EOMES and T-BET are related T-box transcription factors that control natural killer (NK) cell development. Here we demonstrate that EOMES and T-BET regulate largely distinct gene sets during this ...process. EOMES is dominantly expressed in immature NK cells and drives early lineage specification by inducing hallmark receptors and functions. By contrast, T-BET is dominant in mature NK cells, where it induces responsiveness to IL-12 and represses the cell cycle, likely through transcriptional repressors. Regardless, many genes with distinct functions are co-regulated by the two transcription factors. By generating two gene-modified mice facilitating chromatin immunoprecipitation of endogenous EOMES and T-BET, we show a strong overlap in their DNA binding targets, as well as extensive epigenetic changes during NK cell differentiation. Our data thus suggest that EOMES and T-BET may distinctly govern, via differential expression and co-factors recruitment, NK cell maturation by inserting partially overlapping epigenetic regulations.
Chromatin topology is intricately linked to gene expression, yet its functional requirement remains unclear. Here, we comprehensively assessed the interplay between genome topology and gene ...expression using highly rearranged chromosomes (balancers) spanning ~75% of the Drosophila genome. Using transheterozyte (balancer/wild-type) embryos, we measured allele-specific changes in topology and gene expression in cis, while minimizing trans effects. Through genome sequencing, we resolved eight large nested inversions, smaller inversions, duplications and thousands of deletions. These extensive rearrangements caused many changes to chromatin topology, disrupting long-range loops, topologically associating domains (TADs) and promoter interactions, yet these are not predictive of changes in expression. Gene expression is generally not altered around inversion breakpoints, indicating that mis-appropriate enhancer-promoter activation is a rare event. Similarly, shuffling or fusing TADs, changing intra-TAD connections and disrupting long-range inter-TAD loops does not alter expression for the majority of genes. Our results suggest that properties other than chromatin topology ensure productive enhancer-promoter interactions.
Chromosomes are folded and organized into topologically associating domains (TADs) which provide a framework for the interaction of enhancers with the promoter of their target gene(s). Structural ...rearrangements observed during evolution or in disease contexts suggest that changes in genome organization strongly affect gene expression and can have drastic phenotypic effects. In this review, I will discuss how recent genomic engineering experiments reveal a more contrasted picture, suggesting that TADs are important but not always essential for gene expression regulation.
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•TADs are evolutionarily conserved regions largely overlapping with regulatory domains.•Deleting TAD boundaries only mildly affects gene expression.•Chromosomal inversions affecting TAD boundaries are usually more deleterious.•Enhancer redundancy and enhancer–promoter specificity might buffer the effect of chromosomal rearrangements.
X chromosome dosage compensation in Drosophila requires chromosome‐wide coordination of gene activation. The male‐specific lethal dosage compensation complex (DCC) identifies and binds to ...X‐chromosomal high‐affinity sites (HAS) from which it boosts transcription. A sub‐class of HAS, PionX sites, represent first contacts on the X. Here, we explored the chromosomal interactions of representative PionX sites by high‐resolution 4C and determined the global chromosome conformation by Hi‐C in sex‐sorted embryos. Male and female X chromosomes display similar nuclear architecture, concordant with clustered, constitutively active genes. PionX sites, like HAS, are evenly distributed in the active compartment and engage in short‐ and long‐range interactions beyond compartment boundaries. Long‐range, inter‐domain interactions between DCC binding sites are stronger in males, suggesting that the complex refines chromatin organization. By de novo induction of DCC in female cells, we monitored the extent of activation surrounding PionX sites. This revealed a remarkable range of DCC action not only in linear proximity, but also at megabase distance if close in space, suggesting that DCC profits from pre‐existing chromosome folding to activate genes.
Synopsis
X chromosome loci with binding sites for the Drosophila MSL dosage compensation complex are engaged in long‐range interactions within the active compartment. The constitutive chromosome folding likely facilitates vital gene activation in male flies.
Dosage compensation profits from the compartment organization of the X chromosome.
Long‐range contacts between loci in the active compartment facilitate interactions between DCC binding sites.
The activation of genes correlates with their 3D proximity to DCC binding sites.
X chromosome loci with binding sites for the Drosophila MSL dosage compensation complex are engaged in long‐range interactions within the active compartment. The constitutive chromosome folding likely facilitates vital gene activation in male flies.
Chromatin modifications are associated with many aspects of gene expression, yet their role in cellular transitions during development remains elusive. Here, we use a new approach to obtain cell ...type-specific information on chromatin state and RNA polymerase II (Pol II) occupancy within the multicellular Drosophila melanogaster embryo. We directly assessed the relationship between chromatin modifications and the spatio-temporal activity of enhancers. Rather than having a unique chromatin state, active developmental enhancers show heterogeneous histone modifications and Pol II occupancy. Despite this complexity, combined chromatin signatures and Pol II presence are sufficient to predict enhancer activity de novo. Pol II recruitment is highly predictive of the timing of enhancer activity and seems dependent on the timing and location of transcription factor binding. Chromatin modifications typically demarcate large regulatory regions encompassing multiple enhancers, whereas local changes in nucleosome positioning and Pol II occupancy delineate single active enhancers. This cell type-specific view identifies dynamic enhancer usage, an essential step in deciphering developmental networks.
Developmental gene expression is tightly regulated through enhancer elements, which initiate dynamic spatio-temporal expression, and Polycomb response elements (PREs), which maintain stable gene ...silencing. These two
-regulatory functions are thought to operate through distinct dedicated elements. By examining the occupancy of the
pleiohomeotic repressive complex (PhoRC) during embryogenesis, we revealed extensive co-occupancy at developmental enhancers. Using an established in vivo assay for PRE activity, we demonstrated that a subset of characterized developmental enhancers can function as PREs, silencing transcription in a Polycomb-dependent manner. Conversely, some classic
PREs can function as developmental enhancers in vivo, activating spatio-temporal expression. This study therefore uncovers elements with dual function: activating transcription in some cells (enhancers) while stably maintaining transcriptional silencing in others (PREs). Given that enhancers initiate spatio-temporal gene expression, reuse of the same elements by the Polycomb group (PcG) system may help fine-tune gene expression and ensure the timely maintenance of cell identities.
Circularized Chromosome Conformation Capture (4C) is a powerful technique for studying the spatial interactions of a specific genomic region called the 'viewpoint' with the rest of the genome, both ...in a single condition or comparing different experimental conditions or cell types. Observed ligation frequencies typically show a strong, regular dependence on genomic distance from the viewpoint, on top of which specific interaction peaks are superimposed. Here, we address the computational task to find these specific peaks and to detect changes between different biological conditions.
We model the overall trend of decreasing interaction frequency with genomic distance by fitting a smooth monotonically decreasing function to suitably transformed count data. Based on the fit, z-scores are calculated from the residuals, and high z-scores are interpreted as peaks providing evidence for specific interactions. To compare different conditions, we normalize fragment counts between samples, and call for differential contact frequencies using the statistical method DESEQ2: adapted from RNA-Seq analysis.
A full end-to-end analysis pipeline is implemented in the R package FourCSeq available at www.bioconductor.org.
felix.klein@embl.de or whuber@embl.de
Supplementary data are available at Bioinformatics online.
T cell development proceeds under the influence of a network of transcription factors (TFs). The precise role of Zeb1, a member of this network, remains unclear. Here, we report that Zeb1 expression ...is induced early during T cell development in CD4
CD8
double-negative (DN) stage 2 (DN2). Zeb1 expression was further increased in the CD4
CD8
double-positive (DP) stage before decreasing in more mature T cell subsets. We performed an exhaustive characterization of T cells in Cellophane mice that bear Zeb1 hypomorphic mutations. The Zeb1 mutation profoundly affected all thymic subsets, especially DN2 and DP cells. Zeb1 promoted the survival and proliferation of both cell populations in a cell-intrinsic manner. In the periphery of Cellophane mice, the number of conventional T cells was near normal, but invariant NKT cells, NK1.1
γδ T cells and Ly49
CD8 T cells were virtually absent. This suggested that Zeb1 regulates the development of unconventional T cell types from DP progenitors. A transcriptomic analysis of WT and Cellophane DP cells revealed that Zeb1 regulated the expression of multiple genes involved in the cell cycle and TCR signaling, which possibly occurred in cooperation with Tcf1 and Heb. Indeed, Cellophane DP cells displayed stronger signaling than WT DP cells upon TCR engagement in terms of the calcium response, phosphorylation events, and expression of early genes. Thus, Zeb1 is a key regulator of the cell cycle and TCR signaling during thymic T cell development. We propose that thymocyte selection is perturbed in Zeb1-mutated mice in a way that does not allow the survival of unconventional T cell subsets.
The BYpass of Ess1 (Bye1) protein is a putative S. cerevisiae transcription factor homologous to the human cancer-associated PHF3/DIDO family of proteins. Bye1 contains a Plant Homeodomain (PHD) and ...a TFIIS-like domain. The Bye1 PHD finger interacts with tri-methylated lysine 4 of histone H3 (H3K4me3) while the TFIIS-like domain binds to RNA polymerase (Pol) II. Here, we investigated the contribution of these structural features to Bye1 recruitment to chromatin as well as its function in transcriptional regulation. Genome-wide analysis of Bye1 distribution revealed at least two distinct modes of association with actively transcribed genes: within the core of Pol II- and Pol III-transcribed genes concomitant with the presence of the TFIIS transcription factor and, additionally, with promoters of a subset of Pol II-transcribed genes. Specific loss of H3K4me3 abolishes Bye1 association to gene promoters, but doesn't affect its binding within gene bodies. Genetic interactions suggested an essential role of Bye1 in cell fitness under stress conditions compensating the absence of TFIIS. Furthermore, BYE1 deletion resulted in the attenuation of GAL genes expression upon galactose-mediated induction indicating its positive role in transcription regulation. Together, these findings point to a bimodal role of Bye1 in regulation of Pol II transcription. It is recruited via its PHD domain to H3K4 tri-methylated promoters at early steps of transcription. Once Pol II is engaged into elongation, Bye1 binds directly to the transcriptional machinery, modulating its progression along the gene.
Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing ...enhancer-promoter interactions and their dynamics during embryogenesis remain unclear. At the β-globin locus, enhancer-promoter interactions appear dynamic and cell-type specific, whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed. The extent to which preformed enhancer-promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. Here we generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. Our results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer-promoter loops through release of paused polymerase.
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