Distal Regulatory Sites Research Articles

High-capacity sample multiplexing for single cell chromatin accessibility profiling

Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.

Temporal and Spatial Epigenome Editing Allows Precise Gene Regulation in Mammalian Cells

Cell-type specific gene expression programs are tightly linked to epigenetic modifications on DNA and histone proteins. Here, we used a novel CRISPR-based epigenome editing approach to control gene expression spatially and temporally. We show that targeting dCas9–p300 complex to distal non-regulatory genomic regions reprograms the chromatin state of these regions into enhancer-like elements. Notably, through controlling the spatial distance of these induced enhancers (i-Enhancer) to the promoter, the gene expression amplitude can be tightly regulated. To better control the temporal persistence of induced gene expression, we integrated the auxin-inducible degron technology with CRISPR tools. This approach allows rapid depletion of the dCas9-fused epigenome modifier complex from the target site and enables temporal control over gene expression regulation. Using this tool, we investigated the temporal persistence of a locally edited epigenetic mark and its functional consequences. The tools and approaches presented here will allow novel insights into the mechanism of epigenetic memory and gene regulation from distal regulatory sites.

Self-consistent theory of transcriptional control in complex regulatory architectures.

Individual regulatory proteins are typically charged with the simultaneous regulation of a battery of different genes. As a result, when one of these proteins is limiting, competitive effects have a significant impact on the transcriptional response of the regulated genes. Here we present a general framework for the analysis of any generic regulatory architecture that accounts for the competitive effects of the regulatory environment by isolating these effects into an effective concentration parameter. These predictions are formulated using the grand-canonical ensemble of statistical mechanics and the fold-change in gene expression is predicted as a function of the number of transcription factors, the strength of interactions between the transcription factors and their DNA binding sites, and the effective concentration of the transcription factor. The effective concentration is set by the transcription factor interactions with competing binding sites within the cell and is determined self-consistently. Using this approach, we analyze regulatory architectures in the grand-canonical ensemble ranging from simple repression and simple activation to scenarios that include repression mediated by DNA looping of distal regulatory sites. It is demonstrated that all the canonical expressions previously derived in the case of an isolated, non-competing gene, can be generalised by a simple substitution to their grand canonical counterpart, which allows for simple intuitive incorporation of the influence of multiple competing transcription factor binding sites. As an example of the strength of this approach, we build on these results to present an analytical description of transcriptional regulation of the lac operon.

Integrative analysis identifies co-dependent gene expression regulation of BRG1 and CHD7 at distal regulatory sites in embryonic stem cells.

DNA binding proteins such as chromatin remodellers, transcription factors (TFs), histone modifiers and co-factors often bind cooperatively to activate or repress their target genes in a cell type-specific manner. Nonetheless, the precise role of cooperative binding in defining cell-type identity is still largely uncharacterized. Here, we collected and analyzed 214 public datasets representing chromatin immunoprecipitation followed by sequencing (ChIP-Seq) of 104 DNA binding proteins in embryonic stem cell (ESC) lines. We classified their binding sites into those proximal to gene promoters and those in distal regions, and developed a web resource called Proximal And Distal (PAD) clustering to identify their co-localization at these respective regions. Using this extensive dataset, we discovered an extensive co-localization of BRG1 and CHD7 at distal but not proximal regions. The comparison of co-localization sites to those bound by either BRG1 or CHD7 alone showed an enrichment of ESC master TFs binding and active chromatin architecture at co-localization sites. Most notably, our analysis reveals the co-dependency of BRG1 and CHD7 at distal regions on regulating expression of their common target genes in ESC. This work sheds light on cooperative binding of TF binding proteins in regulating gene expression in ESC, and demonstrates the utility of integrative analysis of a manually curated compendium of genome-wide protein binding profiles in our online resource PAD. PAD is freely available at http://pad.victorchang.edu.au/ and its source code is available via an open source GPL 3.0 license at https://github.com/VCCRI/PAD/. pengyi.yang@sydney.edu.au or j.ho@victorchang.edu.au. Supplementary data are available at Bioinformatics online.

CD74 is a novel transcription regulator

CD74 is a cell-surface receptor for the cytokine macrophage migration inhibitory factor. Macrophage migration inhibitory factor binding to CD74 induces its intramembrane cleavage and the release of its cytosolic intracellular domain (CD74-ICD), which regulates cell survival. In the present study, we characterized the transcriptional activity of CD74-ICD in chronic lymphocytic B cells. We show that following CD74 activation, CD74-ICD interacts with the transcription factors RUNX (Runt related transcription factor) and NF-κB and binds to proximal and distal regulatory sites enriched for genes involved in apoptosis, immune response, and cell migration. This process leads to regulation of expression of these genes. Our results suggest that identifying targets of CD74 will help in understanding of essential pathways regulating B-cell survival in health and disease.

Enhancer Turnover Is Associated with a Divergent Transcriptional Response to Glucocorticoid in Mouse and Human Macrophages.

Phenotypic differences between individuals and species are controlled in part through differences in expression of a relatively conserved set of genes. Genes expressed in the immune system are subject to especially powerful selection. We have investigated the evolution of both gene expression and candidate enhancers in human and mouse macrophages exposed to glucocorticoid (GC), a regulator of innate immunity and an important therapeutic agent. Our analyses revealed a very limited overlap in the repertoire of genes responsive to GC in human and mouse macrophages. Peaks of inducible binding of the GC receptor (GR) detected by chromatin immunoprecipitation-Seq correlated with induction, but not repression, of target genes in both species, occurred at distal regulatory sites not promoters, and were strongly enriched for the consensus GR-binding motif. Turnover of GR binding between mice and humans was associated with gain and loss of the motif. There was no detectable signal of positive selection at species-specific GR binding sites, but clear evidence of purifying selection at the small number of conserved sites. We conclude that enhancer divergence underlies the difference in transcriptional activation after GC treatment between mouse and human macrophages. Only the shared inducible loci show evidence of selection, and therefore these loci may be important for the subset of responses to GC that is shared between species.

The Yin and Yang of enhancer-like RNAs

Long noncoding RNAs are emerging as a critical component of transcriptional activation. Over the past few years, there has been a number of examples where long noncoding RNAs expressed from distal sites exert their activating functions on neighbouring protein‐coding genes. A new study in The EMBO Journal (Lu et al , 2013) now extends this paradigm by revealing a role for the transcription factor Yin Yang 1 (YY1) in the regulation of the expression of long noncoding RNAs (termed Yams) during myogenesis. Importantly, the authors demonstrate a functional role for Yam‐1 in muscle differentiation through activation of a neighbouring micoRNA‐715 leading to downregulation of Wnt7b. There is converging evidence that distal regulatory sites commonly known as ‘enhancers’ are transcribed in a stimulus‐dependent manner as long noncoding RNAs (known as enhancer RNAs, eRNAs) and such eRNAs contribute to transcriptional activation of their neighbouring genes (De Santa et al , 2010; Kim et al , 2010; Hah et al , 2013; Lam et al , 2013; Li et al , 2013; Melo et al , 2013). Concomitantly, studies of long noncoding RNAs located in intergenic sites (termed lincRNAs, large intergenic noncoding RNAs) have uncovered a role for a class of lincRNAs (named noncoding RNA‐activating, ncRNA‐a) having enhancer‐like functions (Orom et al , 2010; Wang et al , 2011). These activating lincRNAs are enriched with transcription factor binding sites at their promoters and display responsiveness to differentiating stimuli (Orom et al, …

DNA methylation of distal regulatory sites characterizes dysregulation of cancer genes

BackgroundAbnormal epigenetic marking is well documented in gene promoters of cancer cells, but the study of distal regulatory siteshas lagged behind.We performed a systematic analysis of DNA methylation sites connected with gene expression profilesacross normal and cancerous human genomes.ResultsUtilizing methylation and expression data in 58 cell types, we developed a model for methylation-expression relationships in gene promoters and extrapolated it to the genome. We mapped numerous sites at which DNA methylation was associated with expression of distal genes. These sites bind transcription factors in a methylation-dependent manner, and carry the chromatin marks of a particular class of transcriptional enhancers. In contrast to the traditional model of one enhancer site per cell type, we found that single enhancer sites may define gradients of expression levels across many different cell types. Strikingly, the identified sites were drastically altered in cancers: hypomethylated enhancer sites associated with upregulation of cancer-related genes and hypermethylated sites with downregulation. Moreover, the association between enhancer methylation and gene deregulation in cancerwas significantly stronger than the association of promoter methylationwith gene deregulation.ConclusionsMethylation of distal regulatory sites is closely related to gene expression levels across the genome. Single enhancers may modulate ranges of cell-specific transcription levels, from constantlyopen promoters. In contrast to the remote relationships between promoter methylation and gene dysregulation in cancer, altered methylation of enhancer sites is closely related to gene expression profiles of transformed cells.

Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers

Enhancers are developmentally controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. In this study, we show by genome-wide mapping that the newly discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells and during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates such as Meis1 in P19 cells and PPARγ in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5-methylcytosine hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes.

A Regulatory Archipelago Controls Hox Genes Transcription in Digits

The evolution of digits was an essential step in the success of tetrapods. Among the key players, Hoxd genes are coordinately regulated in developing digits, where they help organize growth and patterns. We identified the distal regulatory sites associated with these genes by probing the three-dimensional architecture of this regulatory unit in developing limbs. This approach, combined with in vivo deletions of distinct regulatory regions, revealed that the active part of the gene cluster contacts several enhancer-like sequences. These elements are dispersed throughout the nearby gene desert, and each contributes either quantitatively or qualitatively to Hox gene transcription in presumptive digits. We propose that this genetic system, which we call a "regulatory archipelago," provides an inherent flexibility that may partly underlie the diversity in number and morphology of digits across tetrapods, as well as their resilience to drastic variations.

ICLIP predicts the dual splicing effects of TIA-RNA interactions.

Author SummaryStudies of splicing regulation have generally focused on RNA elements located close to alternative exons. Recently, it has been suggested that splicing of alternative exons can also be regulated by distal regulatory sites, but the underlying mechanism is not clear. The TIA proteins are key splicing regulators that enhance the recognition of 5′ splice sites, and their distal effects have remained unexplored so far. Here, we use a new method to map the positions of TIA-RNA interactions with high resolution on a transcriptome-wide scale. The identified binding positions successfully predict the local enhancing and distal silencing effects of TIA proteins. In particular, we show that TIA proteins can regulate distal alternative 3′ splice sites by binding at the 5′ splice site of the preceding exon. This result suggests that alternative splicing is affected by the timing of alternative exon definition relative to the recognition of the preceding 5′ splice site. These findings highlight the importance of analysing distal regulatory sites in order to fully understand the regulation of alternative splicing.

Beta-catenin binds to the downstream region and regulates the expression C-reactive protein gene.

C-Reactive protein (CRP) is a major acute-phase response protein, which is activated by various cytokines. We investigated the mechanism of TNF-α-induced CRP expression and found that the p50 subunit of NF-κB was responsible for the transcriptional activation of CRP. Since the p50 protein acts as a positive regulator of CRP expression without an inherent transactivation domain, we looked for an interaction partner that could provide p50 with such a domain. We found that β-catenin enhanced the expression of a CRP mRNA in concert with p50 subunit. Protein–protein interaction between p50 and β-catenin was important for CRP expression and their interactions to CRP promoter were induced after TNF-a treatment. Since gene expression depends upon the proximity of promoters and distal regulatory sites, we explored the long-range genomic interaction at the CRP locus by chromosome conformation capture (3C). We identified a binding site for β-catenin in the downstream of CRP gene by 3C and confirmed TNF-α-induced association of β-catenin and p50 by chromatin immunoprecipitation and co-immunoprecipitation assays. Our findings provide evidence that transcription of the CRP gene depends upon p50 and β-catenin proteins, which is accompanied by close proximity between promoter and the downstream region of CRP gene.

Evolution of allostery in the cyclic nucleotide binding module

Analysis of cyclic nucleotide binding (CNB) domains shows that they have evolved to sense a wide variety of second messenger signals; a mechanism for allosteric regulation by CNB domains is proposed.

Communication Pathways Between the Nucleotide Pocket and Distal Regulatory Sites in Protein Kinases

AbstractFor Abstract see ChemInform Abstract in Full Text.

Communication pathways between the nucleotide pocket and distal regulatory sites in protein kinases.

Protein kinases control many cellular processes via the ATP-dependent phosphorylation of specific amino acids on target proteins. Despite the availability of the three-dimensional structures of a variety of protein kinases, it has been particularly difficult to explain how noncatalytic domains removed from the active site regulate catalytic function. In this review, we describe how solution methodologies complement the available structural data and help explain how protein kinases may utilize medium-to-long-range effects to regulate substrate phosphorylation. For illustration, two protein kinases, cAMP-dependent protein kinase and the C-terminal Src kinase, are presented as paradigms for the serine/threonine- and tyrosine-specific families. While active-site residues provide an optimal environment for fast phosphoryl group transfer in these and other kinases, the overall rate of protein phosphorylation is limited by nucleotide binding and associated structural changes. Hydrogen-deuterium exchange studies reveal that nucleotide binding induces changes that radiate from a central structural assembly composed of the catalytic loop, glycine-rich loop, and helix alpha C to unique peripheral regions inside and outside the kinase core. This collection of conserved and unique elements delivers information from the active site to distal regions and possibly provides information flow back to the active site. This "push-pull" hypothesis offers a means for understanding how protein kinases can be regulated by protein-protein interactions far from the active site.

Rounding up active cis-elements in the triple C corral: Combining conservation, cleavage and conformation capture for the analysis of regulatory gene domains

Identification and functional analysis of potential cis-regulatory elements is a laborious process that often depends on removing putative elements from their natural context to study their activity. While such methods provide valuable information about the isolated element, they disregard the potential role of an element's interaction(s) with other regulatory sequences and the three-dimensional structure of an active gene locus. Here, two novel methods are discussed--chromosome conformation capture (3C) and RNA-TRAP--that can be used to detect interactions between distal regulatory sites and which thus indicate the chromosomal conformation that is adopted by a gene locus in various states of transcriptional activity. Combined with comparative genomics and traditional DNase I hypersensitive site mapping, these methods form a powerful approach for the study of the mechanisms of long-range transcriptional regulation.

Complex interactions between SP1 bound to multiple distal regulatory sites and HNF-4 bound to the proximal promoter lead to transcriptional activation of liver-specific human APOCIII gene.

Footprinting analysis of the human apoCIII promoter identified a set of four proximal (A-D) and six distal (E-J) regulatory elements between nucleotides -792 and -25 [Ogami, K., et al. (1990) J. Biol. Chem. 265, 9808-9815]. The distal regulatory elements of the apoCIII gene increase by 10-fold the strength of the homologous as well as of heterologous proximal promoters. Required for such transcriptional enhancement is the presence of an intact hormone response element (HRE) on the proximal promoter which binds a variety of nuclear hormone receptors. To understand the mechanism of this transcriptional activation, we identified the nature and the importance of the factors which bind to the upstream regulatory elements of the apoCIII promoter by DNA binding, competition, supershift, and transient transfection assays. These analyses showed that the upstream apoCIII promoter contains multiple binding sites for the ubiquitous transcription factor SP1, which recognizes the regulatory elements F, H, and I. The regulatory element G represents a specialized HRE which is recognized by the orphan receptors ARP-1 and EAR-3 but not by HNF-4. A single activity designated CIII J1 binds to the regulatory element J. The same or a similar activity binds as a minor component to the regulatory elements F and I where SP1 is the predominant binding activity. Finally, a minor activity designated CIII 15 binds to the regulatory element I.(ABSTRACT TRUNCATED AT 250 WORDS)