The influence of evolutionary history on human health and disease.

Nearly all genetic variants that influence disease risk have human-specific origins; however, the systems they influence have ancient roots that often trace back to evolutionary events long before the origin of humans. Here, we review how advances in our understanding of the genetic architectures of diseases, recent human evolution and deep evolutionary history can help explain how and why humans in modern environments become ill. Human populations exhibit differences in the prevalence of many common and rare genetic diseases. These differences are largely the result of the diverse environmental, cultural, demographic and genetic histories of modern human populations. Synthesizing our growing knowledge of evolutionary history with genetic medicine, while accounting for environmental and social factors, will help to achieve the promise of personalized genomics and realize the potential hidden in an individual's DNA sequence to guide clinical decisions. In short, precision medicine is fundamentally evolutionary medicine, and integration of evolutionary perspectives into the clinic will support the realization of its full potential.

BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels.

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modifications likely contributes to disease states and cancer. Depletion of the chromatin-binding protein BRWD3 (Bromodomain and WD repeat-containing protein 3), a known substrate-specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, results in increased H3K4me1 (H3 lysine 4 monomethylation) levels. The underlying mechanism linking BRWD3 and H3K4 methylation, however, has yet to be defined. Here, we show that depleting BRWD3 not only causes an increase in H3K4me1 levels but also causes a decrease in H3K4me3 (H3 lysine 4 trimethylation) levels, indicating that BRWD3 influences H3K4 methylation more broadly. Using immunoprecipitation coupled to quantitative mass spectrometry, we identified an interaction between BRWD3 and the H3K4-specific lysine demethylase 5 (KDM5/Lid), an enzyme that removes tri- and dimethyl marks from H3K4. Moreover, analysis of ChIP-seq (chromatin immunoprecipitation sequencing) data revealed that BRWD3 and KDM5 are significantly colocalized throughout the genome and H3K4me3 are highly enriched at BRWD3 binding sites. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels.

GSEL: a fast, flexible python package for detecting signatures of diverse evolutionary forces on genomic regions.

SUMMARY: GSEL is a computational framework for calculating the enrichment of signatures of diverse evolutionary forces in a set of genomic regions. GSEL can flexibly integrate any sequence-based evolutionary metric and analyze sets of human genomic regions identified by genome-wide assays (e.g. GWAS, eQTL, *-seq). The core of GSEL's approach is the generation of empirical null distributions tailored to the allele frequency and linkage disequilibrium structure of the regions of interest. We illustrate the application of GSEL to variants identified from a GWAS of body mass index, a highly polygenic trait. AVAILABILITY AND IMPLEMENTATION: GSEL is implemented as a fast, flexible and user-friendly python package. It is available with demonstration data at https://github.com/abraham-abin13/gsel_vec. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genome-wide enhancer annotations differ significantly in genomic distribution, evolution, and function.

BACKGROUND: Non-coding gene regulatory enhancers are essential to transcription in mammalian cells. As a result, a large variety of experimental and computational strategies have been developed to identify cis-regulatory enhancer sequences. Given the differences in the biological signals assayed, some variation in the enhancers identified by different methods is expected; however, the concordance of enhancers identified by different methods has not been comprehensively evaluated. This is critically needed, since in practice, most studies consider enhancers identified by only a single method. Here, we compare enhancer sets from eleven representative strategies in four biological contexts. RESULTS: All sets we evaluated overlap significantly more than expected by chance; however, there is significant dissimilarity in their genomic, evolutionary, and functional characteristics, both at the element and base-pair level, within each context. The disagreement is sufficient to influence interpretation of candidate SNPs from GWAS studies, and to lead to disparate conclusions about enhancer and disease mechanisms. Most regions identified as enhancers are supported by only one method, and we find limited evidence that regions identified by multiple methods are better candidates than those identified by a single method. As a result, we cannot recommend the use of any single enhancer identification strategy in all settings. CONCLUSIONS: Our results highlight the inherent complexity of enhancer biology and identify an important challenge to mapping the genetic architecture of complex disease. Greater appreciation of how the diverse enhancer identification strategies in use today relate to the dynamic activity of gene regulatory regions is needed to enable robust and reproducible results.

Ethanol consumption in non-human primates alters plasma markers of bone turnover but not tibia architecture.

Ethanol consumption is associated with positive, negative, and neutral effects on the skeletal system. Our previous work using a nonhuman primate model of voluntary ethanol consumption showed that chronic ethanol use has an impact on skeletal attributes, most notably on biochemical markers of bone turnover. However, these studies were limited by small sample sizes and resulting lack of statistical power. Here, we applied a machine learning framework to integrate data from 155 monkeys (100 ethanol and 55 controls) to identify the bone features associated with chronic ethanol use. Specifically, we analyzed the influence of ethanol consumption on biomarkers of bone turnover and cancellous and cortical bone architecture in tibia. We hypothesized that chronic ethanol use for 6 months to 2.5 years would result in measurable changes to cancellous features and the biochemical markers compared to control animals. We observed a decrease in bone turnover in monkeys exposed to ethanol; however, we did not find that ethanol consumption resulted in measurable changes in bone architecture.

Six months of voluntary alcohol consumption in male cynomolgus macaques reduces intracortical bone porosity without altering mineralization or mechanical properties.

Chronic heavy alcohol consumption is a risk factor for low trauma bone fracture. Using a non-human primate model of voluntary alcohol consumption, we investigated the effects of 6 months of ethanol intake on cortical bone in cynomolgus macaques (Macaca fascicularis). Young adult (6.4 ± 0.1 years old, mean ± SE) male cynomolgus macaques (n = 17) were subjected to a 4-month graded ethanol induction period, followed by voluntary self-administration of water or ethanol (4 % w/v) for 22 h/d, 7 d/wk. for 6 months. Control animals (n = 6) consumed an isocaloric maltose-dextrin solution. Tibial response was evaluated using densitometry, microcomputed tomography, histomorphometry, biomechanical testing, and Raman spectroscopy. Global bone response was evaluated using biochemical markers of bone turnover. Monkeys in the ethanol group consumed an average of 2.3 ± 0.2 g/kg/d ethanol resulting in a blood ethanol concentration of 90 ± 12 mg/dl in longitudinal samples taken 7 h after the daily session began. Ethanol consumption had no effect on tibia length, mass, density, mechanical properties, or mineralization (p > 0.642). However, compared to controls, ethanol intake resulted in a dose-dependent reduction in intracortical bone porosity (Spearman rank correlation = -0.770; p < 0.0001) and compared to baseline, a strong tendency (p = 0.058) for lower plasma CTX, a biochemical marker of global bone resorption. These findings are important because suppressed cortical bone remodeling can result in a decrease in bone quality. In conclusion, intracortical bone porosity was reduced to subnormal values 6 months following initiation of voluntary ethanol consumption but other measures of tibia architecture, mineralization, or mechanics were not altered.

Intersecting Pathways in Bioinformatics and Translational Informatics: A One Health Perspective on Key Contributions and Future Directions.

OBJECTIVES: To identify and summarize the top bioinformatics and translational informatics (BTI) papers published in 2022 for the International Medical Informatics Association (IMIA) Yearbook 2023. METHODS: We conducted a comprehensive literature search to identify the top BTI papers, resulting in a set of ten candidate papers. The candidates were reviewed by the section co-editors and external reviewers to select the top three papers from 2022. RESULTS: From a total of 558 papers, we identified a final candidate list of ten BTI papers for peer-review. These papers apply new statistical frameworks and experimental designs to better capture individual variability in disease and incorporate data that captures differences between single cells and across environmental exposures. In addition, they highlight the importance of model generalization across diverse cohorts and scalability to large medical centers. CONCLUSIONS: We note several important trends in the candidate top BTI papers this year, including a continued focus on developing accurate and scalable computational models to predict disease risk across diverse cohorts and new strategies to capture the molecular heterogeneity of disease.

Cis-regulatory Landscape Size, Constraint, and Tissue Specificity Associate with Gene Function and Expression.

Multiple distal cis-regulatory elements (CREs) often cooperate to regulate gene expression, and the presence of multiple CREs for a gene has been proposed to provide redundancy and robustness to variation. However, we do not understand how attributes of a gene's distal CRE landscape-the CREs that contribute to its regulation-relate to its expression and function. Here, we integrate three-dimensional chromatin conformation and functional genomics data to quantify the CRE landscape composition genome-wide across ten human tissues and relate their attributes to the function, constraint, and expression patterns of genes. Within each tissue, we find that expressed genes have larger CRE landscapes than nonexpressed genes and that genes with tissue-specific CREs are more likely to have tissue-specific expression. Controlling for the association between expression level and CRE landscape size, we also find that CRE landscapes around genes under strong constraint (e.g., loss-of-function intolerant and housekeeping genes) are not significantly smaller than other expressed genes as previously proposed; however, they do have more evolutionarily conserved sequences than CREs of expressed genes overall. We also show that CRE landscape size does not associate with expression variability across individuals; nonetheless, genes with larger CRE landscapes have a relative depletion for variants that influence expression levels (expression quantitative trait loci). Overall, this work illustrates how differences in gene function, expression, and evolutionary constraint are reflected in features of CRE landscapes. Thus, considering the CRE landscape of a gene is vital for understanding gene expression dynamics across biological contexts and interpreting the effects of noncoding genetic variants.

A comprehensive map of hotspots of de novo telomere addition in Saccharomyces cerevisiae.

Telomere healing occurs when telomerase, normally restricted to chromosome ends, acts upon a double-strand break to create a new, functional telomere. De novo telomere addition on the centromere-proximal side of a break truncates the chromosome but, by blocking resection, may allow the cell to survive an otherwise lethal event. We previously identified several sequences in the baker’s yeast, Saccharomyces cerevisiae , that act as hotspots of de novo telomere addition (termed Sites of Repair-associated Telomere Addition or SiRTAs), but the distribution and functional relevance of SiRTAs is unclear. Here, we describe a high-throughput sequencing method to measure the frequency and location of telomere addition within sequences of interest. Combining this methodology with a computational algorithm that identifies SiRTA sequence motifs, we generate the first comprehensive map of telomere-addition hotspots in yeast. Putative SiRTAs are strongly enriched in subtelomeric regions where they may facilitate formation of a new telomere following catastrophic telomere loss. In contrast, outside of subtelomeres, the distribution and orientation of SiRTAs appears random. Since truncating the chromosome at most SiRTAs would be lethal, this observation argues against selection for these sequences as sites of telomere addition per se. We find, however, that sequences predicted to function as SiRTAs are significantly more prevalent across the genome than expected by chance. Sequences identified by the algorithm bind the telomeric protein Cdc13, raising the possibility that association of Cdc13 with single-stranded regions generated during the response to DNA damage may facilitate DNA repair more generally.

A comprehensive map of hotspots of de novo telomere addition in Saccharomyces cerevisiae.

Telomere healing occurs when telomerase, normally restricted to chromosome ends, acts upon a double-strand break to create a new, functional telomere. De novo telomere addition (dnTA) on the centromere-proximal side of a break truncates the chromosome but, by blocking resection, may allow the cell to survive an otherwise lethal event. We previously identified several sequences in the baker's yeast, Saccharomyces cerevisiae, that act as hotspots of dnTA [termed Sites of Repair-associated Telomere Addition (SiRTAs)], but the distribution and functional relevance of SiRTAs is unclear. Here, we describe a high-throughput sequencing method to measure the frequency and location of telomere addition within sequences of interest. Combining this methodology with a computational algorithm that identifies SiRTA sequence motifs, we generate the first comprehensive map of telomere-addition hotspots in yeast. Putative SiRTAs are strongly enriched in subtelomeric regions where they may facilitate formation of a new telomere following catastrophic telomere loss. In contrast, outside of subtelomeres, the distribution and orientation of SiRTAs appears random. Since truncating the chromosome at most SiRTAs would be lethal, this observation argues against selection for these sequences as sites of telomere addition per se. We find, however, that sequences predicted to function as SiRTAs are significantly more prevalent across the genome than expected by chance. Sequences identified by the algorithm bind the telomeric protein Cdc13, raising the possibility that association of Cdc13 with single-stranded regions generated during the response to DNA damage may facilitate DNA repair more generally.

Diverse functions associate with non-coding polymorphisms shared between humans and chimpanzees.

BACKGROUND: Long-term balancing selection (LTBS) can maintain allelic variation at a locus over millions of years and through speciation events. Variants shared between species in the state of identity-by-descent, hereafter "trans-species polymorphisms", can result from LTBS, often due to host-pathogen interactions. For instance, the major histocompatibility complex (MHC) locus contains TSPs present across primates. Several hundred candidate LTBS regions have been identified in humans and chimpanzees; however, because many are in non-protein-coding regions of the genome, the functions and potential adaptive roles for most remain unknown. RESULTS: We integrated diverse genomic annotations to explore the functions of 60 previously identified regions with multiple shared polymorphisms (SPs) between humans and chimpanzees, including 19 with strong evidence of LTBS. We analyzed genome-wide functional assays, expression quantitative trait loci (eQTL), genome-wide association studies (GWAS), and phenome-wide association studies (PheWAS) for all the regions. We identify functional annotations for 59 regions, including 58 with evidence of gene regulatory function from GTEx or functional genomics data and 19 with evidence of trait association from GWAS or PheWAS. As expected, the SPs associate in humans with many immune system phenotypes, including response to pathogens, but we also find associations with a range of other phenotypes, including body size, alcohol intake, cognitive performance, risk-taking behavior, and urate levels. CONCLUSIONS: The diversity of traits associated with non-coding regions with multiple SPs support previous hypotheses that functions beyond the immune system are likely subject to LTBS. Furthermore, several of these trait associations provide support and candidate genetic loci for previous hypothesis about behavioral diversity in human and chimpanzee populations, such as the importance of variation in risk sensitivity.

2021 Bioinformatics and Translational Informatics Best Papers.

OBJECTIVES: To identify and summarize the top bioinformatics and translational informatics papers published in 2021 for the IMIA Yearbook. METHODS: We performed a broad literature search to retrieve Bioinformatics and Translational Informatics (BTI) papers and coupled this with a series of editorial and peer reviews to identity the top papers in the area. RESULTS: We identified a final candidate list of 15 BTI papers for peer-review; from these candidates, the top three papers were chosen to highlight in this synopsis. These papers expand the integration of multi-omics data with electronic health records and use advanced machine learning approaches to tailor models to individual patients. In addition, our honorable mention paper foreshadows the growing impact of BTI research on precision medicine through the continued development of large clinical consortia. CONCLUSION: In the top BTI papers this year, we observed several important trends, including the use of deep-learning approaches to analyse diverse data types, the development of integrative and web-accessible bioinformatics pipelines, and a continued focus on the power of individual genome sequencing for precision health.

R-loop Mapping and Characterization During Drosophila Embryogenesis Reveals Developmental Plasticity in R-loop Signatures.

R-loops are involved in transcriptional regulation, DNA and histone post-translational modifications, genome replication and genome stability. To what extent R-loop abundance and genome-wide localization is actively regulated during metazoan embryogenesis is unknown. Drosophila embryogenesis provides a powerful system to address these questions due to its well-characterized developmental program, the sudden onset of zygotic transcription and available genome-wide data sets. Here, we measure the overall abundance and genome localization of R-loops in early and late-stage embryos relative to Drosophila cultured cells. We demonstrate that absolute R-loop levels change during embryogenesis and that RNaseH1 catalytic activity is critical for embryonic development. R-loop mapping by strand-specific DRIP-seq reveals that R-loop localization is plastic across development, both in the genes which form R-loops and where they localize relative to gene bodies. Importantly, these changes are not driven by changes in the transcriptional program. Negative GC skew and absolute changes in AT skew are associated with R-loop formation in Drosophila. Furthermore, we demonstrate that while some chromatin binding proteins and histone modifications such as H3K27me3 are associated with R-loops throughout development, other chromatin factors associated with R-loops in a developmental specific manner. Our findings highlight the importance and developmental plasticity of R-loops during Drosophila embryogenesis.

Nucleoporins facilitate ORC loading onto chromatin.

The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we perform immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we find that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis reveals that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, decreases ORC2 loading onto chromatin. Depleting Elys also sensitizes cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a connection between ORC, replication initiation, and nucleoporins, suggesting a function for nucleoporins in metazoan replication initiation.

Dose-response effects of alcohol on biochemical markers of bone turnover in non-human primates: Effects of species, sex and age of onset of drinking.

PURPOSE: Alcohol consumption suppressed bone turnover in male non-human primates; however, it is unclear the extent to which this effect depends upon biological variables. Using archived plasma samples, we investigated whether sex, age of onset of alcohol intake, and species influence the effects of graded increases in alcohol consumption on bone turnover markers. METHODS: 91 male and female macaques (rhesus and cynomolgus), ranging in age from 4 years (adolescent) to 10 years (adult) were required to increase their consumption of ethanol in 30-day increments: 0 g/kg/day, followed by 0.5 g/kg/day, 1.0 g/kg/day, and, finally, 1.5 g/kg/day. Plasma osteocalcin (formation), plasma CTX (resorption) and osteocalcin to CTX ratio (turnover balance) were measured during these intervals to assess the dose-response effects of alcohol. RESULTS: We detected no relationship between dose and osteocalcin when all monkeys were combined, but there was a significant effect of sex (lower levels in females) and interactions between alcohol dose and sex (osteocalcin levels increased with dose in rhesus females). In contrast, we detected a negative linear dose-response relationship for ethanol and CTX. We did not detect a relationship between dose and osteocalcin to CTX ratio overall, but there was a significant positive relationship detected in females (no change in males). Increased age predicted lower biomarker levels for both osteocalcin and CTX. Species was a significant predictor for osteocalcin and the osteocalcin to CTX ratio in these models. CONCLUSION: These findings indicate that age, sex, and species influence bone turnover and support the concept that factors beyond quantity of alcohol affect skeletal response to alcohol consumption.

CTCF Expression and Dynamic Motif Accessibility Modulates Epithelial-Mesenchymal Gene Expression.

Epithelial-mesenchymal transition (EMT) and its reversal, mesenchymal-epithelial transition (MET) drive tissue reorganization critical for early development. In carcinomas, processing through EMT, MET, or partial states promotes migration, invasion, dormancy, and metastatic colonization. As a reversible process, EMT is inherently regulated at epigenetic and epigenomic levels. To understand the epigenomic nature of reversible EMT and its partial states, we characterized chromatin accessibility dynamics, transcriptomic output, protein expression, and cellular phenotypes during stepwise reversible EMT. We find that the chromatin insulating protein machinery, including CTCF, is suppressed and re-expressed, coincident with broad alterations in chromatin accessibility, during EMT/MET, and is lower in triple-negative breast cancer cell lines with EMT features. Through an analysis of chromatin accessibility using ATAC-seq, we identify that early phases of EMT are characterized by enrichment for AP-1 family member binding motifs, but also by a diminished enrichment for CTCF binding motifs. Through a loss-of-function analysis, we demonstrate that the suppression of CTCF alters cellular plasticity, strengthening the epithelial phenotype via the upregulation of epithelial markers E-cadherin/CDH1 and downregulation of N-cadherin/CDH2. Conversely, the upregulation of CTCF leads to the upregulation of EMT gene expression and an increase in mesenchymal traits. These findings are indicative of a role of CTCF in regulating epithelial-mesenchymal plasticity and gene expression.

Ethanol alters the relationship between IGF-1 and bone turnover in male macaques.

Insulin-like growth factor 1 (IGF-1) influences bone turnover. Transient decreases in IGF-I levels and/or bioavailability may contribute to the detrimental effects of alcohol on bone. The goals of this non-human primate study were to i) evaluate the 20-h response of bone turnover markers to ethanol consumption and ii) assess how ethanol consumption influences the relationship between IGF-1 and these markers. Osteocalcin (bone formation), carboxyterminal cross-linking telopeptide of type 1 collagen (CTX, bone resorption), IGF-1, and IGF binding protein 1 (IGFBP-1) were measured in plasma from male rhesus macaques (N = 10, 8.4 ± 0.3 years) obtained at 12:00, 16:00, and 06:00 h during two phases: pre-ethanol (alcohol-naïve) and ethanol access. During the ethanol access phase, monkeys consumed 1.5 g/kg/day ethanol (4% w/v) beginning at 10:00 h. Osteocalcin and CTX were lower, and the ratio of osteocalcin to CTX was higher at each time point during ethanol access compared to the pre-ethanol phase. Pre-ethanol marker levels did not vary across time points, but markers varied during ethanol access. IGF-1 levels, but not IGFBP-1 levels, varied during the pre-ethanol phase. In contrast, IGF-1 levels were stable during ethanol access but IGFBP-1 levels varied. There were positive relationships between IGF-1 and turnover markers during the pre-ethanol phase, but not during ethanol access. In conclusion, chronic ethanol consumption reduces levels of bone turnover markers and blocks the normal positive relationship between IGF-1 and turnover markers and alters the normal relationship between IGF-1 and IGFBP-1. These findings support the hypothesis that chronic alcohol consumption leads to growth hormone/IGF-1 resistance.

Predictions, Pivots, and a Pandemic: a Review of 2020's Top Translational Bioinformatics Publications.

OBJECTIVES: Provide an overview of the emerging themes and notable papers which were published in 2020 in the field of Bioinformatics and Translational Informatics (BTI) for the International Medical Informatics Association Yearbook. METHODS: A team of 16 individuals scanned the literature from the past year. Using a scoring rubric, papers were evaluated on their novelty, importance, and objective quality. 1,224 Medical Subject Headings (MeSH) terms extracted from these papers were used to identify themes and research focuses. The authors then used the scoring results to select notable papers and trends presented in this manuscript. RESULTS: The search phase identified 263 potential papers and central themes of coronavirus disease 2019 (COVID-19), machine learning, and bioinformatics were examined in greater detail. CONCLUSIONS: When addressing a once in a centruy pandemic, scientists worldwide answered the call, with informaticians playing a critical role. Productivity and innovations reached new heights in both TBI and science, but significant research gaps remain.

Functional annotation of rare structural variation in the human brain.

Structural variants (SVs) contribute to many disorders, yet, functionally annotating them remains a major challenge. Here, we integrate SVs with RNA-sequencing from human post-mortem brains to quantify their dosage and regulatory effects. We show that genic and regulatory SVs exist at significantly lower frequencies than intergenic SVs. Functional impact of copy number variants (CNVs) stems from both the proportion of genic and regulatory content altered and loss-of-function intolerance of the gene. We train a linear model to predict expression effects of rare CNVs and use it to annotate regulatory disruption of CNVs from 14,891 independent genome-sequenced individuals. Pathogenic deletions implicated in neurodevelopmental disorders show significantly more extreme regulatory disruption scores and if rank ordered would be prioritized higher than using frequency or length alone. This work shows the deleteriousness of regulatory SVs, particularly those altering CTCF sites and provides a simple approach for functionally annotating the regulatory consequences of CNVs.

Epo reprograms the epigenome of erythroid cells.

The hormone erythropoietin (Epo) is required for erythropoiesis, yet its molecular mechanism of action remains poorly understood, particularly with respect to chromatin dynamics. To investigate how Epo modulates the erythroid epigenome, we performed epigenetic profiling using an ex vivo murine cell system that undergoes synchronous erythroid maturation in response to Epo stimulation. Our findings define the repertoire of Epo-modulated enhancers, illuminating a new facet of Epo signaling. First, a large number of enhancers rapidly responded to Epo stimulation, revealing a cis-regulatory network of Epo-responsive enhancers. In contrast, most of the other identified enhancers remained in an active acetylated state during Epo signaling, suggesting that most erythroid enhancers are established at an earlier precursor stage. Second, we identified several hundred super-enhancers that were linked to key erythroid genes, such as Tal1, Bcl11a, and Mir144/451. Third, experimental and computational validation revealed that many predicted enhancer regions were occupied by TAL1 and enriched with DNA-binding motifs for GATA1, KLF1, TAL1/E-box, and STAT5. Additionally, many of these cis-regulatory regions were conserved evolutionarily and displayed correlated enhancer:promoter acetylation. Together, these findings define a cis-regulatory enhancer network for Epo signaling during erythropoiesis, and provide the framework for future studies involving the interplay of epigenetics and Epo signaling.