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Author(s): Meng How Tan (corresponding author) [1, 2, 3]; Qin Li [1]; Raghuvaran Shanmugam [2, 3]; Robert Piskol [1]; Jennefer Kohler [1]; Amy N. Young [1]; Kaiwen Ivy Liu [3]; [...]

Carcinogenesis and neoplastic progression are mediated by the accumulation of somatic mutations. Here we report that the local density of somatic mutations in cancer genomes is highly reduced specifically in accessible regulatory DNA defined by DNase I hypersensitive sites. This reduction is independent of any known factors influencing somatic mutation density and is observed in diverse cancer types, suggesting a general mechanism. By analyzing individual cancer genomes, we show that the reduced local mutation density within regulatory DNA is linked to intact global genome repair machinery, with nearly complete abrogation of the hypomutation phenomenon in individual cancers that possess mutations in components of the nucleotide excision repair system. Together, our results connect chromatin structure, gene regulation and cancer-associated somatic mutation. [ABSTRACT FROM AUTHOR]

It is currently not possible to resolve the genome-wide relationship of transcription factors (TFs) and nucleosomes at the level of individual chromatin templates despite rapidly increasing data on TF and nucleosome occupancy in the human genome. Here we describe DNase I-released fragment-length analysis of hypersensitivity (DNase-FLASH), an approach that directly couples mapping of TF occupancy, via quantification of DNA microfragments released from individual TF recognition sites in regulatory DNA, to the surrounding nucleosome architecture, via analysis of larger DNA fragments, in a single assay. DNase-FLASH enables coupling of individual TF footprints to nucleosome occupancy, identifying TFs that precisely demarcate the regulatory DNA-nucleosome interface. [ABSTRACT FROM AUTHOR]

Background: Mapping of DNase I hypersensitive sites (DHSs) is a powerful tool to experimentally identify cisregulatory elements (CREs). Among CREs, enhancers are abundant and predominantly act in driving cell-specific gene expression. Krüppel-like factors (KLFs) are a family of eukaryotic transcription factors. Several KLFs have been demonstrated to play important roles in hematopoiesis. However, transcriptional regulation of KLFs via CREs, particularly enhancers, in erythroid cells has been poorly understood. Results: In this study, 23 erythroid-specific or putative erythroid-specific DHSs were identified by DNase-seq in the genomic regions of 17 human KLFs, and their enhancer activities were evaluated using dual-luciferase reporter (DLR) assay. Of the 23 erythroid-specific DHSs, the enhancer activities of 15 DHSs were comparable to that of the classical enhancer HS2 in driving minimal promoter (minP). Fifteen DHSs, some overlapping those that increased minP activities, acted as enhancers when driving the corresponding KLF promoters (KLF-Ps) in erythroid cells; of these, 10 DHSs were finally characterized as erythroid-specific KLF enhancers. These 10 erythroid-specific KLF enhancers were further confirmed using chromatin immunoprecipitation coupled to sequencing (ChIP-seq) data-based bioinformatic and biochemical analyses. Conclusion: Our present findings provide a feasible strategy to extensively identify gene- and cell-specific enhancers from DHSs obtained by high-throughput sequencing, which will help reveal the transcriptional regulation and biological functions of genes in some specific cells [ABSTRACT FROM AUTHOR]

Human embryonic stem cells provide an alternative to using human embryos for studying developmentally regulated gene expression. The co-expression of high levels of embryonic ε and fetal γ globin by the hESC-derived erythroblasts allows the interrogation of ε globin regulation at the transcriptional and epigenetic level which could only be attained previously by studying cell lines or transgenic mice. In this study, we compared the histone modifications across the β globin locus of the undifferentiated hESCs and hESC-, FL-, and mobilized PB CD34 cells-derived erythroblasts, which have distinct globin expression patterns corresponding to their developmental stages. We demonstrated that the histone codes employed by the β globin locus are conserved throughout development. Furthermore, in spite of the close proximity of the ε globin promoter, as compared to the β or γ globin promoter, with the LCR, a chromatin loop was also formed between the LCR and the active ε globin promoter, similar to the loop that forms between the β or γ globin promoters and the LCR, in contrary to the previously proposed tracking mechanism. [ABSTRACT FROM AUTHOR]

Cell-selective glucocorticoid receptor (GR) binding to distal regulatory elements is associated with cell type-specific regions of locally accessible chromatin. These regions can either pre-exist in chromatin (pre-programmed) or be induced by the receptor (de novo). Mechanisms that create and maintain these sites are not well understood. We observe a global enrichment of CpG density for pre-programmed elements, and implicate their demethylated state in the maintenance of open chromatin in a tissue-specific manner. In contrast, sites that are actively opened by GR (de novo) are characterized by low CpG density, and form a unique class of enhancers devoid of suppressive effect of agglomerated methyl-cytosines. Furthermore, treatment with glucocorticoids induces rapid changes in methylation levels at selected CpGs within de novo sites. Finally, we identify GR-binding elements with CpGs at critical positions, and show that methylation can affect GR-DNA interactions in vitro. The findings present a unique link between tissue-specific chromatin accessibility, DNA methylation and transcription factor binding and show that DNA methylation can be an integral component of gene regulation by nuclear receptors. [ABSTRACT FROM AUTHOR]

Background: Acute myeloid leukemia (AML) is a heterogeneous disease with an overall poor prognosis. Gene expression profiling studies of patients with AML has provided key insights into disease pathogenesis while exposing potential diagnostic and prognostic markers and therapeutic targets. A systematic comparison of the large body of gene expression profiling studies in AML has the potential to test the extensibility of conclusions based on single studies and provide further insights into AML. Methodology/Principal Findings: In this study, we systematically compared 25 published reports of gene expression profiling in AML. There were a total of 4,918 reported genes of which one third were reported in more than one study. We found that only a minority of reported prognostically-associated genes (9.6%) were replicated in at least one other study. In a combined analysis, we comprehensively identified both gene sets and functional gene categories and pathways that exhibited significant differential regulation in distinct prognostic categories, including many previously unreported associations. Conclusions/Significance: We developed a novel approach for granular, cross-study analysis of gene-by-gene data and their relationships with established prognostic features and patient outcome. We identified many robust novel prognostic molecular features in AML that were undetected in prior studies, and which provide insights into AML pathogenesis with potential diagnostic, prognostic, and therapeutic implications. Our database and integrative analysis are available online (http://gat.stamlab.org). [ABSTRACT FROM AUTHOR]

It is widely assumed that human noncoding sequences comprise a substantial reservoir for functional variants impacting gene regulation and other chromosomal processes. Evolutionarily conserved noncoding sequences (CNSs) in the human genome have attracted considerable attention for their potential to simplify the search for functional elements and phenotypically important human alleles. A major outstanding question is whether functionally significant human noncoding variation is concentrated in CNS5 or distributed more broadly across the genome. Here, we combine whole- genome sequence data from four nonhuman species (chimp, dog, mouse, and rat) with recently available comprehensive human polymorphism data to analyze selection at single-nucleotide resolution. We show that a substantial fraction of active purifying selection in human noncoding sequences occurs outside of CNSs and is diffusely distributed across the genome. This finding suggests the existence of a large complement of human noncoding variants that may impact gene expression and phenotypic traits, the majority of which will escape detection with current approaches to genome analysis. [ABSTRACT FROM AUTHOR]

The study of gene regulation on a genomic scale has been constrained by the modest pace with which new trans-regulatory factors have been identified and by the fact that cis-regulatory sequences have to date been described even in part for only a small fraction of vertebrate genes. An indirect approach for assessing the significance of cis- and trans-regulatory mechanisms on a global scale is to utilize gene expression as a surrogate for transcriptional regulation and to combine genome-scale transcriptional profiling with studies of genetic variation, classical genetic techniques such as linkage analysis, and examination of allelic expression patterns that reveal cis-regulatory variability. A number of recent studies employing these methods provide insight into questions of central importance to our understanding of the larger role of transcriptional regulation in the organization of the human and other complex genomes. [Copyright &y& Elsevier]

The article offers information on transcription initiation by RNA polymerase II in eukaryotic gene regulation. The process was earlier reportedly regarded as a stereotyped process but due to large-scale sequencing of RNA species the perception has changed. cDNA transcripts generated by reverse transciptase is the main tool to understand RNA sequence. It states that the distribution of transcription start site (TSS) in a promoter region and biological behavior has a correlation.

Gene regulation is essential to placental function and fetal development. We built a genome-scale transcriptional regulatory network (TRN) of the human placenta using digital genomic footprinting and transcriptomic data. We integrated 475 transcriptomes and 12 DNase hypersensitivity datasets from placental samples to globally and quantitatively map transcription factor (TF)-target gene interactions. In an independent dataset, the TRN model predicted target gene expression with an out-of- sample R² greater than 0.25 for 73% of target genes. We performed siRNA knockdowns of four TFs and achieved concordance between the predicted gene targets in our TRN and differences in expression of knockdowns with an accuracy of >0.7 for three of the four TFs. Our final model contained 113,158 interactions across 391 TFs and 7712 target genes and is publicly available. We identified 29 TFs which were significantly enriched as regulators for genes previously associated with preterm birth, and eight of these TFs were decreased in preterm placentas. [ABSTRACT FROM AUTHOR]

Gene regulation is chiefly determined at the level of individual linear chromatin molecules, yet our current understanding of cis-regulatory architectures derives from fragmented sampling of large numbers of disparate molecules. We developed an approach for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using nonspecific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (Fiber-seq). Fiber-seq exposed widespread plasticity in the linear organization of individual chromatin fibers and illuminated principles guiding regulatory DNA actuation, the coordinated actuation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. Our approach and results open new vistas on the primary architecture of gene regulation. [ABSTRACT FROM AUTHOR]

Summary Major features of transcription by human RNA polymerase II (Pol II) remain poorly defined due to a lack of quantitative approaches for visualizing Pol II progress at nucleotide resolution. We developed a simple and powerful approach for performing native elongating transcript sequencing (NET-seq) in human cells that globally maps strand-specific Pol II density at nucleotide resolution. NET-seq exposes a mode of antisense transcription that originates downstream and converges on transcription from the canonical promoter. Convergent transcription is associated with a distinctive chromatin configuration and is characteristic of lower-expressed genes. Integration of NET-seq with genomic footprinting data reveals stereotypic Pol II pausing coincident with transcription factor occupancy. Finally, exons retained in mature transcripts display Pol II pausing signatures that differ markedly from skipped exons, indicating an intrinsic capacity for Pol II to recognize exons with different processing fates. Together, human NET-seq exposes the topography and regulatory complexity of human gene expression. [ABSTRACT FROM AUTHOR]

Summary: Gene-distal enhancers are critical for tissue-specific gene expression, but their genomic determinants within a specific lineage at different stages of development are unknown. Here we profile chromatin state maps, transcription factor occupancy, and gene expression profiles during human erythroid development at fetal and adult stages. Comparative analyses of human erythropoiesis identify developmental stage-specific enhancers as primary determinants of stage-specific gene expression programs. We find that erythroid master regulators GATA1 and TAL1 act cooperatively within active enhancers but confer little predictive value for stage specificity. Instead, a set of stage-specific coregulators collaborates with master regulators and contributes to differential gene expression. We further identify and validate IRF2, IRF6, and MYB as effectors of an adult-stage expression program. Thus, the combinatorial assembly of lineage-specific master regulators and transcriptional coregulators within developmental stage-specific enhancers determines gene expression programs and temporal regulation of transcriptional networks in a mammalian genome. [ABSTRACT FROM AUTHOR]

Summary: Directed differentiation of human embryonic stem cells (ESCs) into cardiovascular cells provides a model for studying molecular mechanisms of human cardiovascular development. Although it is known that chromatin modification patterns in ESCs differ markedly from those in lineage-committed progenitors and differentiated cells, the temporal dynamics of chromatin alterations during differentiation along a defined lineage have not been studied. We show that differentiation of human ESCs into cardiovascular cells is accompanied by programmed temporal alterations in chromatin structure that distinguish key regulators of cardiovascular development from other genes. We used this temporal chromatin signature to identify regulators of cardiac development, including the homeobox gene MEIS2. Using the zebrafish model, we demonstrate that MEIS2 is critical for proper heart tube formation and subsequent cardiac looping. Temporal chromatin signatures should be broadly applicable to other models of stem cell differentiation to identify regulators and provide key insights into major developmental decisions. [ABSTRACT FROM AUTHOR]

Summary: The combinatorial cross-regulation of hundreds of sequence-specific transcription factors (TFs) defines a regulatory network that underlies cellular identity and function. Here we use genome-wide maps of in vivo DNaseI footprints to assemble an extensive core human regulatory network comprising connections among 475 sequence-specific TFs and to analyze the dynamics of these connections across 41 diverse cell and tissue types. We find that human TF networks are highly cell selective and are driven by cohorts of factors that include regulators with previously unrecognized roles in control of cellular identity. Moreover, we identify many widely expressed factors that impact transcriptional regulatory networks in a cell-selective manner. Strikingly, in spite of their inherent diversity, all cell-type regulatory networks independently converge on a common architecture that closely resembles the topology of living neuronal networks. Together, our results provide an extensive description of the circuitry, dynamics, and organizing principles of the human TF regulatory network. [ABSTRACT FROM AUTHOR]

The spatial organization of genes in the interphase nucleus plays an important role in establishment and regulation of gene expression. Contradicting results have been reported to date, with little consensus about the dynamics of nuclear organization and the features of the contact loci. In this study, we investigated the properties and dynamics of genomic loci that are in contact with glucocorticoid receptor (GR)-responsive loci. We took a systematic approach, combining genome-wide interaction profiling by the chromosome conformation capture on chip (4C) technology with expression, protein occupancy, and chromatin accessibility profiles. This approach allowed a comprehensive analysis of how distinct features of the linear genome are organized in the three-dimensional nuclear space in the context of rapid gene regulation. We found that the transcriptional response to GR occurs without dramatic nuclear reorganization. Moreover, contrary to the view of transcription-driven organization, even genes with opposite transcriptional responses colocalize. Regions contacting GR-regulated genes are not particularly enriched for GR-regulated loci or for any functional group of genes, suggesting that these subnuclear environments are not organized to respond to a specific factor. The contact regions are, however, highly enriched for DNase I-hypersensitive sites that comprehensively mark cell-type-specific regulatory sites. These findings indicate that the nucleus is pre-organized in a conformation allowing rapid transcriptional reprogramming, and this organization is significantly correlated with cell-type-specific chromatin sites accessible to regulatory factors. Numerous open chromatin loci may be arranged in nuclear domains that are poised to respond to diverse signals in general and to permit efficient gene regulation. [ABSTRACT FROM AUTHOR]