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.
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► Extensive transcription factor regulatory networks for 41 human cell and tissue types ► Regulatory networks are highly cell selective and expose regulators of cellular identity ► Network analysis identifies cell-selective functions for commonly expressed regulators ► The circuitry of human transcription factor networks mirrors living neuronal networks
The circuitry, dynamics, and organizing principles of human transcription factor regulatory networks are revealed by extensive network mapping in 41 diverse human cell types. Networks are highly cell type specific and demonstrate how common transcription factors modulate cell-selective functions.
In its first production phase, The ENCODE Project Consortium (ENCODE) has generated thousands of genome-scale data sets, resulting in a genomic "parts list" that encompasses transcripts, sites of ...transcription factor binding, and other functional features that now number in the millions of distinct elements. These data are reshaping many long-held beliefs concerning the information content of the human and other complex genomes, including the very definition of the gene. Here I discuss and place in context many of the leading findings of ENCODE, as well as trends that are shaping the generation and interpretation of ENCODE data. Finally, I consider prospects for the future, including maximizing the accuracy, completeness, and utility of ENCODE data for the community.
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.
Although DNA methylation is commonly invoked as a mechanism for transcriptional repression, the extent to which it actively silences transcription factor (TF) occupancy sites in vivo is unknown. To ...study the role of DNA methylation in the active modulation of TF binding, we quantified the effect of DNA methylation depletion on the genomic occupancy patterns of CTCF, an abundant TF with known methylation sensitivity that is capable of autonomous binding to its target sites in chromatin. Here, we show that the vast majority (>98.5%) of the tens of thousands of unoccupied, methylated CTCF recognition sequences remain unbound upon abrogation of DNA methylation. The small fraction of sites that show methylation-dependent binding in vivo are in turn characterized by highly variable CTCF occupancy across cell types. Our results suggest that DNA methylation is not a primary groundskeeper of genomic TF landscapes, but rather a specialized mechanism for stabilizing intrinsically labile sites.
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•Most CTCF binding in vivo is unaltered by genomic abrogation of DNA methylation•A limited set of CTCF sites from other cell lineages is methylation sensitive•Methylation-sensitive CTCF sites are highly variable across cell types•Key sequence and chromatin features predict methylation sensitivity of CTCF binding
Alterations of DNA methylation in malignancy and development are frequently interpreted as affecting transcriptional activity. Maurano et al. find that, upon genomic abrogation of DNA methylation, binding of the canonically methylation-sensitive transcriptional regulator CTCF is largely unaffected. Their results suggest that a limited set of methylation-sensitive CTCF sites are variable across cell types and that key sequence and chromatin features predict methylation sensitivity of CTCF binding.
Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with coregulators. The role of these interactions in determining selective binding of ...GR to regulatory elements remains unclear. Recent findings indicate that a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model in which the basal occupancy of transcription factors acts to prime chromatin and direct inducible transcription factors to select regions in the genome.
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► AP1 and GR binding are universally associated with open chromatin ► GR binding is substantially associated with AP1 occupancy ► AP1 binding maintains chromatin accessibility at regulatory elements genome-wide ► AP1 establishes genomic binding patterns for signal-dependent factors such as GR
Human Mitochondrial Transcriptome Mercer, Tim R; Neph, Shane; Dinger, Marcel E ...
Cell,
08/2011, Letnik:
146, Številka:
4
Journal Article
Recenzirano
Odprti dostop
The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs, and rRNAs. ...Here, we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single-nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA and provides a resource for future studies of mitochondrial function (accessed at http://mitochondria.matticklab.com).
Regulatory T (Treg) cells, whose identity and function are defined by the transcription factor Foxp3, are indispensable for immune homeostasis. It is unclear whether Foxp3 exerts its Treg lineage ...specification function through active modification of the chromatin landscape and establishment of new enhancers or by exploiting a pre-existing enhancer landscape. Analysis of the chromatin accessibility of Foxp3-bound enhancers in Treg and Foxp3-negative T cells showed that Foxp3 was bound overwhelmingly to preaccessible enhancers occupied by its cofactors in precursor cells or a structurally related predecessor. Furthermore, the bulk of Foxp3-bound Treg cell enhancers lacking in Foxp3− CD4+ cells became accessible upon T cell receptor activation prior to Foxp3 expression, and only a small subset associated with several functionally important genes were exclusively Treg cell specific. Thus, in a late cellular differentiation process, Foxp3 defines Treg cell functionality in an “opportunistic” manner by largely exploiting the preformed enhancer network instead of establishing a new enhancer landscape.
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► Treg cell specification factor Foxp3 binds to preaccessible enhancer ► Foxp3 binds to sites occupied by cofactors in precursor cell ► Structural homolog Foxo1 serves as a Foxp3 predecessor in precursor cells ► TCR activation confers accessibility to the majority of Treg-specific enhancers
The transcription factor Foxp3, which specifies regulatory T cell fate, promotes differentiation not by making chromatin more accessible to specific factors but instead by modifying transcriptional activity at pre-existing enhancers, which are already accessible due to the binding of cofactors and paralogs in precursor cells.
The glucocorticoid receptor (GR), like other eukaryotic transcription factors, regulates gene expression by interacting with chromatinized DNA response elements. Photobleaching experiments in living ...cells indicate that receptors transiently interact with DNA on the time scale of seconds and predict that the response elements may be sparsely occupied on average. Here, we show that the binding of one receptor at the glucocorticoid response element (GRE) does not reduce the steady-state binding of another receptor variant to the same GRE. Mathematical simulations reproduce this noncompetitive state using short GR/GRE residency times and relatively long times between DNA binding events. At many genomic sites where GR binding causes increased chromatin accessibility, concurrent steady-state binding levels for the variant receptor are actually increased, a phenomenon termed assisted loading. Temporally sparse transcription factor-DNA interactions induce local chromatin reorganization, resulting in transient access for binding of secondary regulatory factors.
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► Many transcription factors exchange with response elements on a time scale of seconds ► Proteins with identical recognition sites fail to exhibit competitive binding in vivo ► Models support dynamic, temporally sparse, factor/response element interactions ► “Assisted loading” results from chromatin remodeling that creates transient access
Genomic footprinting Vierstra, Jeff; Stamatoyannopoulos, John A
Nature methods,
03/2016, Letnik:
13, Številka:
3
Journal Article
Recenzirano
The advent of DNA footprinting with DNase I more than 35 years ago enabled the systematic analysis of protein-DNA interactions, and the technique has been instrumental in the decoding of ...cis-regulatory elements and the identification and characterization of transcription factors and other DNA-binding proteins. The ability to analyze millions of individual genomic cleavage events via massively parallel sequencing has enabled in vivo DNase I footprinting on a genomic scale, offering the potential for global analysis of transcription factor occupancy in a single experiment. Genomic footprinting has opened unique vistas on the organization, function and evolution of regulatory DNA; however, the technology is still nascent. Here we discuss both prospects and challenges of genomic footprinting, as well as considerations for its application to complex genomes.
Cellular-state information between generations of developing cells may be propagated via regulatory regions. We report consistent patterns of gain and loss of DNase I-hypersensitive sites (DHSs) as ...cells progress from embryonic stem cells (ESCs) to terminal fates. DHS patterns alone convey rich information about cell fate and lineage relationships distinct from information conveyed by gene expression. Developing cells share a proportion of their DHS landscapes with ESCs; that proportion decreases continuously in each cell type as differentiation progresses, providing a quantitative benchmark of developmental maturity. Developmentally stable DHSs densely encode binding sites for transcription factors involved in autoregulatory feedback circuits. In contrast to normal cells, cancer cells extensively reactivate silenced ESC DHSs and those from developmental programs external to the cell lineage from which the malignancy derives. Our results point to changes in regulatory DNA landscapes as quantitative indicators of cell-fate transitions, lineage relationships, and dysfunction.
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•Cell fate and lineage relationships can be derived from DHS patterns•The proportion of a cell’s DHSs shared with ESCs is a benchmark of maturity•Developmentally stable DHSs encode binding sites for self-regulating TFs•Cancer cells reactivate both ESC DHSs and those from noncognate lineages
Gains and losses of DNase I-hypersensitive sites (DHSs) during cellular differentiation provide a measure of developmental maturity and lineage relationships between cell types. Whereas developing cells exhibit a steady pruning of DHSs shared with ESCs, cancer cells show a disordered acquisition of multiple unrelated DHSs.
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