Nuclear organisation and architecture are essential for the maintenance of genomic integrity as well as for the epigenetic regulations and gene expression. Disruption of lamin B1, major structural and functional member of the nuclear lamina, is observed in human laminopathies and in sporadic cancers, and leads to chromosomal rearrangements and alterations of gene expression. The tumour suppressor p53 has been shown to direct specific transcriptional programmes by regulating lamin A/C, however its relationship with lamin B1 has remained elusive. Here, we show that loss of p53 correlates with increased expression of members belonging to the nuclear pore complex and nuclear lamina and directly regulates transcription of lamin B1. We show that the genomic loci of a fraction of p53-dependent genes physically interact with lamin B1 and Nup210. This observation provides a possible mechanistic explanation for the p53-depedent changes of chromatin accessibility, with the consequent influence of expression and rearrangement of these genomic sites in pancreatic cancer. Overall, these data suggest a potential functional and biochemical regulatory network connecting p53 and nuclear architecture. 

/pdf/p53-regulates-expression-of-nuclear-envelope-components-in-24npmtqg.pdf

p53 regulates expression of nuclear envelope components in cancer cells

T cells develop from multipotent progenitors by a gradual process dependent on intrathymic Notch signaling and coupled with extensive proliferation. The stages leading them to T-cell lineage commitment are well characterized by single-cell and bulk RNA analyses of sorted populations and by direct measurements of precursor-product relationships. This process depends not only on Notch signaling but also on multiple transcription factors, some associated with stemness and multipotency, some with alternative lineages, and others associated with T-cell fate. These factors interact in opposing or semi-independent T cell gene regulatory network (GRN) subcircuits that are increasingly well defined. A newly comprehensive picture of this network has emerged. Importantly, because key factors in the GRN can bind to markedly different genomic sites at one stage than they do at other stages, the genes they significantly regulate are also stage-specific. Global transcriptome analyses of perturbations have revealed an underlying modular structure to the T-cell commitment GRN, separating decisions to lose “stem-ness” from decisions to block alternative fates. Finally, the updated network sheds light on the intimate relationship between the T-cell program, which depends on the thymus, and the innate lymphoid cell (ILC) program, which does not.

/pdf/multi-modular-structure-of-the-gene-regulatory-network-for-3fwmpm1w.pdf

Multi-modular structure of the gene regulatory network for specification and commitment of murine T cells

In recent decades, the detrimental effects of environmental contaminants on human health have become a serious public concern. Organophosphate (OP) pesticides are widely used in agriculture, and the negative impacts of OP and its metabolites on human health have been demonstrated. We hypothesized that exposure to OPs during pregnancy could impose damaging effects on the fetus by affecting various processes. We analyzed sex-specific epigenetic responses in the placenta samples obtained from the mother-child PELAGIE cohort. We assayed the telomere length and mitochondrial copy numbers using genomic DNA. We analyzed H3K4me3 by using chromatin immunoprecipitation followed by qPCR (ChIP‒qPCR) and high-throughput sequencing (ChIP-seq). The human study was confirmed with mouse placenta tissue analysis. Our study revealed a higher susceptibility of male placentas to OP exposure. Specifically, we observed telomere length shortening and an increase in γH2AX levels, a DNA damage marker. We detected lower histone H3K9me3 occupancy at telomeres in diethylphosphate (DE)-exposed male placentas than in nonexposed placentas. We found an increase in H3K4me3 occupancy at the promoters of thyroid hormone receptor alpha (THRA), 8-oxoguanine DNA glycosylase (OGG1) and insulin-like growth factor (IGF2) in DE-exposed female placentas. H3K4me3 occupancy at PPARG was increased in both male and female placentas exposed to dimethylphosphate (DM). The genome-wide sequencing of selected samples revealed sex-specific differences induced by DE exposure. Specifically, we found alterations in H3K4me3 in genes related to the immune system in female placenta samples. In DE-exposed male placentas, a decrease in H3K4me3 occupancy at development-related, collagen and angiogenesis-related genes was observed. Finally, we observed a high number of NANOG and PRDM6 binding sites in regions with altered histone occupancy, suggesting that the effects were possibly mediated via these factors. Our data suggest that in utero exposure to organophosphate metabolites affects normal placental development and could potentially impact late childhood. 

/pdf/genome-wide-analysis-of-sex-specific-differences-in-the-271qx8qz.pdf

Genome-wide analysis of sex-specific differences in the mother–child PELAGIE cohort exposed to organophosphate metabolites

Epigenetic mechanisms regulate the multilineage differentiation capacity of hematopoietic stem cells (HSCs) into a variety of blood and immune cells. Mapping the chromatin dynamics of functionally defined cell populations will shed mechanistic insight on two major, unanswered questions in stem cell biology: how does epigenetic identity contribute to a cell type's lineage potential, and how do cascades of chromatin remodeling dictate ensuing fate decisions? Our recent work revealed evidence of multilineage gene priming in HSCs, where open cis-regulatory elements (CREs) exclusively shared between HSCs and unipotent lineage cells were enriched for DNA binding motifs of known lineage-specific transcription factors. Oligopotent progenitor populations operating between the HSCs and unipotent cells play essential roles in effecting hematopoietic homeostasis. To test the hypothesis that selective HSC-primed lineage-specific CREs remain accessible throughout differentiation, we used ATAC-seq to map the temporal dynamics of chromatin remodeling during progenitor differentiation. We observed epigenetic-driven clustering of oligopotent and unipotent progenitors into distinct erythromyeloid and lymphoid branches, with multipotent HSCs and MPPs associating with the erythromyeloid lineage. We mapped the dynamics of lineage-primed CREs throughout hematopoiesis and identified both unique and shared CREs as potential lineage reinforcement mechanisms at fate branch points. Additionally, quantification of genome-wide peak count and size revealed overall greater chromatin accessibility in HSCs, allowing us to identify HSC-unique peaks as putative regulators of self-renewal and multilineage potential. Finally, CRISPRi-mediated targeting of ATACseq- identified putative CREs in HSCs allowed us to demonstrate the functional role of selective CREs in lineage-specific gene expression. These findings provide insight into the regulation of stem cell multipotency and lineage commitment throughout hematopoiesis and serve as a resource to test functional drivers of hematopoietic lineage fate.

Dynamics of Chromatin Accessibility during Hematopoietic Stem Cell Differentiation into Progressively Lineage-Committed Progeny.

The assay for transposase-accessible chromatin with sequencing (ATAC-seq) is a common assay to identify chromatin accessible regions by using a Tn5 transposase that can access, cut, and ligate adapters to DNA fragments for subsequent amplification and sequencing. These sequenced regions are quantified and tested for enrichment in a process referred to as “peak calling”. Most unsupervised peak calling methods are based on simple statistical models and suffer from elevated false positive rates. Newly developed supervised deep learning methods can be successful, but they rely on high quality labeled data for training, which can be difficult to obtain. Moreover, though biological replicates are recognized to be important, there are no established approaches for using replicates in the deep learning tools, and the approaches available for traditional methods either cannot be applied to ATAC-seq, where control samples may be unavailable, or are post-hoc and do not capitalize on potentially complex, but reproducible signal in the read enrichment data. Here, we propose a novel peak caller that uses unsupervised contrastive learning to extract shared signals from multiple replicates. Raw coverage data are encoded to obtain low-dimensional embeddings and optimized to minimize a contrastive loss over biological replicates. These embeddings are passed to another contrastive loss for learning and predicting peaks and decoded to denoised data under an autoencoder loss. We compared our Replicative Contrastive Learner (RCL) method with other existing methods on ATAC-seq data, using annotations from ChromHMM genome and transcription factor ChIP-seq as noisy truth. RCL consistently achieved the best performance.

/pdf/unsupervised-contrastive-peak-caller-for-atac-seq-1rdcen1d.pdf

Unsupervised Contrastive Peak Caller for ATAC-seq

We present WhichTF, a computational method to identify functionally important transcription factors (TFs) from chromatin accessibility measurements. To rank TFs, WhichTF applies an ontology-guided functional approach to compute novel enrichment by integrating accessibility measurements, high-confidence pre-computed conservation-aware TF binding sites, and putative gene-regulatory models. Comparison with prior sheer abundance-based methods reveals the unique ability of WhichTF to identify context-specific TFs with functional relevance, including NF-κB family members in lymphocytes and GATA factors in cardiac cells. To distinguish the transcriptional regulatory landscape in closely related samples, we apply differential analysis and demonstrate its utility in lymphocyte, mesoderm developmental, and disease cells. We find suggestive, under-characterized TFs, such as RUNX3 in mesoderm development and GLI1 in systemic lupus erythematosus. We also find TFs known for stress response, suggesting routine experimental caveats that warrant careful consideration. WhichTF yields biological insight into known and novel molecular mechanisms of TF-mediated transcriptional regulation in diverse contexts, including human and mouse cell types, cell fate trajectories, and disease-associated cells. 

Leanne Chang

Papers

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