How cells adopt different identities has long fascinated biologists. Signal transduction in response to environmental cues results in the activation of transcription factors that determine the ...gene-expression program characteristic of each cell type. Technological advances in the study of 3D chromatin folding are bringing the role of genome conformation in transcriptional regulation to the fore. Characterizing this role of genome architecture has profound implications, not only for differentiation and development but also for diseases including developmental malformations and cancer. Here we review recent studies indicating that the interplay between transcription and genome conformation is a driving force for cell-fate decisions.
Accurate control of gene expression in the right cell at the right moment is of fundamental importance to animal development and homeostasis. At the heart of gene regulation lie the enhancers, a ...class of gene regulatory elements that ensures precise spatiotemporal activation of gene transcription. Mammalian genomes are littered with enhancers, which are frequently organized in cooperative clusters such as locus control regions and superenhancers. Here, we discuss our current knowledge of enhancer biology, including an overview of the discovery of the various enhancer subsets and the mechanistic models used to explain their gene regulatory function.
Chromosome structure is a crucial regulatory factor for a wide range of nuclear processes. Chromosome conformation capture (3C)-based experiments combined with computational modelling are pivotal for ...unveiling 3D chromosome structure. Here, we introduce TADdyn, a tool that integrates time-course 3C data, restraint-based modelling, and molecular dynamics to simulate the structural rearrangements of genomic loci in a completely data-driven way. We apply TADdyn on in situ Hi-C time-course experiments studying the reprogramming of murine B cells to pluripotent cells, and characterize the structural rearrangements that take place upon changes in the transcriptional state of 21 genomic loci of diverse expression dynamics. By measuring various structural and dynamical properties, we find that during gene activation, the transcription starting site contacts with open and active regions in 3D chromatin domains. We propose that these 3D hubs of open and active chromatin may constitute a general feature to trigger and maintain gene transcription.
HIV-1 recurrently targets active genes and integrates in the proximity of the nuclear pore compartment in CD4
T cells. However, the genomic features of these genes and the relevance of their ...transcriptional activity for HIV-1 integration have so far remained unclear. Here we show that recurrently targeted genes are proximal to super-enhancer genomic elements and that they cluster in specific spatial compartments of the T cell nucleus. We further show that these gene clusters acquire their location during the activation of T cells. The clustering of these genes along with their transcriptional activity are the major determinants of HIV-1 integration in T cells. Our results provide evidence of the relevance of the spatial compartmentalization of the genome for HIV-1 integration, thus further strengthening the role of nuclear architecture in viral infection.
Chromosomal architecture is known to influence gene expression, yet its role in controlling cell fate remains poorly understood. Reprogramming of somatic cells into pluripotent stem cells (PSCs) by ...the transcription factors (TFs) OCT4, SOX2, KLF4 and MYC offers an opportunity to address this question but is severely limited by the low proportion of responding cells. We have recently developed a highly efficient reprogramming protocol that synchronously converts somatic into pluripotent stem cells. Here, we used this system to integrate time-resolved changes in genome topology with gene expression, TF binding and chromatin-state dynamics. The results showed that TFs drive topological genome reorganization at multiple architectural levels, often before changes in gene expression. Removal of locus-specific topological barriers can explain why pluripotency genes are activated sequentially, instead of simultaneously, during reprogramming. Together, our results implicate genome topology as an instructive force for implementing transcriptional programs and cell fate in mammals.
Innate lymphoid cells (ILCs) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCPs). How ILCPs give rise to mature tissue-resident ILCs ...remains unclear. Here, we identify circulating and tissue ILCPs in humans that fail to express the transcription factors and cytokine outputs of mature ILCs but have these signature loci in an epigenetically poised configuration. Human ILCPs robustly generate all ILC subsets in vitro and in vivo. While human ILCPs express low levels of retinoic acid receptor (RAR)-related orphan receptor C (RORC) transcripts, these cells are found in RORC-deficient patients and retain potential for EOMES+ natural killer (NK) cells, interferon gamma-positive (IFN-γ+) ILC1s, interleukin (IL)-13+ ILC2s, and for IL-22+, but not for IL-17A+ ILC3s. Our results support a model of tissue ILC differentiation (“ILC-poiesis”), whereby diverse ILC subsets are generated in situ from systemically distributed ILCPs in response to local environmental signals.
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•Discovery of circulating human ILC precursors (ILCPs)•Blood-borne ILCPs generate cytotoxic and helper ILCs in vitro and in vivo•ILCPs are present in lymphoid and non-lymphoid human tissues•Multi-potent ILCPs are present in RORC-deficient patients
Human innate lymphoid cell progenitors circulate systemically, differentiating into diverse subtypes in specific tissues in response to localized cues.
Here, we report DNA methylation and hydroxymethylation dynamics at nucleotide resolution using C/EBPα-enhanced reprogramming of B cells into induced pluripotent cells (iPSCs). We observed successive ...waves of hydroxymethylation at enhancers, concomitant with a decrease in DNA methylation, suggesting active demethylation. Consistent with this finding, ablation of the DNA demethylase Tet2 almost completely abolishes reprogramming. C/EBPα, Klf4, and Tfcp2l1 each interact with Tet2 and recruit the enzyme to specific DNA sites. During reprogramming, some of these sites maintain high levels of 5hmC, and enhancers and promoters of key pluripotency factors become demethylated as early as 1 day after Yamanaka factor induction. Surprisingly, methylation changes precede chromatin opening in distinct chromatin regions, including Klf4 bound sites, revealing a pioneer factor activity associated with alteration in DNA methylation. Rapid changes in hydroxymethylation similar to those in B cells were also observed during compound-accelerated reprogramming of fibroblasts into iPSCs, highlighting the generality of our observations.
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•Base-resolution profiling of DNA (hydroxy)methylation during iPSC reprogramming•Major contribution of Tet2-mediated demethylation throughout reprogramming•C/EBPα, Klf4, and Tfcp2l1 drive Tet2-mediated enhancer demethylation and activation•Klf4 induces enhancer demethylation in the absence of nucleosome repositioning
Using a highly efficient reprogramming system, Sardina et al. examined the dynamics of DNA methylation and hydroxymethylation. They found that throughout the process several transcription factors can recruit Tet2 to specific sites, leading to demethylation. Some of these sites became demethylated before chromatin opening.
Targeted functional genomics represents a powerful approach for studying gene function in vivo and in vitro. However, its application to gene expression studies in human mast cells has been hampered ...by low yields of human mast cell cultures and their poor transfection efficiency. We developed an imaging system in which mast cell degranulation can be visualized in single cells subjected to shRNA knockdown or CRISPR-Cas9 gene editing. By using high-resolution confocal microscopy and a fluorochrome-labeled avidin probe, one can directly assess the alteration of functional responses, i.e., degranulation, in single human mast cells (10-12 weeks old). The elimination of a drug or marker selection step avoids the use of potentially toxic treatment procedures, and the brief hands-on time of the functional analysis step enables high-throughput screening of shRNA or CRISPR-Cas9 constructs to identify genes that regulate human mast cell degranulation. The ability to analyze single cells substantially reduces the total number of cells required and enables the parallel visualization of the degranulation profiles of both edited and non-edited mast cells, offering a consistent internal control not found in other protocols. Moreover, our protocol offers a flexible choice between RNA interference (RNAi) and CRISPR-Cas9 genome editing for perturbation of gene expression using our human mast cell single-cell imaging system. Perturbation of gene expression, acquisition of microscopy data and image analysis can be completed within 5 d, requiring only standard laboratory equipment and expertise.
Recently, human ILCs that express CD117 and CD127 but lack CRTH2 and NKp44 have been shown to contain precursors of ILC1, ILC2, and ILC3. However, these ILCs have not been extensively characterized. ...We performed an unbiased hierarchical stochastic neighbor embedding (HSNE) analysis of the phenotype of peripheral blood CD117
ILCs, which revealed the presence of three major subsets: the first expressed NKp46, the second expressed both NKp46 and CD56, and the third expressed KLRG1, but not NKp46 or CD56. Analysis of their cytokine production profiles and transcriptome revealed that NKp46
ILCs predominantly develop into ILC3s; some of them can differentiate into ILC1/NK-like cells, but they are unable to develop into ILC2s. In contrast, KLRG1
ILCs predominantly differentiate into ILC2s. Single-cell cultures demonstrate that KLRG1
ILCs can also differentiate into other ILC subsets depending on the signals they receive. Epigenetic profiling of KLRG1
ILCs is consistent with the broad differentiation potential of these cells.
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