MethMotif.Org 2024: a database integrating context-specific transcription factor-binding motifs with DNA methylation patterns.

MethMotif (https://methmotif.org) is a publicly available database that provides a comprehensive repository of transcription factor (TF)-binding profiles, enriched with DNA methylation patterns. In this release, we have enhanced the platform, expanding our initial collection to over 700 position weight matrices (PWM), all of which include DNA methylation profiles. One of the key advancements in this release is the segregation of TF-binding motifs based on their cofactors and DNA methylation status. We have previously demonstrated that gene ontology (GO) enriched terms associated with TF target genes may differ based on their association with alternative cofactors and DNA methylation status. MethMotif provides precomputed GO annotations for each human TF of interest, as well as for TF-co-TF complexes, enabling a comprehensive analysis of TF functions in the context of their co-factors. Additionally, MethMotif has been updated to encompass data for two new species, Mus musculus and Arabidopsis thaliana, widening its applicability to a broader community. MethMotif stands out as the first and only TF-binding motifs database to incorporate context-specific PWM coupled with epigenetic information, thereby enlightening context-specific TF functions. This enhancement allows the community to explore and gain deeper insights into the regulatory mechanisms governing transcriptional processes.

Correspondence on NanoVar's performance outlined by Jiang T. et al. in "Long-read sequencing settings for efficient structural variation detection based on comprehensive evaluation".

A recent paper by Jiang et al. in BMC Bioinformatics presented guidelines on long-read sequencing settings for structural variation (SV) calling, and benchmarked the performance of various SV calling tools, including NanoVar. In their simulation-based benchmarking, NanoVar was shown to perform poorly compared to other tools, mostly due to low SV recall rates. To investigate the causes for NanoVar's poor performance, we regenerated the simulation datasets (3× to 20×) as specified by Jiang et al. and performed benchmarking for NanoVar and Sniffles. Our results did not reflect the findings described by Jiang et al. In our analysis, NanoVar displayed more than three times the F1 scores and recall rates as reported in Jiang et al. across all sequencing coverages, indicating a previous underestimation of its performance. We also observed that NanoVar outperformed Sniffles in calling SVs with genotype concordance by more than 0.13 in F1 scores, which is contrary to the trend reported by Jiang et al. Besides, we identified multiple detrimental errors encountered during the analysis which were not addressed by Jiang et al. We hope that this commentary clarifies NanoVar's validity as a long-read SV caller and provides assurance to its users and the scientific community.

Autosomal dominant non-syndromic hearing loss maps to DFNA33 (13q34) and co-segregates with splice and frameshift variants in ATP11A, a phospholipid flippase gene.

Sequencing exomes/genomes have been successful for identifying recessive genes; however, discovery of dominant genes including deafness genes (DFNA) remains challenging. We report a new DFNA gene, ATP11A, in a Newfoundland family with a variable form of bilateral sensorineural hearing loss (SNHL). Genome-wide SNP genotyping linked SNHL to DFNA33 (LOD = 4.77), a locus on 13q34 previously mapped in a German family with variable SNHL. Whole-genome sequencing identified 51 unremarkable positional variants on 13q34. Continuous clinical ascertainment identified several key recombination events and reduced the disease interval to 769 kb, excluding all but one variant. ATP11A (NC_000013.11: chr13:113534963G>A) is a novel variant predicted to be a cryptic donor splice site. RNA studies verified in silico predictions, revealing the retention of 153 bp of intron in the 3' UTR of several ATP11A isoforms. Two unresolved families from Israel were subsequently identified with a similar, variable form of SNHL and a novel duplication (NM_032189.3:c.3322_3327+2dupGTCCAGGT) in exon 28 of ATP11A extended exon 28 by 8 bp, leading to a frameshift and premature stop codon (p.Asn1110Valfs43Ter). ATP11A is a type of P4-ATPase that transports (flip) phospholipids from the outer to inner leaflet of cell membranes to maintain asymmetry. Haploinsufficiency of ATP11A, the phospholipid flippase that specially transports phosphatidylserine (PS) and phosphatidylethanolamine (PE), could leave cells with PS/PE at the extracellular side vulnerable to phagocytic degradation. Given that surface PS can be pharmaceutically targeted, hearing loss due to ATP11A could potentially be treated. It is also likely that ATP11A is the gene underlying DFNA33.

ß-Catenin-TCF/LEF signaling promotes steady-state and emergency granulopoiesis via G-CSF receptor upregulation.

The canonical Wnt signaling pathway is mediated by interaction of ß-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and subsequent transcription activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of ß-catenin that yielded contradictory results. Here, we used a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) that specifically abrogates ß-catenin-TCF/LEF interaction. Disruption of the ß-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced granulocyte colony-stimulating factor (G-CSF) receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of ß-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candida albicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of ß-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the ß-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the ß-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.

Hematopoietic stem cells acquire survival advantage by loss of RUNX1 methylation identified in familial leukemia.

RUNX1 is among the most frequently mutated genes in human leukemia, and the loss or dominant-negative suppression of RUNX1 function is found in myelodysplastic syndrome and acute myeloid leukemia (AML). How posttranslational modifications (PTMs) of RUNX1 affect its in vivo function, however, and whether PTM dysregulation of RUNX1 can cause leukemia are largely unknown. We performed targeted deep sequencing on a family with 3 occurrences of AML and identified a novel RUNX1 mutation, R237K. The mutated R237 residue is a methylation site by protein arginine methyltransferase 1, and loss of methylation reportedly impairs the transcriptional activity of RUNX1 in vitro. To explore the biologic significance of RUNX1 methylation in vivo, we used RUNX1 R233K/R237K double-mutant mice, in which 2 arginine-to-lysine mutations precluded RUNX1 methylation. Genetic ablation of RUNX1 methylation led to loss of quiescence and expansion of hematopoietic stem cells (HSCs), and it changed the genomic and epigenomic signatures of phenotypic HSCs to a poised progenitor state. Furthermore, loss of RUNX1 R233/R237 methylation suppressed endoplasmic reticulum stress-induced unfolded protein response genes, including Atf4, Ddit3, and Gadd34; the radiation-induced p53 downstream genes Bbc3, Pmaip1, and Cdkn1a; and subsequent apoptosis in HSCs. Mechanistically, activating transcription factor 4 was identified as a direct transcriptional target of RUNX1. Collectively, defects in RUNX1 methylation in HSCs confer resistance to apoptosis and survival advantage under stress conditions, a hallmark of a preleukemic clone that may predispose affected individuals to leukemia. Our study will lead to a better understanding of how dysregulation of PTMs can contribute to leukemogenesis.

Lysine acetyltransferase Tip60 is required for hematopoietic stem cell maintenance.

Hematopoietic stem cells (HSCs) have the potential to replenish the blood system for the lifetime of the organism. Their 2 defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Using conditional gene-knockout models, we demonstrated a critical requirement of lysine acetyltransferase 5 (Kat5, also known as Tip60) for murine HSC maintenance in both the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling in murine hematopoietic stem and progenitor cells revealed that Tip60 colocalizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell cycling, and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin, and Tip60 deletion induced a robust reduction in the acH2A.Z/H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance, at least in part through Tip60-dependent H2A.Z acetylation to activate Myc target genes.

TFregulomeR reveals transcription factors' context-specific features and functions.

Transcription factors (TFs) are sequence-specific DNA binding proteins, fine-tuning spatiotemporal gene expression. Since genomic occupancy of a TF is highly dynamic, it is crucial to study TF binding sites (TFBSs) in a cell-specific context. To date, thousands of ChIP-seq datasets have portrayed the genomic binding landscapes of numerous TFs in different cell types. Although these datasets can be browsed via several platforms, tools that can operate on that data flow are still lacking. Here, we introduce TFregulomeR (https://github.com/benoukraflab/TFregulomeR), an R-library linked to an up-to-date compendium of cistrome and methylome datasets, implemented with functionalities that facilitate integrative analyses. In particular, TFregulomeR enables the characterization of TF binding partners and cell-specific TFBSs, along with the study of TF's functions in the context of different partnerships and DNA methylation levels. We demonstrated that TFs' target gene ontologies can differ notably depending on their partners and, by re-analyzing well characterized TFs, we brought to light that numerous leucine zipper TFBSs derived from ChIP-seq experiments documented in current databases were inadequately characterized, due to the fact that their position weight matrices were assembled using a mixture of homodimer and heterodimer binding sites. Altogether, analyses of context-specific transcription regulation with TFregulomeR foster our understanding of regulatory network-dependent TF functions.

Phosphorylation of Tet3 by cdk5 is critical for robust activation of BRN2 during neuronal differentiation.

Tet3 regulates the dynamic balance between 5-methylcyotsine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA during brain development and homeostasis. However, it remains unclear how its functions are modulated in a context-dependent manner during neuronal differentiation. Here, we show that cyclin-dependent kinase 5 (cdk5) phosphorylates Tet3 at the highly conserved serine 1310 and 1379 residues within its catalytic domain, changing its in vitro dioxygenase activity. Interestingly, when stably expressed in Tet1, 2, 3 triple-knockout mouse embryonic stem cells (ESCs), wild-type Tet3 induces higher level of 5hmC and concomitant expression of genes associated with neurogenesis whereas phosphor-mutant (S1310A/S1379A) Tet3 causes elevated 5hmC and expression of genes that are linked to metabolic processes. Consistent with this observation, Tet3-knockout mouse ESCs rescued with wild-type Tet3 have higher level of 5hmC at the promoter of neuron-specific gene BRN2 when compared to cells that expressed phosphor-mutant Tet3. Wild-type and phosphor-mutant Tet3 also exhibit differential binding affinity to histone variant H2A.Z. The differential 5hmC enrichment and H2A.Z occupancy at BRN2 promoter is correlated with higher gene expression and more efficient neuronal differentiation of ESCs that expressed wild-type Tet3. Taken together, our results suggest that cdk5-mediated phosphorylation of Tet3 is required for robust activation of neuronal differentiation program.

MethMotif: an integrative cell specific database of transcription factor binding motifs coupled with DNA methylation profiles.

Several recent studies have portrayed DNA methylation as a new player in the recruitment of transcription factors (TF) within chromatin, highlighting a need to connect TF binding sites (TFBS) with their respective DNA methylation profiles. However, current TFBS databases are restricted to DNA binding motif sequences. Here, we present MethMotif, a two-dimensional TFBS database that records TFBS position weight matrices along with cell type specific CpG methylation information computed from a combination of ChIP-seq and whole genome bisulfite sequencing datasets. Integrating TFBS motifs with TFBS DNA methylation better portrays the features of DNA loci recognised by TFs. In particular, we found that DNA methylation patterns within TFBS can be cell specific (e.g. MAFF). Furthermore, for a given TF, different DNA methylation profiles are associated with different DNA binding motifs (e.g. REST). To date, MethMotif database records over 500 TFBSs computed from over 2000 ChIP-seq datasets in 11 different cell types. MethMotif portal is accessible through an open source web interface (https://bioinfo-csi.nus.edu.sg/methmotif) that allows users to intuitively explore the entire dataset and perform both single, and batch queries.

TIP60 represses activation of endogenous retroviral elements.

TIP60 is a lysine acetyltransferase and is known to be a haplo-insufficient tumor suppressor. TIP60 downregulation is an early event in tumorigenesis which has been observed in several cancer types including breast and colorectal cancers. However, the mechanism by which it regulates tumor progression is not well understood. In this study, we identified the role of TIP60 in the silencing of endogenous retroviral elements (ERVs). TIP60-mediated silencing of ERVs is dependent on BRD4. TIP60 and BRD4 positively regulate the expression of enzymes, SUV39H1 and SETDB1 and thereby, the global H3K9 trimethylation (H3K9me3) level. In colorectal cancer, we found that the loss of TIP60 de-represses retrotransposon elements genome-wide, which in turn activate the cellular response to pathogens, mediated by STING, culminating in an induction of Interferon Regulatory Factor 7 (IRF7) and associated inflammatory response. In summary, this study has identified a unique mechanism of ERV regulation in cancer cells mediated by TIP60 and BRD4 through regulation of histone H3 K9 trimethylation, and a new tumor suppressive role of TIP60 in vivo.

DNMT1-interacting RNAs block gene-specific DNA methylation.

DNA methylation was first described almost a century ago; however, the rules governing its establishment and maintenance remain elusive. Here we present data demonstrating that active transcription regulates levels of genomic methylation. We identify a novel RNA arising from the CEBPA gene locus that is critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extend the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene-selective demethylation of therapeutic targets in human diseases.

miREM: an expectation-maximization approach for prioritizing miRNAs associated with gene-set.

BACKGROUND: The knowledge of miRNAs regulating the expression of sets of mRNAs has led to novel insights into numerous and diverse cellular mechanisms. While a single miRNA may regulate many genes, one gene can be regulated by multiple miRNAs, presenting a complex relationship to model for accurate predictions. RESULTS: Here, we introduce miREM, a program that couples an expectation-maximization (EM) algorithm to the common approach of hypergeometric probability (HP), which improves the prediction and prioritization of miRNAs from gene-sets of interest. miREM has been made available through a web-server ( https://bioinfo-csi.nus.edu.sg/mirem2/ ) that can be accessed through an intuitive graphical user interface. The program incorporates a large compendium of human/mouse miRNA-target prediction databases to enhance prediction. Users may upload their genes of interest in various formats as an input and select whether to consider non-conserved miRNAs, amongst filtering options. Results are reported in a rich graphical interface that allows users to: (i) prioritize predicted miRNAs through a scatterplot of HP p-values and EM scores; (ii) visualize the predicted miRNAs and corresponding genes through a heatmap; and (iii) identify and filter homologous or duplicated predictions by clustering them according to their seed sequences. CONCLUSION: We tested miREM using RNAseq datasets from two single "spiked" knock-in miRNA experiments and two double knock-out miRNA experiments. miREM predicted these manipulated miRNAs as having high EM scores from the gene set signatures (i.e. top predictions for single knock-in and double knock-out miRNA experiments). Finally, we have demonstrated that miREM predictions are either similar or better than results provided by existing programs.

Methodological aspects of whole-genome bisulfite sequencing analysis.

The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation beyond CpG sites and CpG islands. These technologies have opened new avenues to understand the interplay between epigenetic events, chromatin plasticity and gene regulation. However, the processing, managing and mining of this huge volume of data require specialized computational tools and statistical methods that are yet to be standardized. Here, we describe a complete bisulfite sequencing analysis workflow, including recently developed programs, highlighting each of the crucial analysis steps required, i.e. sequencing quality control, reads alignment, methylation scoring, methylation heterogeneity assessment, genomic features annotation, data visualization and determination of differentially methylated cytosines. Moreover, we discuss the limitations of these technologies and considerations to perform suitable analyses.

Pasha: a versatile R package for piling chromatin HTS data.

UNLABELLED: We describe an R package designed for processing aligned reads from chromatin-oriented high-throughput sequencing experiments. Pasha (preprocessing of aligned sequences from HTS analyses) allows easy manipulation of aligned reads from short-read sequencing technologies (ChIP-seq, FAIRE-seq, MNase-Seq, …) and offers innovative approaches such as ChIP-seq reads elongation, nucleosome midpoint piling strategy for positioning analyses, or the ability to subset paired-end reads by groups of insert size that can contain biologically relevant information. AVAILABILITY AND IMPLEMENTATION: Pasha is a multi-platform R package, available on CRAN repositories under GPL-3 license (https://cran.r-project.org/web/packages/Pasha/). CONTACTS: rfenouil@gmail.com or jean-christophe.andrau@igmm.cnrs.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

H3K4 tri-methylation provides an epigenetic signature of active enhancers.

Combinations of post-translational histone modifications shape the chromatin landscape during cell development in eukaryotes. However, little is known about the modifications exactly delineating functionally engaged regulatory elements. For example, although histone H3 lysine 4 mono-methylation (H3K4me1) indicates the presence of transcriptional gene enhancers, it does not provide clearcut information about their actual position and stage-specific activity. Histone marks were, therefore, studied here at genomic loci differentially expressed in early stages of T-lymphocyte development. The concomitant presence of the three H3K4 methylation states (H3K4me1/2/3) was found to clearly reflect the activity of bona fide T-cell gene enhancers. Globally, gain or loss of H3K4me2/3 at distal genomic regions correlated with, respectively, the induction or the repression of associated genes during T-cell development. In the Tcrb gene enhancer, the H3K4me3-to-H3K4me1 ratio decreases with the enhancer's strength. Lastly, enhancer association of RNA-polymerase II (Pol II) correlated with the presence of H3K4me3 and Pol II accumulation resulted in local increase of H3K4me3. Our results suggest the existence of functional links between Pol II occupancy, H3K4me3 enrichment and enhancer activity.

C/EBPα is required for development of dendritic cell progenitors.

Dendritic cells (DCs) are master regulators of the immune system, but molecular regulation of early DC differentiation has been poorly understood. Here, we report that the transcription factor C/EBPα coordinates the development of progenitor cells required for production of multiple categories of DCs. C/EBPα was needed for differentiation from stem/progenitor cells to common DC progenitors (CDPs), but not for transition of CDP to mature DCs. C/EBPα deletion in mature DCs did not affect their numbers or function, suggesting that this transcription factor is not needed for maintenance of DCs in lymphoid tissues. ChIP-seq and microarrays were used to identify candidate genes regulated by C/EBPα and required for DC formation. Genes previously shown to be critical for DC formation were bound by C/EBPα, and their expression was decreased in the earliest hematopoietic compartments in the absence of C/EBPα. These data indicate that C/EBPα is important for the earliest stages of steady-state DC differentiation.

GBSA: a comprehensive software for analysing whole genome bisulfite sequencing data.

High-throughput sequencing is increasingly being used in combination with bisulfite (BS) assays to study DNA methylation at nucleotide resolution. Although several programmes provide genome-wide alignment of BS-treated reads, the resulting information is not readily interpretable and often requires further bioinformatic steps for meaningful analysis. Current post-alignment BS-sequencing programmes are generally focused on the gene-specific level, a restrictive feature when analysis in the non-coding regions, such as enhancers and intergenic microRNAs, is required. Here, we present Genome Bisulfite Sequencing Analyser (GBSA-http://ctrad-csi.nus.edu.sg/gbsa), a free open-source software capable of analysing whole-genome bisulfite sequencing data with either a gene-centric or gene-independent focus. Through analysis of the largest published data sets to date, we demonstrate GBSA's features in providing sequencing quality assessment, methylation scoring, functional data management and visualization of genomic methylation at nucleotide resolution. Additionally, we show that GBSA's output can be easily integrated with other high-throughput sequencing data, such as RNA-Seq or ChIP-seq, to elucidate the role of methylated intergenic regions in gene regulation. In essence, GBSA allows an investigator to explore not only known loci but also all the genomic regions, for which methylation studies could lead to the discovery of new regulatory mechanisms.

Feasibility of low-throughput next generation sequencing for germline DNA screening.

BACKGROUND: Next generation sequencing (NGS) promises many benefits for clinical diagnostics. However, current barriers to its adoption include suboptimal amenability for low clinical throughputs and uncertainty over data accuracy and analytical procedures. We assessed the feasibility and performance of low-throughput NGS for detecting germline mutations for Lynch syndrome (LS). METHODS: Sequencing depth, time, and cost of 6 formats on the MiSeq and Personal Genome Machine platforms at 1-12 samples/run were calculated. Analytical performance was assessed from 3 runs of 3 DNA samples annotated for 7500 nucleotides by BeadChip arrays. The clinical performance of low-throughput NGS and 9 analytical processes were assessed through blinded analysis of DNA samples from 12 LS cases confirmed by Sanger sequencing, and 3 control cases. RESULTS: The feasibility analysis revealed different formats were optimal at different throughputs. Detection was reproducible for 2619/2635 (99.39%) replicate variants, and sensitivity and specificity to array annotation were 99.42% and 99.99% respectively. Eleven of 16 inconsistently detected variants could be specifically identified by having allele frequencies ≤ 0.15, strand biases >-35, or genotype quality scores ≤ 80. Positive selection for variants in the Human Genome Mutation Database (colorectal cancer, nonpolyposis) and variants with ≤ 5% frequency in the Asian population gave the best clinical performance (92% sensitivity, 67% specificity). CONCLUSIONS: Low-throughput NGS can be a cost-efficient and reliable approach for screening germline variants; however, its clinical utility is subject to the quality of annotation of clinically relevant variants.

The gene signature in CCAAT-enhancer-binding protein α dysfunctional acute myeloid leukemia predicts responsiveness to histone deacetylase inhibitors.

C/EPBα proteins, encoded by the CCAAT-enhancer-binding protein α gene, play a crucial role in granulocytic development, and defects in this transcription factor have been reported in acute myeloid leukemia. Here, we defined the C/EBPα signature characterized by a set of genes up-regulated upon C/EBPα activation. We analyzed expression of the C/EBPα signature in a cohort of 525 patients with acute myeloid leukemia and identified a subset characterized by low expression of this signature. We referred to this group of patients as the C/EBPα dysfunctional subset. Remarkably, a large percentage of samples harboring C/EBPα biallelic mutations clustered within this subset. We hypothesize that re-activation of the C/EBPα signature in the C/EBPα dysfunctional subset could have therapeutic potential. In search for small molecules able to reverse the low expression of the C/EBPα signature we applied the connectivity map. This analysis predicted positive connectivity between the C/EBPα activation signature and histone deacetylase inhibitors. We showed that these inhibitors reactivate expression of the C/EBPα signature and promote granulocytic differentiation of primary samples from the C/EBPα dysfunctional subset harboring biallelic C/EBPα mutations. Altogether, our study identifies histone deacetylase inhibitors as potential candidates for the treatment of certain leukemias characterized by down-regulation of the C/EBPα signature.

A unique H3K4me2 profile marks tissue-specific gene regulation.

Characterization of the epigenetic landscape fundamentally contributes toward deciphering the regulatory mechanisms that govern gene expression. However, despite an increasing flow of newly generated data, no clear pattern of chromatin modifications has so far been linked to specific modes of transcriptional regulation. Here, we used high-throughput genomic data from CD4(+) T lymphocytes to provide a comprehensive analysis of histone H3 lysine 4 dimethylation (H3K4me2) enrichment in genomic regions surrounding transcriptional start sites (TSSs). We discovered that a subgroup of genes linked to T cell functions displayed high levels of H3K4me2 within their gene body, in sharp contrast to the TSS-centered profile typical of housekeeping genes. Analysis of additional chromatin modifications and DNase I hypersensitive sites (DHSS) revealed a combinatorial chromatin signature characteristic of this subgroup. We propose that this epigenetic feature reflects the activity of an as yet unrecognized, intragenic cis-regulatory platform dedicated to refining tissue-specificity in gene expression.