Chromatin priming elements direct tissue-specific gene activity before hematopoietic specification.

Tissue-specific gene regulation during development involves the interplay between transcription factors and epigenetic regulators binding to enhancer and promoter elements. The pattern of active enhancers defines the cellular differentiation state. However, developmental gene activation involves a previous step called chromatin priming which is not fully understood. We recently developed a genome-wide functional assay that allowed us to functionally identify enhancer elements integrated in chromatin regulating five stages spanning the in vitro differentiation of embryonic stem cells to blood. We also measured global chromatin accessibility, histone modifications, and transcription factor binding. The integration of these data identified and characterised cis-regulatory elements which become activated before the onset of gene expression, some of which are primed in a signalling-dependent fashion. Deletion of such a priming element leads to a delay in the up-regulation of its associated gene in development. Our work uncovers the details of a complex network of regulatory interactions with the dynamics of early chromatin opening being at the heart of dynamic tissue-specific gene expression control.

Epigenetic regulation during cancer transitions across 11 tumour types.

Chromatin accessibility is essential in regulating gene expression and cellular identity, and alterations in accessibility have been implicated in driving cancer initiation, progression and metastasis1-4. Although the genetic contributions to oncogenic transitions have been investigated, epigenetic drivers remain less understood. Here we constructed a pan-cancer epigenetic and transcriptomic atlas using single-nucleus chromatin accessibility data (using single-nucleus assay for transposase-accessible chromatin) from 225 samples and matched single-cell or single-nucleus RNA-sequencing expression data from 206 samples. With over 1 million cells from each platform analysed through the enrichment of accessible chromatin regions, transcription factor motifs and regulons, we identified epigenetic drivers associated with cancer transitions. Some epigenetic drivers appeared in multiple cancers (for example, regulatory regions of ABCC1 and VEGFA; GATA6 and FOX-family motifs), whereas others were cancer specific (for example, regulatory regions of FGF19, ASAP2 and EN1, and the PBX3 motif). Among epigenetically altered pathways, TP53, hypoxia and TNF signalling were linked to cancer initiation, whereas oestrogen response, epithelial-mesenchymal transition and apical junction were tied to metastatic transition. Furthermore, we revealed a marked correlation between enhancer accessibility and gene expression and uncovered cooperation between epigenetic and genetic drivers. This atlas provides a foundation for further investigation of epigenetic dynamics in cancer transitions.

[Methylation of Regulatory Regions of DNA Repair Genes in Carotid Atherosclerosis].

The status of DNA methylation in the human genome changes during the pathogenesis of common diseases and acts as a predictor of life expectancy. Therefore, it is of interest to investigate the methylation level of regulatory regions of genes responsible for general biological processes that are potentially significant for the development of age-associated diseases. Among them there are genes encoding proteins of DNA repair system, which are characterized by pleiotropic effects. Here, results of the targeted methylation analysis of two regions of the human genome (the promoter of the MLH1 gene and the enhancer near the ATM gene) in different tissues of patients with carotid atherosclerosis are present. Analysis of the methylation profiles of studied genes in various tissues of the same individuals demonstrated marked differences between leukocytes and tissues of the vascular wall. Differences in methylation levels between normal and atherosclerotic tissues of the carotid arteries were revealed only for two studied CpG sites (chr11:108089866 and chr11:108090020, GRCh37/hg19 assembly) in the ATM gene. Based on this, we can assume the involvement of ATM in the development of atherosclerosis. "Overload" of the studied regions with transcription factor binding sites (according to ReMapp2022 data) indicate that the tissue-specific nature of methylation of the regulatory regions of the MLH1 and ATM may be associated with expression levels of these genes in a particular tissue. It has been shown that inter-individual differences in the methylation levels of CpG sites are associated with sufficiently distant nucleotide substitutions.

Phylogenetic modeling of enhancer shifts in African mole-rats reveals regulatory changes associated with tissue-specific traits.

Changes in gene regulation are thought to underlie most phenotypic differences between species. For subterranean rodents such as the naked mole-rat, proposed phenotypic adaptations include hypoxia tolerance, metabolic changes, and cancer resistance. However, it is largely unknown what regulatory changes may associate with these phenotypic traits, and whether these are unique to the naked mole-rat, the mole-rat clade, or are also present in other mammals. Here, we investigate regulatory evolution in the heart and liver from two African mole-rat species and two rodent outgroups using genome-wide epigenomic profiling. First, we adapted and applied a phylogenetic modeling approach to quantitatively compare epigenomic signals at orthologous regulatory elements and identified thousands of promoter and enhancer regions with differential epigenomic activity in mole-rats. These elements associate with known mole-rat adaptations in metabolic and functional pathways and suggest candidate genetic loci that may underlie mole-rat innovations. Second, we evaluated ancestral and species-specific regulatory changes in the study phylogeny and report several candidate pathways experiencing stepwise remodeling during the evolution of mole-rats, such as the insulin and hypoxia response pathways. Third, we report nonorthologous regulatory elements overlap with lineage-specific repetitive elements and appear to modify metabolic pathways by rewiring of HNF4 and RAR/RXR transcription factor binding sites in mole-rats. These comparative analyses reveal how mole-rat regulatory evolution informs previously reported phenotypic adaptations. Moreover, the phylogenetic modeling framework we propose here improves upon the state of the art by addressing known limitations of inter-species comparisons of epigenomic profiles and has broad implications in the field of comparative functional genomics.

Identification of oil palm cis-regulatory elements based on DNA free energy and single nucleotide polymorphism density.

Transcription control through cis-regulatory elements (CREs) is one of important regulators of gene expression. This study aimed to identify the location of CREs in oil palm (Elaeis guineensis Jacq.) using the combination of DNA free energy and single nucleotide polymorphism (SNP) density approaches. Promoter region sequences were extracted oil palm genome spanning from 1500 nucleotides (nt) upstream to 1000 nt downstream of every annotated transcription start sites (TSS). Free energy profiles of each promoter region were calculated using PromPredict software. Raw reads from the deep sequencing of 59 oil palm origins were used to calculate SNP density of each promoter region. The result showed that the average free energy (AFE) on the upstream region of TSS is about 1.5 kcal/mol higher compared to the downstream region. Using DNA free energy method, 16,281 regions of CREs were predicted. Most of predicted CREs was located between 1 and 500 nt upstream of TSS. Anti-correlation pattern between free energy and SNP density was observed on the predicted regions of CREs. This anti-correlated pattern was also observed on an experimentally determined promoter of the oil palm metallothionein gene, EgMSP1. Considering the increasing use of promoter information on plant biotechnology, an easy and accurate promoter prediction using the combination of free energy and SNP density method could be recommended.

H4K16ac activates the transcription of transposable elements and contributes to their cis-regulatory function.

Mammalian genomes harbor abundant transposable elements (TEs) and their remnants, with numerous epigenetic repression mechanisms enacted to silence TE transcription. However, TEs are upregulated during early development, neuronal lineage, and cancers, although the epigenetic factors contributing to the transcription of TEs have yet to be fully elucidated. Here, we demonstrate that the male-specific lethal (MSL)-complex-mediated histone H4 acetylation at lysine 16 (H4K16ac) is enriched at TEs in human embryonic stem cells (hESCs) and cancer cells. This in turn activates transcription of subsets of full-length long interspersed nuclear elements (LINE1s, L1s) and endogenous retrovirus (ERV) long terminal repeats (LTRs). Furthermore, we show that the H4K16ac-marked L1 and LTR subfamilies display enhancer-like functions and are enriched in genomic locations with chromatin features associated with active enhancers. Importantly, such regions often reside at boundaries of topologically associated domains and loop with genes. CRISPR-based epigenetic perturbation and genetic deletion of L1s reveal that H4K16ac-marked L1s and LTRs regulate the expression of genes in cis. Overall, TEs enriched with H4K16ac contribute to the cis-regulatory landscape at specific genomic locations by maintaining an active chromatin landscape at TEs.

cis-Regulatory Elements in Plant Development, Adaptation, and Evolution.

cis-Regulatory elements encode the genomic blueprints that ensure the proper spatiotemporal patterning of gene expression necessary for appropriate development and responses to the environment. Accumulating evidence implicates changes to gene expression as a major source of phenotypic novelty in eukaryotes, including acute phenotypes such as disease and cancer in mammals. Moreover, genetic and epigenetic variation affecting cis-regulatory sequences over longer evolutionary timescales has become a recurring theme in studies of morphological divergence and local adaptation. Here, we discuss the functions of and methods used to identify various classes of cis-regulatory elements, as well as their role in plant development and response to the environment. We highlight opportunities to exploit cis-regulatory variants underlying plant development and environmental responses for crop improvement efforts. Although a comprehensive understanding of cis-regulatory mechanisms in plants has lagged behind that in animals, we showcase several breakthrough findings that have profoundly influenced plant biology and shaped the overall understanding of transcriptional regulation in eukaryotes.

Characterizing chromatin interactions of regulatory elements and nucleosome positions, using Hi-C, Micro-C, and promoter capture Micro-C.

BACKGROUND: Regulatory elements such as promoters, enhancers, and insulators interact each other to mediate molecular processes. To capture chromatin interactions of regulatory elements, 3C-derived methods such as Hi-C and Micro-C are developed. Here, we generated and analyzed Hi-C, Micro-C, and promoter capture Micro-C datasets with different sequencing depths to study chromatin interactions of regulatory elements and nucleosome positions in human prostate cancer cells. RESULTS: Compared to Hi-C, Micro-C identifies more high-resolution loops, including ones around structural variants. By evaluating the effect of sequencing depth, we revealed that more than 2 billion reads of Micro-C are needed to detect chromatin interactions at 1 kb resolution. Moreover, we found that deep-sequencing identifies additional long-range loops that are longer than 1 Mb in distance. Furthermore, we found that more than 50% of the loops are involved in insulators while less than 10% of the loops are promoter-enhancer loops. To comprehensively capture chromatin interactions that promoters are involved in, we performed promoter capture Micro-C. Promoter capture Micro-C identifies loops near promoters with a lower amount of sequencing reads. Sequencing of 160 million reads of promoter capture Micro-C resulted in reaching a plateau of identifying loops. However, there was still a subset of promoters that are not involved in loops even after deep-sequencing. By integrating Micro-C with NOMe-seq and ChIP-seq, we found that active promoters involved in loops have a more accessible region with lower levels of DNA methylation and more highly phased nucleosomes, compared to active promoters that are not involved in loops. CONCLUSION: We determined the required sequencing depth for Micro-C and promoter capture Micro-C to generate high-resolution chromatin interaction maps and loops. We also investigated the effect of sequencing coverage of Hi-C, Micro-C, and promoter capture Micro-C on detecting chromatin loops. Our analyses suggest the presence of distinct regulatory element groups, which are differently involved in nucleosome positions and chromatin interactions. This study does not only provide valuable insights on understanding chromatin interactions of regulatory elements, but also present guidelines for designing research projects on chromatin interactions among regulatory elements.

Non-coding variants disrupting a tissue-specific regulatory element in HK1 cause congenital hyperinsulinism.

Gene expression is tightly regulated, with many genes exhibiting cell-specific silencing when their protein product would disrupt normal cellular function1. This silencing is largely controlled by non-coding elements, and their disruption might cause human disease2. We performed gene-agnostic screening of the non-coding regions to discover new molecular causes of congenital hyperinsulinism. This identified 14 non-coding de novo variants affecting a 42-bp conserved region encompassed by a regulatory element in intron 2 of the hexokinase 1 gene (HK1). HK1 is widely expressed across all tissues except in the liver and pancreatic beta cells and is thus termed a 'disallowed gene' in these specific tissues. We demonstrated that the variants result in a loss of repression of HK1 in pancreatic beta cells, thereby causing insulin secretion and congenital hyperinsulinism. Using epigenomic data accessed from public repositories, we demonstrated that these variants reside within a regulatory region that we determine to be critical for cell-specific silencing. Importantly, this has revealed a disease mechanism for non-coding variants that cause inappropriate expression of a disallowed gene.

chromMAGMA: regulatory element-centric interrogation of risk variants.

Candidate causal risk variants from genome-wide association studies reside almost exclusively in noncoding regions of the genome and innovative approaches are necessary to understand their biological function. Multi-marker analysis of genomic annotation (MAGMA) is a widely used program that nominates candidate risk genes by mapping single-nucleotide polymorphism summary statistics from genome-wide association studies to gene bodies. We augmented MAGMA to create chromatin-MAGMA (chromMAGMA), a method to nominate candidate risk genes based on the presence of risk variants within noncoding regulatory elements (REs). We applied chromMAGMA to a genetic susceptibility dataset for epithelial ovarian cancer (EOC), a rare gynecologic malignancy characterized by high mortality. This identified 155 unique candidate EOC risk genes across five EOC histotypes; 83% (105/127) of high-grade serous ovarian cancer risk genes had not previously been implicated in this EOC histotype. Risk genes nominated by chromMAGMA converged on mRNA splicing and transcriptional dysregulation pathways. chromMAGMA is a pipeline that nominates candidate risk genes through a gene regulation-focused approach and helps interpret the biological mechanism of noncoding risk variants for complex diseases.

[Mapping Regulatory Elements within 5' and 3' UTRs of SIGLEC15 with a Use of Reporter System].

Siglec-15 is an immune suppressor with broad upregulation on various cancer types and has emerged as a potential target for cancer immunotherapy. However, it remains unclear how SIGLEC15 expression is controlled in normal or cancer cells. In this work, we utilized reporter assays to evaluate the impact of the 5' UTR and the 3' UTR of SIGLEC15 mRNA on gene expression. We found that the 3' UTR dramatically reduced reporter protein production, whereas the 5' UTR showed modest inhibitory effect. Quantification of steady-state mRNA revealed the good coupling of protein amount and mRNA abundance that was associated with the 3' UTR. In contrast, the 5' UTR had little effect on mRNA abundance compared with the empty control. By measuring mRNA half-life, we showed that the 3' UTR markedly promoted mRNA degradation. Testing shortened 3' UTR fragments demonstrated five out of the six having notable inhibitory effect, with the one spanning 993-1317 had the most robust activity. More interestingly, the 993-1317 region contains a predicted 43-nt stem-loop structure that showed apparent inhibitory activity in four cell lines tested. These results suggested that the 3' UTR inhibited reporter gene expression by accelerating mRNA decay possibly via multiple cis-regulatory elements, but the 5' UTR repressed gene expression by inhibiting translation. Thus, our findings provided a clue to the molecular mechanism underlying the regulation of SIGLEC15 expression.

Association and functional significance of genetic variants present in regulatory elements of SERPINB5 gene in gallbladder cancer.

SERPINB5 is a mammary serine protease inhibitor, which is involved in various cellular functions. The aberrant expression of SERPINB5 is reported in many cancers along with GBC but limited information is available about its role in genetic predisposition for GBC. We carried out case-control study in 206 cases and 219 controls. Promoter SNPs were genotyped by Sanger's sequencing. In-silico promoter analysis and luciferase reporter assay were done to elucidate the role of promoter variants in regulation of SERPINB5 expression. Out of four SNPs, three SERPINB5 promoter variants showed association with GBC in different models. The 'C' allele of variant rs17071138 was found to be significantly associated with GBC (p = 0.017). The 'T' allele of rs3744940 significantly increased the risk for GBC in dominant (p = 0.035) and additive models (p = 0.005). Also, rs3744941 'T' allele increased the risk for GBC by dominant (p = 0.042) as well as additive models (p = 0.016). In-silico promoter analysis and luciferase reporter assay revealed the probable regulatory role of the SERPINB5 promoter variant rs17071138 on the expression. Overall, our study reveals the genetic association of SERPINB5 promoter variants with GBC and possible role of rs17071138 in the regulation of expression.

Regulatory Single Nucleotide Polymorphism of the Bovine IFITM3 Gene Induces Differential Transcriptional Capacities of Hanwoo and Holstein Cattle.

Interferon-induced transmembrane protein 3 (IFITM3), a crucial effector of the host's innate immune system, prohibits an extensive range of viruses. Previous studies have reported that single nucleotide polymorphisms (SNPs) of the IFITM3 gene are associated with the expression level and length of the IFITM3 protein and can impact susceptibility to infectious viruses and the severity of infection with these viruses. However, there have been no studies on polymorphisms of the bovine IFITM3 gene. In the present study, we finely mapped the bovine IFITM3 gene and annotated the identified polymorphisms. We investigated polymorphisms of the bovine IFITM3 gene in 108 Hanwoo and 113 Holstein cattle using direct sequencing and analyzed genotype, allele, and haplotype frequencies and linkage disequilibrium (LD) between the IFITM3 genes of the two cattle breeds. In addition, we analyzed transcription factor-binding sites and transcriptional capacity using PROMO and luciferase assays, respectively. Furthermore, we analyzed the effect of a nonsynonymous SNP of the IFITM3 gene using PolyPhen-2, PANTHER, and PROVEAN. We identified 23 polymorphisms in the bovine IFITM3 gene and found significantly different genotype, allele, and haplotype frequency distributions and LD scores between polymorphisms of the bovine IFITM3 gene in Hanwoo and Holstein cattle. In addition, the ability to bind the transcription factor Nkx2-1 and transcriptional capacities were significantly different depending on the c.-193T > C allele. Furthermore, nonsynonymous SNP (F121L) was predicted to be benign. To the best of our knowledge, this is the first genetic study of bovine IFITM3 polymorphisms.

Identifying tumorigenic non-coding mutations through altered cis-regulation.

Identification of non-coding mutations driving tumorigenesis requires alternative approaches to coding mutations. Enriched associations between mutated regulatory elements and altered cis-regulation in tumors are a promising approach to stratify candidate non-coding driver mutations. Here we provide a bioinformatics pipeline to mine data from the Cancer Genomic Commons (GDC) for such associations. The pipeline integrates RNA and whole-genome sequencing with genotyping data to reveal putative non-coding driver mutations by cancer type. For complete information on the generation and use of this protocol, please refer to Cheng et al. (2021).

Epigenetic Analysis of the Chromatin Landscape Identifies a Repertoire of Murine Eosinophil-Specific PU.1-Bound Enhancers.

Eosinophils develop in the bone marrow from hematopoietic progenitors into mature cells capable of a plethora of immunomodulatory roles via the choreographed process of eosinophilopoiesis. However, the gene regulatory elements and transcription factors (TFs) orchestrating this process remain largely unknown. The potency and resulting diversity fundamental to an eosinophil's complex immunomodulatory functions and tissue specialization likely result from dynamic epigenetic regulation of the eosinophil genome, a dynamic eosinophil regulome. In this study, we applied a global approach using broad-range, next-generation sequencing to identify a repertoire of eosinophil-specific enhancers. We identified over 8200 active enhancers located within 1-20 kB of expressed eosinophil genes. TF binding motif analysis revealed PU.1 (Spi1) motif enrichment in eosinophil enhancers, and chromatin immunoprecipitation coupled with massively parallel sequencing confirmed PU.1 binding in likely enhancers of genes highly expressed in eosinophils. A substantial proportion (>25%) of these PU.1-bound enhancers were unique to murine, culture-derived eosinophils when compared among enhancers of highly expressed genes of three closely related myeloid cell subsets (macrophages, neutrophils, and immature granulocytes). Gene ontology analysis of eosinophil-specific, PU.1-bound enhancers revealed enrichment for genes involved in migration, proliferation, degranulation, and survival. Furthermore, eosinophil-specific superenhancers were enriched in genes whose homologs are associated with risk loci for eosinophilia and allergic diseases. Our collective data identify eosinophil-specific enhancers regulating key eosinophil genes through epigenetic mechanisms (H3K27 acetylation) and TF binding (PU.1).

Discovering single nucleotide variants and indels from bulk and single-cell ATAC-seq.

Genetic variants and de novo mutations in regulatory regions of the genome are typically discovered by whole-genome sequencing (WGS), however WGS is expensive and most WGS reads come from non-regulatory regions. The Assay for Transposase-Accessible Chromatin (ATAC-seq) generates reads from regulatory sequences and could potentially be used as a low-cost 'capture' method for regulatory variant discovery, but its use for this purpose has not been systematically evaluated. Here we apply seven variant callers to bulk and single-cell ATAC-seq data and evaluate their ability to identify single nucleotide variants (SNVs) and insertions/deletions (indels). In addition, we develop an ensemble classifier, VarCA, which combines features from individual variant callers to predict variants. The Genome Analysis Toolkit (GATK) is the best-performing individual caller with precision/recall on a bulk ATAC test dataset of 0.92/0.97 for SNVs and 0.87/0.82 for indels within ATAC-seq peak regions with at least 10 reads. On bulk ATAC-seq reads, VarCA achieves superior performance with precision/recall of 0.99/0.95 for SNVs and 0.93/0.80 for indels. On single-cell ATAC-seq reads, VarCA attains precision/recall of 0.98/0.94 for SNVs and 0.82/0.82 for indels. In summary, ATAC-seq reads can be used to accurately discover non-coding regulatory variants in the absence of whole-genome sequencing data and our ensemble method, VarCA, has the best overall performance.

Separating the Wheat from the Chaff: The Use of Upstream Regulator Analysis to Identify True Differential Expression of Single Genes within Transcriptomic Datasets.

The development of DNA microarray and RNA-sequencing technology has led to an explosion in the generation of transcriptomic differential expression data under a wide range of biologic systems including those recapitulating the monogenic muscular dystrophies. Data generation has increased exponentially due in large part to new platforms, improved cost-effectiveness, and processing speed. However, reproducibility and thus reliability of data remain a central issue, particularly when resource constraints limit experiments to single replicates. This was observed firsthand in a recent rare disease drug repurposing project involving RNA-seq-based transcriptomic profiling of primary cerebrocortical cultures incubated with clinic-ready blood-brain penetrant drugs. Given the low validation rates obtained for single differential expression genes, alternative approaches to identify with greater confidence genes that were truly differentially expressed in our dataset were explored. Here we outline a method for differential expression data analysis in the context of drug repurposing for rare diseases that incorporates the statistical rigour of the multigene analysis to bring greater predictive power in assessing individual gene modulation. Ingenuity Pathway Analysis upstream regulator analysis was applied to the differentially expressed genes from the Care4Rare Neuron Drug Screen transcriptomic database to identify three distinct signaling networks each perturbed by a different drug and involving a central upstream modulating protein: levothyroxine (DIO3), hydroxyurea (FOXM1), dexamethasone (PPARD). Differential expression of upstream regulator network related genes was next assessed in in vitro and in vivo systems by qPCR, revealing 5× and 10× increases in validation rates, respectively, when compared with our previous experience with individual genes in the dataset not associated with a network. The Ingenuity Pathway Analysis based gene prioritization may increase the predictive value of drug-gene interactions, especially in the context of assessing single-gene modulation in single-replicate experiments.

Reactivation of tumour suppressor in breast cancer by enhancer switching through NamiRNA network.

Dysfunction of Tumour Suppressor Genes (TSGs) is a common feature in carcinogenesis. Epigenetic abnormalities including DNA hypermethylation or aberrant histone modifications in promoter regions have been described for interpreting TSG inactivation. However, in many instances, how TSGs are silenced in tumours are largely unknown. Given that miRNA with low expression in tumours is another recognized signature, we hypothesize that low expression of miRNA may reduce the activity of TSG related enhancers and further lead to inactivation of TSG during cancer development. Here, we reported that low expression of miRNA in cancer as a recognized signature leads to loss of function of TSGs in breast cancer. In 157 paired breast cancer and adjacent normal samples, tumour suppressor gene GPER1 and miR-339 are both downregulated in Luminal A/B and Triple Negative Breast Cancer subtypes. Mechanistic investigations revealed that miR-339 upregulates GPER1 expression in breast cancer cells by switching on the GPER1 enhancer, which can be blocked by enhancer deletion through the CRISPR/Cas9 system. Collectively, our findings reveal novel mechanistic insights into TSG dysfunction in cancer development, and provide evidence that reactivation of TSG by enhancer switching may be a promising alternative strategy for clinical breast cancer treatment.

ChIP-GSM: Inferring active transcription factor modules to predict functional regulatory elements.

Transcription factors (TFs) often function as a module including both master factors and mediators binding at cis-regulatory regions to modulate nearby gene transcription. ChIP-seq profiling of multiple TFs makes it feasible to infer functional TF modules. However, when inferring TF modules based on co-localization of ChIP-seq peaks, often many weak binding events are missed, especially for mediators, resulting in incomplete identification of modules. To address this problem, we develop a ChIP-seq data-driven Gibbs Sampler to infer Modules (ChIP-GSM) using a Bayesian framework that integrates ChIP-seq profiles of multiple TFs. ChIP-GSM samples read counts of module TFs iteratively to estimate the binding potential of a module to each region and, across all regions, estimates the module abundance. Using inferred module-region probabilistic bindings as feature units, ChIP-GSM then employs logistic regression to predict active regulatory elements. Validation of ChIP-GSM predicted regulatory regions on multiple independent datasets sharing the same context confirms the advantage of using TF modules for predicting regulatory activity. In a case study of K562 cells, we demonstrate that the ChIP-GSM inferred modules form as groups, activate gene expression at different time points, and mediate diverse functional cellular processes. Hence, ChIP-GSM infers biologically meaningful TF modules and improves the prediction accuracy of regulatory region activities.

Structural basis for glucocorticoid receptor recognition of both unmodified and methylated binding sites, precursors of a modern recognition element.

The most common form of DNA methylation involves the addition of a methyl group to a cytosine base in the context of a cytosine-phosphate-guanine (CpG) dinucleotide. Genomes from more primitive organisms are more abundant in CpG sites that, through the process of methylation, deamination and subsequent mutation to thymine-phosphate-guanine (TpG) sites, can produce new transcription factor binding sites. Here, we examined the evolutionary history of the over 36 000 glucocorticoid receptor (GR) consensus binding motifs in the human genome and identified a subset of them in regulatory regions that arose via a deamination and subsequent mutation event. GR can bind to both unmodified and methylated pre-GR binding sequences (GBSs) that contain a CpG site. Our structural analyses show that CpG methylation in a pre-GBS generates a favorable interaction with Arg447 mimicking that made with a TpG in a GBS. This methyl-specific recognition arose 420 million years ago and was conserved during the evolution of GR and likely helps fix the methylation on the relevant cytosines. Our study provides the first genetic, biochemical and structural evidence of high-affinity binding for the likely evolutionary precursor of extant TpG-containing GBS.