and hormone therapy for transmen, which elevates androgens greatly to achieve levels similar to those in cisgender men. The event announcement says: "The role that these two different concentrations play in cardiovascular physiology and pathophysiology remains unclear. Gaps and opportunities in basic research and clinical practice will be highlighted." The speaker will be Licy Yanes Cardozo, a physician-scientist at the University of Mississippi Medical Center. Learn more and register.

In May, the Howard Hughes Medical Institute launched a roughly $1.5 billion program to "help build a scientific workforce that more fully reflects our increasingly diverse country." The Freeman Hrabowski Scholars Program will fund 30 scholars every other year, and each appointment can last up to 10 years. That represents up to $8.6 million in total support per scholar. HHMI is accepting applications from researchers "who are strongly committed to advancing diversity, equity, and inclusion in science." Learn more.

Save the date for the ASBMB Career Expo. This virtual event aims to highlight the diversity of career choices available to modern biomedical researchers. No matter your career stage, this expo will provide a plethora of career options for you to explore while simultaneously connecting you with knowledgeable professionals in these careers. Each 60-minute session will focus on a different career path and will feature breakout rooms with professionals in those paths. Attendees can choose to meet in a small group with a single professional for the entire session or move freely between breakout rooms to sample advice from multiple professionals. Sessions will feature the following five sectors: industry, government, science communication, science policy and other. The expo will be held from 11 a.m. to 5 p.m. Eastern on Nov. 2. Stay tuned for a link to register!

The Journal of Science Policy & Governanceand the National Science Policy Network issued a call for papersfor an issue containingpolicy ideas from the next generation of scientists. The submission deadline is Nov. 6. Theyencourage submissions "that highlight policy opportunities and audiences related to the 2022 U.S. midterm elections at the local, stateor national level as well as related foreign policy issues."Read the press release.

The ASBMB provides members with a virtual platform to share scientific research and accomplishments and to discuss emerging topics and technologies with the BMB community.

The ASBMB will manage the technical aspects, market the event to tens of thousands of contacts and present the digital event live to a remote audience. Additional tools such as polling, Q&A, breakout rooms and post event Twitter chats may be used to facilitate maximum engagement.

Seminars are typically one to two hours long. A workshop or conference might be longer and even span several days.

Prospective organizers may submit proposals at any time. Decisions are usually made within four to six weeks.

Propose an event.

If you are a graduate student, postdoc or early-career investigator interested in hosting a #LipidTakeover, fill out this application. You can spend a day tweeting from the Journal of Lipid Research's account (@JLipidRes) about your favorite lipids and your work.

The International Union of Biochemistry and Molecular Biology is offering $500 to graduate students and postdocs displaced from their labs as a result of natural disaster, war or "other events beyond their control that interrupt their training." The money is for travel and settling in. Learn more and spread the word to those who could use assistance.

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Calendar of events, awards and opportunities - ASBMB Today

Engineering | Health and medicine | Science | UW News blog

July 15, 2022

Another beautiful day on the University of Washingtons Seattle campus.University of Washington

Seven professors at the University of Washington are among 25 new members of the Washington State Academy of Sciences, according to a July 15 announcement. Joining the academy is a recognition of their outstanding record of scientific and technical achievement, and their willingness to work on behalf of the Academy to bring the best available science to bear on issues within the State of Washington.

Twenty of the incoming members for 2022 were selected by current WSAS members, while the other five were chosen by virtue of recently joining one of the National Academies.

UW faculty selected by current Academy members are:

In addition, Dr. Jay Shendure, UW professor of genome sciences, investigator with the Howard Hughes Medical Institute and faculty member in the Molecular Engineering & Sciences Institute, was selected by virtue of his election to the National Academy of Sciences for pioneering a variety of genome sequencing and analysis methods, including exome sequencing and its earliest applications to gene discovery for Mendelian disorders and autism; cell-free DNA diagnostics for cancer and reproductive medicine; massively parallel reporter assays; saturation genome editing; whole organism lineage tracing; and massively parallel molecular profiling of single cells.

New members to the Washington State Academy of Sciences will be formally inducted in September.

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Seven UW faculty members elected to the Washington State Academy of Sciences - University of Washington

A recent study led by the University of Wisconsin Madison has found that some species of copepods such as Eurytemora affinis tiny crustaceans measuring about a millimeter in length and roaming coastal waters of oceans and estuaries around the world in massive numbers can evolve fast enough to survive in the face of rapid climate change.

This is a dominant coastal species, serving as very abundant and highly nutritious fish food, said study senior author Carol Eunmi Lee, a professor of Integrative Biology at UW Madison. But theyre vulnerable to climate change.

Since ocean salinity is dropping rapidly as ice melts and precipitation patterns change, this saltwater species that evolved over the ages in waters high in salinity, now needs to adapt to much fresher water in their environment.

In order to study how copepods respond to drops in salinity, the scientists kept a population of Eurytemora affinisfrom the Baltic Sea in their laboratory and observed them over a few generations. After splitting the copepods into 14 groups of a few thousands each, they placed four of these groups in environments similar to the Baltic, while exposing the other ten groups to declining salinity levels that simulated the type of pressure caused by climate change. For a total of ten generations, these groups had their water gradually reduced to lower salinity levels.

To track evolutionary changes across the genomes of the tiny crustaceans, the researchers sequenced the genome of each line of copepods at the beginning of the experiment, as well as after six and ten generations.

The analysis revealed that the strongest signals of natural selection where changes were largest and more frequent across the groups exposed to low salinity levels were in areas of the genome that are important in regulating ions, such as sodium transporters.

In saltwater, there are a lot of ions, like sodium, that are essential for survival. But when you get to freshwater, these ions are precious, Professor Lee explained. So, the copepods need to suck them up from the environment and hang on to them, and the ability to do that relies on these ion transporters that we found undergoing natural selection.

At the end of the experiment, the copepods with certain genetic combinations of the ion transporter were more likely to survive, even as the salinity of their water decreased. According to the researchers, the gene variants found in the copepods that managed to survive the salinity decline in the laboratory are also common among copepods living in the fresher regions of the Baltic Sea.

This copepod gives us an idea of what it takes, an idea of what conditions are needed, that enable a population to evolve rapidly in response to climate change. It also shows how important evolution is for understanding our changing planet and how or even whether populations and ecosystems will survive, Professor Lee concluded.

The study is published in the journal Nature Communications.

By Andrei Ionescu, Earth.com Staff Writer

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Tiny crustaceans have what it takes to survive climate change - Earth.com

We have long been baffled as to why men live around five years less than women, on average. But now a new study suggests that, beyond the age of 60, the main culprit is a genetic defect: the loss of the Y chromosome, which determines sex at birth.

Its clear that men are more fragile, the question is why, explains Lars Forsberg, a researcher at Uppsala University in Sweden.

For decades it was thought that the male Y chromosomes only function was to generate sperm that determine the sex of a newborn. A boy carries one X chromosome from the mother and one Y from the father, while a girl carries two Xs, one from each parent.

In 1963, a team of scientists discovered that as men age, their blood cells lose the Y chromosome due to a copying error that happens when the mother cell divides to produce a daughter cell. In 2014, Forsberg analyzed the life expectancy of older men based on whether their blood cells had lost the Y chromosome, a mutation called mLOY. The effect recorded was mindblowing, the researcher recalls.

Men with fewer Y chromosomes had a higher risk of cancer and lived five and a half years less than those who retained this part of the genome. Three years later, Forsberg discovered that this mutation makes getting Alzheimers three times as likely. What is most worrying is the enormous prevalence of this defect. Twenty percent of men over the age of 60 have the mutation. The rate rises to 40% in those over 70 and 57% in those over 90, according to Forsbergs previous studies. It is undoubtedly the most common mutation in humans, he says.

Until now, nobody knew whether the gradual disappearance of the Y chromosome in the blood played a pivotal role in diseases associated with aging. In a study just published in the journal Science, Forsberg and scientists from Japan and the US demonstrate for the first time that this mutation increases the risk of heart problems, immune system failure and premature death.

The researchers have created the first animal model without a Y chromosome in their blood stem cells: namely, mice modified with the gene-editing tool CRISPR. The study showed that these rodents develop scarring of the heart in the form of fibrosis, one of the most common cardiovascular ailments in humans, and die earlier than normal mice. The authors then analyzed the life expectancy recorded in nearly 15,700 patients with cardiovascular disease whose data are stored in the UK public biobank. The analysis shows that loss of the Y chromosome in the blood is associated with a 30% increased risk of dying from cardiovascular disease.

This genetic factor can explain more than 75% of the difference in life expectancy between men and women over the age of 60, explains biochemist Kenneth Walsh, a researcher at the University of Virginia in the US and co-author of the study. In other words, this mutation would explain four of the five years lower life expectancy in men. Walshs estimate links to a previous study in which men with a high mLOY load live about four years less than those without it.

It is well known that men die earlier than women because they smoke and drink more and are more prone to recklessness. But, beyond the age of 60, genetics becomes the main culprit in the deterioration of their health: It seems as if men age earlier than women, Walsh points out.

The study reveals the molecular keys to the damage associated with the mLOY mutation. Within the large group of blood cells can be found the immune systems white blood cells responsible for defending the body against viruses and other pathogens. The loss of the Y chromosome triggers aberrant behavior in macrophages, a type of white blood cell, causing them to scar heart tissue, which in turn increases the risk of heart failure. Researchers have shown that the damage can be reversed if they give mice pirfenidone, a drug approved to treat humans with idiopathic pulmonary fibrosis, a condition in which the lungs become scarred and breathing becomes increasingly difficult.

There are three factors that increase the risk of Y chromosome loss. The first is the inevitable ageing process. The longer one lives, the more cell divisions occur in the body and the greater the likelihood of mutations occurring in the genome copying process. The second is smoking. Smoking causes you to lose the Y chromosome in your blood at an accelerated rate; if you stop smoking, healthy cells once again become the majority, says Walsh. But the third is also inevitable: other inherited genetic mutations can increase the gradual loss of the Y chromosome in the blood by a factor of five, explains Forsberg.

Both Forsberg and Walsh believe that this study opens up an enormous field of research. Still to be studied is whether men with this mutation also have cardiac fibrosis and whether this is behind their heart attacks and other cardiac ailments. We also need to better understand why losing the Y chromosome damages health. For now, we have shown that the Y chromosome is not just there for reproduction, but is is also important for our health, says Forsberg. The next step is to identify which genes are responsible for the phenomenon.

The loss of this chromosome has been detected in all organs and tissues of the body and at all ages, although it is more evident after 60. It is abundant in the blood because this is a tissue that produces millions of new cells every day from blood stem cells. Healthy stem cells produce healthy daughter cells and mutated ones produce daughter cells with mLOY.

A previous study showed that this mutation of the Y chromosome disrupts the function of up to 500 genes located elsewhere in the genome. It has also been shown to damage lymphocytes and natural killer cells, evident in men with prostate cancer and Alzheimers disease, respectively.

There are hardly any tests for mLOY at present. But Forsberg and his colleagues have designed a PCR test that measures the level of this mutation in the blood and could serve to determine which levels of this mutation are harmful to health. Right now, we see people in their 80s with 80% of their blood cells mutated, but we dont know what impact this has on their health, says Walsh.

Another unanswered question is why men lose the genetic mark of the male with age. The evolutionary logic, argue the authors of the paper, is that men are biologically designed to have offspring as soon as possible and to live 40 to 50 years at most. The spectacular increase in life expectancy in the last century has meant that men and women live to an advanced age 80 and 86 years in Spain, respectively which makes the effect of these mutations more evident. Another fact which possibly has some bearing on the issue: the vast majority of people who reach 100 are women.

To transform all these discoveries into treatments, we first need to better understand this phenomenon, says Forsberg. We men are not designed to live forever, but perhaps we can increase our life expectancy by a few more years.

Biochemist Jos Javier Fuster, who studies pathological mutations in blood cells at the National Center for Cardiovascular Research, stresses the importance of the work. Until now it was not clear whether the loss of Y was the cause of cancer, Alzheimers disease and heart failure, he explains. This is the first demonstration in animals that it has a causal role. The human Y chromosome is different from the mouse chromosome, so the priority now is to accumulate more data in humans. This is a great first step in understanding this new mechanism behind aging-linked diseases, he adds.

The cells of the human body group their DNA into 23 pairs of chromosomes that pair up one by one when a cell copies its genome to generate a daughter cell. The Y is the only one that does not have a symmetrical partner to pair up with: instead, it does so with an X chromosome; and the entire Y chromosome is often lost, explains Luis Alberto Prez Jurado from Pompeu Fabra University in Barcelona. For now, six genes have been identified within the Y chromosome that would be responsible for an impact on health, he says. All of them are related to the proper functioning of the immune system. In part, this would also explain the greater vulnerability of males to viral infections, including Covid-19.

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mLOY: The genetic defect that explains why men have shorter lives than woman - EL PAS USA

When Josefin Stiller was growing up in Berlin, she loved reading about Greek gods in an encyclopedia of mythology. She often lost track of their relationships, howevertheir feuds, trysts, and betrayalsas she flipped among the entries. Frustrated, she wrote each name on a card and started to arrange children beneath parents on a desk in her bedroom. As lineages became clear, so did family dramas. Sons killed fathers; uncles kidnapped nieces; siblings fell in love. I wonder if this experience of reconstructing a family tree primed me to appreciate trees and the powerful insights they hold, Stiller told me in a recent e-mail.

Years later, as a graduate student in biology, Stiller worked on an evolutionary tree for seahorses and their relatives, using DNA to understand the ancestry of different species. Then, in 2017, she moved to the University of Copenhagen and joined B10K, a scientific collaboration that aims to sequence the genome of every bird speciesmore than ten thousand in alland to reveal their connections in a comprehensive tree. The amount of data and computing power required for this mission is almost unfathomable, but the final product should be as simple in principle as the diagram Stiller had assembled as a child. Everything in biology has a history, and we can show this history as a bifurcating tree, she said.

Birds are the most diverse vertebrates on land, and they have always been central to ideas about the natural world. In 1837, a taxonomist in London told Charles Darwin that the finches he had shot and carelessly lumped together in the Galpagos Islands were, in fact, many different species. Darwin wondered whether the finches might have shared a common ancestor from mainland South Americawhether all of life might have evolved through a process of descent with modificationand he drew a rudimentary tree in his private notebook, beneath the words I think. The tree showed how a single ancestral population could branch into many species, each with its own evolutionary path. On the Origin of Species, published twenty-two years later, includes only one diagram: an evolutionary tree. The tree of life became for biology what the periodic table was for chemistryboth a foundation and an emblem for the field. Thetime will come I believe, though I shall not live to see it, when we shall have fairly true genealogical treesofeach great kingdomofnature, Darwin wrote to a friend.

The rise of genome sequencing, at the turn of the twenty-first century, seemed to bring Darwins dream within reach. It is now realistic to conceive of reconstructing the entire Tree of Lifeeventually to include all of the living and extinct species, Joel Cracraft, the curator of birds at the American Museum of Natural History, wrote, in 2004. The naturalist E. O. Wilson predicted that such a tree could unify biology. Its value to such fields as agriculture, conservation, and medicine would be incalculable; evolutionary trees have already deepened our understanding of SARS-CoV-2, the virus that causes COVID-19. By mapping a major branch on the tree of life, B10K aims to light the way.

When Stiller joined the project, her colleagues were combing through museums and laboratories to sample three hundred and sixty-three bird species, chosen carefully to represent the diversity of living birds. With help from four supercomputers in three different countries, they began to compare each birds DNA to figure out how they were related. I think there was always this idea that, once we sequence full genomes, we will be able to solve it, Stiller told me. But, early in the process, she encountered an evolutionary enigma called Opisthocomus hoazin. I was completely amazed by this bird, she said.

Hoatzins, which live along oxbow lakes in tropical South America, have blood-red eyes, blue cheeks, and crests of spiky auburn feathers. Their chicks have primitive claws on their tiny wings and respond to danger by plunging into water and then clawing their way back to their nestsa trait that inspired some ornithologists to link them to dinosaurs. Other taxonomists argued that the hoatzin is closely related to pheasants, cuckoos, pigeons, and a group of African birds called turacos. Alejandro Grajal, the director of Seattles Woodland Park Zoo, said that the bird looks like a punk-rock chicken, and smells like manure because it digests leaves through bacterial fermentation, similar to a cow.

DNA research has not solved the mysteries of the hoatzin; it has deepened them. One 2014 analysis suggested that the birds closest living relatives are cranes and shorebirds such as gulls and plovers. Another, in 2020, concluded that this clumsy flier is a sister species to a group that includes tiny, hovering hummingbirds and high-speed swifts. Frankly, there is no one in the world who knows what hoatzins are, Cracraft, who is now a member of B10K, said. The hoatzin may be more than a missing piece of the evolutionary puzzle. It may be a sphinx with a riddle that many biologists are reluctant to consider: What if the pattern of evolution is not actually a tree?

Fossils that resemble hoatzins have been found in Europe and Africa, but today the birds can be found only in the river basins of the Amazon and Orinoco of South America. I live in Germany, so I visited them in Berlins Museum of Natural History, where cabinets are filled with thousands of stuffed birds. Sylke Frahnert, the bird curator, kept two taxidermy hoatzins on a shelf near the cuckoos and turacos, which seems as good a place as any. Over the years, there have been so many conflicting trees of birds, she told me. You would have been crazy to change the collection with every one. One of the museums hoatzins was shot in Brazil more than two centuries ago, and the years have drained the color from its face. I had heard that even the specimens smell like manure, but Frahnert warned me not to sniff them, since birds were once preserved with arsenic.

In the eighteenth century, natural-history museums started using anatomical similarities to classify plants and animals into increasingly specific categories: class, order, family, genus, species. Darwin realized that species share traits because their ancestors were one and the same. Fish, amphibians, reptiles, birds, and mammals all have spines, but not because God had given them to each creature separately; rather, the spine suggested a common parent living long ago. The construction of evolutionary trees was dubbed phylogeny, literally meaning the generation of species, by the zoologist Ernst Haeckel. The more traits two species shared, the theory went, the more recently they had shared a common ancestor. Human beings and other great apes evolved from a common ancestor millions of years ago, but even human beings and bacteria have a common ancestorthe first known living organisms, which date to three and a half billion years ago.

Hoatzinsin some respects the most aberrant of birds, according to one Victorian ornithologistwere a problem from the beginning. Early European naturalists described them as pheasants, and the first major tree for birds, published in 1888 by Max Frbringer, placed them on the fowl branch. But, by the early nineteen-hundreds, some scientists were comparing hoatzins and cuckoos on the basis of traits such as jaws and feathers, and others were noting similarities between hoatzins and turacos, pigeons, barn owls, and rails. Even the hoatzins parasites defied classification: they hosted feather lice found on no other birds.

One crucial problem in phylogeny was convergent evolution. Sometimes natural selection nudges two organisms toward the same trait. Birds and bats independently evolved the ability to fly. Swifts and swallows each evolved into aerodynamic insectivores with nearly identical silhouettes, but traits such as their vocal organs and foot bones reveal that they are only distantly related. Because taxonomists often disagreed about things such as how to distinguish common ancestry from convergent evolution, the literature grew thick with conflicting trees, to the point that some twentieth-century biologists seemed ready to give up. The construction of phylogenetic trees has opened the door to a wave of uninhibited speculation, one wrote in 1959. Science ends where comparative morphology, comparative physiology, comparative ethology have failed us.

Phylogeny made a comeback in the seventies and eighties, after the German entomologist Willi Hennig developed more rigorous criteria for identifying common ancestry and drawing evolutionary trees. These innovations laid a foundation for a new wave of research that did not rely solely on physical specimens but, rather, on the emerging science of DNA. Organisms are related to one another by the degree to which they share genetic information, two ornithologists wrote in the early nineties, adding that genetics could reveal a different view of the process of evolution and its effects. The typical bird genome is a string of more than a billion base pairs that mutate randomly over time. Scientists can compare the same parts of the genome across multiple species to estimate their evolutionary closeness. Typically, species that share mutations have a more recent common ancestor, and species that do not are more distantly related.

Early sequencing was expensive and tedious, but, by the beginning of the twenty-first century, a signal was emerging from the noise. The journal Nature published an article about the promise of a single unified tree of life. But its author also identified a complication: each genome contains many different genes, and each one could generate a different evolutionary tree.

In 2001, a paper in the Proceedings of the Royal Society identified a pair of bird siblings as unlikely as Arnold Schwarzenegger and Danny DeVito: the flamingos closest relative was a little diving bird called a grebe. That was probably the single most astounding result that anybodys ever gotten, Peter Houde, an avian biologist from New Mexico State University, told me. Ornithologists had always reasoned that grebes were closely related to short-legged loons, whereas tall wading birds such as flamingos, storks, and herons probably had a long-legged common ancestor.

That was the first domino to fall. In 2008, Science published a new avian tree based on DNA. Research led by Shannon Hackett, Rebecca Kimball, and Sushma Reddy, scientists affiliated with the Field Museum and the University of Florida, examined nineteen parts of the genomes of a hundred and sixty-nine avian species. The root of their tree resembled trees based on physical specimens: large, flightless birds such as ostriches, emus, and kiwisknown collectively as ratiteswere first to diverge from all the others, followed by land fowl and waterfowl. The remaining ninety-five per cent of living birds, from parrots to penguins and pigeons, are known as modern birds and descended from a common ancestor, probably around the time that an asteroid hit the earth, sixty-six million years ago, and the dinosaurs went extinct. The youngest orderpasserines, which include all songbirdsbranched out into a staggering six thousand species in the span of tens of millions of years. The genetic tree for modern birds was decked with relationships that few, if any, taxonomists had guessed from anatomy; key groups such as parrots, owls, woodpeckers, vultures, and cranes shifted places.

Scientists had long assumed, for example, that daytime hunters such as hawks, eagles, and falcons all descended from a single bird of prey. But, in the genetic tree, hawks and eagles shared a branch with vultures, yet falcons turned out to be closer relatives of passerines and parrots. This meant that the peregrine falcon is more closely related to colorful macaws and tiny sparrows than to any hawk or eagle. The traditional explanation for flightlessness in ratitesthat a common ancestor diverged into ostriches, emus, rheas, cassowaries, and kiwis after the southern continents split apartalso collapsed. DNA showed that the ratites also included flying birds called tinamous, suggesting that the group evolved flightlessness at least three separate times. That study revolutionized our understanding of how the major groups of living birds are related to each other, Daniel J. Field, an avian paleontologist at the University of Cambridge, said. Bird-watching guides had to reorganize their contents to reflect the new relationships.

Excerpt from:
The Bizarre Bird Thats Breaking the Tree of Life - The New Yorker