Inaccessible Chromatin Research Articles

The HSV-1 encoded CCCTC-binding factor, CTRL2, impacts the nature of viral chromatin during HSV-1 lytic infection.

HSV-1 genomes are rapidly heterochromatinized following entry by host cells to limit viral gene expression. Efficient HSV-1 genome replication requires mechanisms that de-repress chromatin associated with the viral genome. CCCTC-binding factors, or CTCF insulators play both silencing and activating roles in cellular transcriptional regulation. Importantly, the HSV-1 genome encodes several CTCF insulators that flank IE genes, implying that individual HSV-1 encoded CTCF insulators regulate IE transcription during all stages of the HSV-1 life cycle. We previously reported that the HSV-1 encoded CTCF insulator located downstream of the LAT (CTRL2) controlled IE gene silencing during latency. To further characterize the role of this insulator during the lytic infection we leveraged a ΔCTRL2 recombinant virus to show that there was a genome replication defect that stemmed from decreased IE gene expression in fibroblasts and epithelial cells at early times following initiation of infection. Further experiments indicated that the defect in gene expression resulted from chromatin inaccessibility in the absence of the insulator. To elucidate how chromatin accessibility was altered in the absence of the CTRL2 insulator, we showed that enrichment of Alpha-thalassemia/mental retardation, X-linked chromatin remodeler (ATRX), and the histone variant H3.3, both of which are known for their roles in maintaining repressive histone markers on the HSV-1 viral genome were increased on IE regions of HSV-1. Finally, both H3K27me3 and H3K9me3 repressive histone marks remained enriched by 4 hours post infection in the absence of the CTRL2 insulator, confirming that the CTRL2 insulator is required for de-repression of IE genes of viral genomes. To our knowledge these are the first data that show that a specific CTCF insulator in the HSV-1 genome (CTRL2) regulates chromatin accessibility during the lytic infection.

Central role of SUMOylation in the regulation of chromatin interactions and transcriptional outputs of the androgen receptor in prostate cancer cells.

The androgen receptor (AR) is pivotal in prostate cancer (PCa) progression and represents a critical therapeutic target. AR-mediated gene regulation involves intricate interactions with nuclear proteins, with many mediating and undergoing post-translational modifications that present alternative therapeutic avenues. Through chromatin proteomics in PCa cells, we identified SUMO ligases together with nuclear receptor coregulators and pioneer transcription factors within the AR's protein network. Intriguingly, this network displayed a significant association with SUMO2/3. To elucidate the influence of SUMOylation on AR chromatin interactions and subsequent gene regulation, we inhibited SUMOylation using ML-792 (SUMOi). While androgens generally facilitated the co-occupancy of SUMO2/3 and AR on chromatin, SUMOi induced divergent effects dependent on the type of AR-binding site (ARB). SUMOi augmented AR's pioneer-like binding on inaccessible chromatin regions abundant in androgen response elements (AREs) and diminished its interaction with accessible chromatin regions sparse in AREs yet rich in pioneer transcription factor motifs. The SUMOi-impacted ARBs divergently influenced AR-regulated genes; those associated with AR-mediated activation played roles in negative regulation of cell proliferation, while those with AR-mediated repression were involved in pattern formation. In conclusion, our findings underscore the pervasive influence of SUMOylation in shaping AR's role in PCa cells, potentially unveiling new therapeutic strategies.

Rhabdomyosarcoma fusion oncoprotein initially pioneers a neural signature in vivo.

Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer molecularly characterized by arrested myogenesis. The defining genetic driver, PAX3::FOXO1, functions as a chimeric gain-of-function transcription factor. An incomplete understanding of PAX3::FOXO1's in vivo epigenetic mechanisms has hindered therapeutic development. Here, we establish a PAX3::FOXO1 zebrafish injection model and semi-automated ChIP-seq normalization strategy to evaluate how PAX3::FOXO1 initially interfaces with chromatin in a developmental context. We investigated PAX3::FOXO1's recognition of chromatin and subsequent transcriptional consequences. We find that PAX3::FOXO1 interacts with inaccessible chromatin through partial/homeobox motif recognition consistent with pioneering activity. However, PAX3::FOXO1-genome binding through a composite paired-box/homeobox motif alters chromatin accessibility and redistributes H3K27ac to activate neural transcriptional programs. We uncover neural signatures that are highly representative of clinical rhabdomyosarcoma gene expression programs that are enriched following chemotherapy. Overall, we identify partial/homeobox motif recognition as a new mode for PAX3::FOXO1 pioneer function and identify neural signatures as a potentially critical PAX3::FOXO1 tumor initiation event.

P-523 Single molecule chromatin footprinting reveals only low occurrence of nucleosomes at multiple loci throughout chromatin of human and mouse spermatozoa

Abstract Study question What fraction of spermatozoa from mouse and human semen contain nucleosomes at specific loci? What chromatin features affect nucleosome retention in spermatozoa? Summary answer On average, 1-2% mouse spermatozoa and 5-9% human spermatozoa contain nucleosomes at sampled genomic loci, with moderately higher frequencies at unmethylated regions of DNA. What is known already Nucleosomes constitute the basic packaging and gene regulatory unit of DNA in eukaryotic cells. During final stages of spermatogenesis in mammals, however, most nucleosomes are replaced by protamines. Previous global chromatin immunoprecipitation-sequencing studies reported the presence of nucleosomes at gene regulatory and other genomic regions. Such nucleosomes were proposed to convey intergenerational inheritance of epigenetic information. Additional studies revealed that infertile men with decreased success of assisted reproductive therapy have protamine deficiency or displayed more widely distributed nucleosomes in their sperm genome. Direct links between altered levels of nuclear proteins and fertilizing potential of sperm have, however, not been established. Study design, size, duration We conducted a case study on nucleosome retention levels in sperm from four selected fertile human samples. This study was complemented in mice, in which we characterized the dynamic changes in nucleosome frequencies at genomic loci during differentiation of haploid spermatids into spermatozoa, and their responses in mutant spermatozoa lacking DNA methylation at certain loci. Participants/materials, setting, methods We used the single-molecule sequencing technique called Nucleosome Occupancy and Methylome Sequencing to quantify nucleosome occupancy rates at selected loci in spermatozoa from ejaculated semen of four fertile semen donors. We also profiled round spermatids, early and late elongating spermatids and spermatozoa of C57BL/6J wild-type mice as well as spermatozoa of mice conditionally deficient for the methyltransferases Dnmt3a and Dnmt3b, lacking DNA methylation at certain genomic regions. Main results and the role of chance To our knowledge, this is the first study to quantify nucleosome occupancy in mouse male gametes and human spermatozoa at single-molecule resolution. In mice, we observed that chromatin remodelling during spermatid elongation resulted in a progressive loss of nucleosomes and their positioning, a transient gain in global chromatin accessibility which was succeeded by increasing chromatin inaccessibility and nuclear compaction. In both human and mouse sperm, we profiled nucleosome occupancy levels at different genomic loci and measured only low percentages, ranging from ∼1-2% in mouse and 5-9% in human. In both organisms we observed a near two-fold higher nucleosome occupancy at loci being unmethylated at their DNA over loci being highly methylated. Mice partially deficient in genomic DNA methylation manifested a similar fold increase in nucleosome occupancy in absence of DNA methylation. These data reveal a moderate modulatory role for DNA methylation established in spermatogonial stages in regulating nucleosome eviction during spermiogenesis. The overall low nucleosome occupancy frequencies in mammalian spermatozoa suggest a stochastic or random rather than a programmed role of nucleosomes in regulating paternal gene expression during ensuing embryonic development. Limitations, reasons for caution Amplicon-based sequencing provides deep sequencing coverage for selected genomic loci. We lack, however, information on the distribution of nucleosomes among loci within the genome of single spermatozoa. In human, we studied a limited number of human donors. Wider implications of the findings This study resolves a long-standing question on the frequency and local distribution of nucleosomes in murine and human spermatozoa. The variability in nucleosome occupancy between spermatozoa within and between samples of human individuals inspires further investigations into the relationship between chromatin states and quality of individual spermatozoa for embryonic development. Trial registration number SNCTP000003654

Abstract 2861: Rhabdomyosarcoma fusion-oncogene alters the chromatin landscape to initially drive a neural signature in vivo

Abstract Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer lacking curative therapies, and outcomes for children with this disease have not improved in decades. The chimeric transcription factor PAX3::FOXO1 is the most common and lethal driver of fusion-positive rhabdomyosarcoma, and it consists of the DNA binding domains of PAX3 fused to the transactivation domain of FOXO1. Despite its well-established essentiality for tumorigenesis, how PAX3::FOXO1 initially generates a tumorigenic cell state has been challenging to study due to the lack of in vivo models expressing the fusion-oncogene at accessible early developmental time points. Here, we developed a novel zebrafish mRNA injection model that ubiquitously expresses PAX3::FOXO1 during gastrulation representing all three germ layers. Using this high-throughput model, we used ChIP-seq to identify initial in vivo PAX3::FOXO1 binding sites and targets. PAX3::FOXO1 has pioneering activity, where it can utilize partial-motif recognition to bind to inaccessible chromatin and alter chromatin structure. With biochemical and 2D chromatin sequencing approaches in our model, we find evidence for in vivo PAX3::FOXO1 pioneering activity. PAX3::FOXO1 consists of two DNA binding domains, a paired and homeobox domain, and we demonstrate a new mode of pioneering activity driven by its homeobox domain given a strong enrichment of homeobox-related motifs at PAX3::FOXO1 binding sites. PAX3::FOXO1 activity in developing zebrafish embryos results in an arrested development phenotype and transcriptional signatures enriched for pathways such as an inhibition of segmentation and myogenic development. Critically, we find that PAX3::FOXO1 directly activates neural-related gene targets by increasing chromatin accessibility and re-distribution of the active histone mark H3K27Ac to these loci. These discoveries are particularly striking because rhabdomyosarcoma is traditionally associated with skeletal muscle characteristics. With our findings, we hypothesize neural gene activation is a critical mechanism for fusion-positive rhabdomyosarcoma tumor initiation and could be a critical pathway across various stages of rhabdomyosarcoma tumorigenesis. This hypothesis agrees with our previous discovery of a novel PAX3::FOXO1 cooperating neural transcription factor, HES3, which we find is directly activated by PAX3::FOXO1 in our mRNA injection model. Our long-term goal is to utilize this versatile model to functionally evaluate cooperation between PAX3::FOXO1 and both canonical rhabdomyosarcoma and neural transcription factors. Our approach will employ a cross-species comparative analysis across this injection model, our zebrafish tumor models, other model systems, and patient data. This work will identify conserved targets and mechanisms of PAX3::FOXO1 tumorigenesis that could become new therapeutic vulnerabilities. Citation Format: Jack Kucinski, Cenny Taslim, Alexi Tallan, Matthew Cannon, Katherine Silvius, Benjamin Stanton, Genevieve Kendall. Rhabdomyosarcoma fusion-oncogene alters the chromatin landscape to initially drive a neural signature in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2861.

HMGA2 directly mediates chromatin condensation in association with neuronal fate regulation

Identification of factors that regulate chromatin condensation is important for understanding of gene regulation. High-mobility group AT-hook (HMGA) proteins 1 and 2 are abundant nonhistone chromatin proteins that play a role in many biological processes including tissue stem-progenitor cell regulation, but the nature of their protein function remains unclear. Here we show that HMGA2 mediates direct condensation of polynucleosomes and forms droplets with nucleosomes. Consistently, most endogenous HMGA2 localized to transposase 5– and DNase I–inaccessible chromatin regions, and its binding was mostly associated with gene repression, in mouse embryonic neocortical cells. The AT-hook 1 domain was necessary for chromatin condensation by HMGA2 in vitro and in cellulo, and an HMGA2 mutant lacking this domain was defective in the ability to maintain neuronal progenitors in vivo. Intrinsically disordered regions of other proteins could substitute for the AT-hook 1 domain in promoting this biological function of HMGA2. Taken together, HMGA2 may regulate neural cell fate by its chromatin condensation activity.

Lineage commitment of dermal fibroblast progenitors is controlled by Kdm6b-mediated chromatin demethylation.

Dermal Fibroblast Progenitors (DFPs) differentiate into distinct fibroblast lineages during skin development. However, the epigenetic mechanisms that regulate DFP differentiation are not known. Our objective was to use multimodal single-cell approaches, epigenetic assays, and allografting techniques to define a DFP state and the mechanism that governs its differentiation potential. Our initial results indicated that the overall transcription profile of DFPs is repressed by H3K27me3 and has inaccessible chromatin at lineage-specific genes. Surprisingly, the repressive chromatin profile of DFPs renders them unable to reform the skin in allograft assays despite their multipotent potential. We hypothesized that chromatin derepression was modulated by the H3K27me3 demethylase, Kdm6b/Jmjd3. Dermal fibroblast-specific deletion of Kdm6b/Jmjd3 in mice resulted in adipocyte compartment ablation and inhibition of mature dermal papilla functions, confirmed by additional single-cell RNA-seq, ChIP-seq, and allografting assays. We conclude that DFPs are functionally derepressed during murine skin development by Kdm6b/Jmjd3. Our studies therefore reveal a multimodal understanding of how DFPs differentiate into distinct fibroblast lineages and provide a novel publicly available multiomics search tool.

Identification of mammalian transcription factors that bind to inaccessible chromatin.

Transcription factors (TFs) are proteins that affect gene expression by binding to regulatory regions of DNA in a sequence specific manner. The binding of TFs to DNA is controlled by many factors, including the DNA sequence, concentration of TF, chromatin accessibility and co-factors. Here, we systematically investigated the binding mechanism of hundreds of TFs by analysing ChIP-seq data with our explainable statistical model, ChIPanalyser. This tool uses as inputs the DNA sequence binding motif; the capacity to distinguish between strong and weak binding sites; the concentration of TF; and chromatin accessibility. We found that approximately one third of TFs are predicted to bind the genome in a DNA accessibility independent fashion, which includes TFs that can open the chromatin, their co-factors and TFs with similar motifs. Our model predicted this to be the case when the TF binds to its strongest binding regions in the genome, and only a small number of TFs have the capacity to bind dense chromatin at their weakest binding regions, such as CTCF, USF2 and CEBPB. Our study demonstrated that the binding of hundreds of human and mouse TFs is predicted by ChIPanalyser with high accuracy and showed that many TFs can bind dense chromatin.

Hi-TrAC detects active sub-TADs and reveals internal organizations of super-enhancers.

The spatial folding of eukaryotic genome plays a key role in genome function. We report here that our recently developed method, Hi-TrAC, which specializes in detecting chromatin loops among accessible genomic regions, can detect active sub-TADs with a median size of 100kb, most of which harbor one or two cell specifically expressed genes and regulatory elements such as super-enhancers organized into nested interaction domains. These active sub-TADs are characterized by highly enriched histone mark H3K4me1 and chromatin-binding proteins, including Cohesin complex. Deletion of selected sub-TAD boundaries have different impacts, such as decreased chromatin interaction and gene expression within the sub-TADs or compromised insulation between the sub-TADs, depending on the specific chromatin environment. We show that knocking down core subunit of the Cohesin complex using shRNAs in human cells or decreasing the H3K4me1 modification by deleting the H3K4 methyltransferase Mll4 gene in mouse Th17 cells disrupted the sub-TADs structure. Our data also suggest that super-enhancers exist as an equilibrium globule structure, while inaccessible chromatin regions exist as a fractal globule structure. In summary, Hi-TrAC serves as a highly sensitive and inexpensive approach to study dynamic changes of active sub-TADs, providing more explicit insights into delicate genome structures and functions.

B lymphocytes in treatment-naive paediatric patients with lupus are epigenetically distinct from healthy children

BackgroundSLE is likely triggered by gene–environment interactions. We have shown that most SLE-associated haplotypes encompass genomic regions enriched for epigenetic marks associated with enhancer function in lymphocytes, suggesting genetic risk...

Stalled oligodendrocyte differentiation in IDH-mutant gliomas

BackgroundRoughly 50% of adult gliomas harbor isocitrate dehydrogenase (IDH) mutations. According to the 2021 WHO classification guideline, these gliomas are diagnosed as astrocytomas, harboring no 1p19q co-deletion, or oligodendrogliomas, harboring 1p19q co-deletion. Recent studies report that IDH-mutant gliomas share a common developmental hierarchy. However, the neural lineages and differentiation stages in IDH-mutant gliomas remain inadequately characterized.MethodsUsing bulk transcriptomes and single-cell transcriptomes, we identified genes enriched in IDH-mutant gliomas with or without 1p19q co-deletion, we also assessed the expression pattern of stage-specific signatures and key regulators of oligodendrocyte lineage differentiation. We compared the expression of oligodendrocyte lineage stage-specific markers between quiescent and proliferating malignant single cells. The gene expression profiles were validated using RNAscope analysis and myelin staining and were further substantiated using data of DNA methylation and single-cell ATAC-seq. As a control, we assessed the expression pattern of astrocyte lineage markers.ResultsGenes concordantly enriched in both subtypes of IDH-mutant gliomas are upregulated in oligodendrocyte progenitor cells (OPC). Signatures of early stages of oligodendrocyte lineage and key regulators of OPC specification and maintenance are enriched in all IDH-mutant gliomas. In contrast, signature of myelin-forming oligodendrocytes, myelination regulators, and myelin components are significantly down-regulated or absent in IDH-mutant gliomas. Further, single-cell transcriptomes of IDH-mutant gliomas are similar to OPC and differentiation-committed oligodendrocyte progenitors, but not to myelinating oligodendrocyte. Most IDH-mutant glioma cells are quiescent; quiescent cells and proliferating cells resemble the same differentiation stage of oligodendrocyte lineage. Mirroring the gene expression profiles along the oligodendrocyte lineage, analyses of DNA methylation and single-cell ATAC-seq data demonstrate that genes of myelination regulators and myelin components are hypermethylated and show inaccessible chromatin status, whereas regulators of OPC specification and maintenance are hypomethylated and show open chromatin status. Markers of astrocyte precursors are not enriched in IDH-mutant gliomas.ConclusionsOur studies show that despite differences in clinical manifestation and genomic alterations, all IDH-mutant gliomas resemble early stages of oligodendrocyte lineage and are stalled in oligodendrocyte differentiation due to blocked myelination program. These findings provide a framework to accommodate biological features and therapy development for IDH-mutant gliomas.

Measuring Inaccessible Chromatin Genome-Wide Using Protect-seq.

Chromatin accessibility has been an immensely powerful metric for identifying and understanding regulatory elements in the genome. Many important regulatory elements, such as enhancers and transcriptional start sites, are characterized by "open" or nucleosome-free regions. Understanding the areas of the genome that are not considered open chromatin has been more difficult. Protect-seq is a genomics technique that aims to identify inaccessible chromatin associated with the nuclear periphery. These regions are enriched for histone modifications associated with transcriptional repression and correlate with loci identified by other techniques measuring heterochromatin and peripheral localization. Here, we discuss the protocol and best practices to perform Protect-seq.

PPARγ lipodystrophy mutants reveal intermolecular interactions required for enhancer activation

Peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of adipocyte differentiation, and mutations that interfere with its function cause lipodystrophy. PPARγ is a highly modular protein, and structural studies indicate that PPARγ domains engage in several intra- and inter-molecular interactions. How these interactions modulate PPARγ’s ability to activate target genes in a cellular context is currently poorly understood. Here we take advantage of two previously uncharacterized lipodystrophy mutations, R212Q and E379K, that are predicted to interfere with the interaction of the hinge of PPARγ with DNA and with the interaction of PPARγ ligand binding domain (LBD) with the DNA-binding domain (DBD) of the retinoid X receptor, respectively. Using biochemical and genome-wide approaches we show that these mutations impair PPARγ function on an overlapping subset of target enhancers. The hinge region-DNA interaction appears mostly important for binding and remodelling of target enhancers in inaccessible chromatin, whereas the PPARγ-LBD:RXR-DBD interface stabilizes the PPARγ:RXR:DNA ternary complex. Our data demonstrate how in-depth analyses of lipodystrophy mutants can unravel molecular mechanisms of PPARγ function.

Epigenetic Reprogramming Leads to Downregulation of CD4 and Functional Changes in African Green Monkey Memory CD4+ T Cells.

African green monkeys (AGMs), Chlorocebus pygerythrus, are a natural host for a lentivirus related to HIV, SIV. SIV-infected AGMs rarely progress to AIDS despite robust viral replication. Though multiple mechanisms are involved, a primary component is the animals' ability to downregulate CD4 expression on mature CD4+ Th cells, rendering these cells resistant to infection by SIV. These CD8αα+ T cells retain functional characteristics of CD4+ Th cells while simultaneously acquiring abilities of cytotoxic CD8αβ+ T cells. To determine mechanisms underlying functional differences between T cell subsets in AGMs, chromatin accessibility in purified populations was determined by assay for transposase-accessible chromatin sequencing. Differences in chromatin accessibility alone were sufficient to cluster cells by subtype, and accessibility at the CD4 locus reflected changes in CD4 expression. DNA methylation at the CD4 locus also correlated with inaccessible chromatin. By associating accessible regions with nearby genes, gene expression was found to correlate with accessibility changes. T cell and immune system activation pathways were identified when comparing regions that changed accessibility from CD4+ T cells to CD8αα+ T cells. Different transcription factor binding sites are revealed as chromatin accessibility changes, and these differences may elicit downstream changes in differentiation. This comprehensive description of the epigenetic landscape of AGM T cells identified genes and pathways that could have translational value in therapeutic approaches recapitulating the protective effects CD4 downregulation.

Integrating chromatin accessibility states in the design of targeted sequencing panels for liquid biopsy

Dying tumor cells shed DNA fragments into the circulation that are known as circulating tumor DNA (ctDNA). Liquid biopsy tests aim to detect cancer using known markers, including genetic alterations and epigenetic profiles of ctDNA. Despite various advantages, the major limitation remains the low fraction of tumor-originating DNA fragments in a high background of normal blood-cell originating fragments in the cell-free DNA (cfDNA) pool in plasma. Deep targeted sequencing of cfDNA allows for enrichment of fragments in known cancer marker-associated regions of the genome, thus increasing the chances of detecting the low fraction variant harboring fragments. Most targeted sequencing panels are designed to include known recurrent mutations or methylation markers of cancer. Here, we propose the integration of cancer-specific chromatin accessibility states into panel designs for liquid biopsy. Using machine learning approaches, we first identify accessible and inaccessible chromatin regions specific to each major human cancer type. We then introduce a score that quantifies local chromatin accessibility in tumor relative to blood cells and show that this metric can be useful for prioritizing marker regions with higher chances of being detected in cfDNA for inclusion in future panel designs.

Epigenetic regulation of genes involved in fluid‐electrolyte balance in the kidney

Recent studies have identified at least 20 different kidney cell types based upon chromatin structure and gene expression. Histone deacetylases (HDACs) are epigenetic transcriptional regulators via deacetylation of histone lysines resulting in inaccessible chromatin. We reported that kidney epithelial HDAC1 and HDAC2 activity is critical for maintaining a healthy kidney and preventing fluid‐electrolyte abnormalities. However, to what extent does Hdac1/Hdac2 knockdown affect chromatin structure and subsequent transcript expression in the kidney? To answer this question, we used single nucleus Assay for Transposase‐Accessible Chromatin‐sequencing (snATAC‐seq) and snRNA‐seq to profile kidney nuclei from adult male and female, control and littermate inducible kidney epithelial‐specific Hdac1/Hdac2 knockdown mice (iKSHdac1/2KO). Compared to littermate control mice iKSHdac1/2KO mice had significant elevations in plasma sodium (male control n=17, PNa= 143.6 ± 1.5 vs KO n=8, 147.6 ± 3.5, P=0.002, female control n=6, 141.8 ± 2.5 vs KO n=8 144.6 ± 1.8, P=0.03). iKSHdac1/2KO mice also had a significant increase in the kidney to body mass ratio (males 11.5 ±1.4 mg/g vs 13.1 ±1.5 P=0.1, females 9.7 ± 1.0 vs 12.3 ± 1.4, P=0.001) but no differences in liver to body mass ratio. Kidney histological analyses determined significant interstitial fibrosis, tubular atrophy and necrosis, dilated tubules, and protein casts in the iKSHdac1/2KO regardless of sex. Kidney structure was normal in all control mice. Survival was also significantly impacted in the iKSHdac1/2KO as all of these mice died (male mice died 27‐28 days after induction of knockdown). At 2 weeks after induction of KO, kidney nuclei from control and iKSHdac1/2KO mice were isolated and snATAC‐seq and snRNA‐seq analyses completed. We identified 26 unique clusters in both datasets that represented epithelial, endothelial, immune, stromal, and podocyte cells. Hdac1/Hdac2 knockdown resulted in significant changes in the chromatin structure predominantly within the promoter region of gene loci involved in fluid‐electrolyte balance such as the aquaporins, with both increased and decreased accessibility captured. Aqp1 expressed in the proximal tubules was reduced 60% and principal cell Aqp2 was reduced 50% in the iKSHdac1/2KO mice compared to controls. However, not all aquaporins were affected as for example, principal cell Aqp4 was not significantly different between the mice (P=0.98). Moreover, sex‐specific transcriptomic and chromatin structure were apparent in the proximal tubule clusters. Hdac1/Hdac2 knockdown resulted in different gene loci being accessible with a corresponding increased transcript number in the kidney, but among all mice only 24‐30% of chromatin accessibility agreed with transcript expression (e.g. open chromatin, increased transcript). To conclude, although chromatin structure does affect transcription, ~70% of the differentially expressed genes cannot be explained by changes in chromatin accessibility and HDAC1/HDAC2 had a minimal effect on these global patterns. Yet, the genes that are targets of HDAC1 and HDAC2 are critically important for maintaining kidney function and survival.

The role of DNA methylation in genome-wide gene regulation during development.

Although it is well known that DNA methylation serves to repress gene expression, precisely how it functions during the process of development remains unclear. Here, we propose that the overall pattern of DNA methylation established in the early embryo serves as a sophisticated mechanism for maintaining a genome-wide network of gene regulatory elements in an inaccessible chromatin structure throughout the body. As development progresses, programmed demethylation in each cell type then provides the specificity for maintaining select elements in an open structure. This allows these regulatory elements to interact with a large range of transcription factors and thereby regulate the gene expression profiles that define cell identity.

The peroxisome proliferator-activated receptor agonist rosiglitazone specifically represses tumour metastatic potential in chromatin inaccessibility-mediated FABP4-deficient gastric cancer.

Background: Efforts to prevent recurrence in gastric cancer (GC) patients are limited by current incomplete understanding of the pathological mechanisms. The present study aimed to identify novel tumour metastasis-associated genes and investigate potential value of these genes in clinical diagnosis and therapy.Methods: RNA sequencing was performed to identify differentially expressed genes related to GC metastasis. The expression and prognostic significance of fatty acid binding protein 4 (FABP4) were evaluated in two independent cohorts of GC patients. Chromatin immunoprecipitation sequencing, diverse mouse models and assays for transposase-accessible chromatin with high-throughput sequencing were used to investigate the roles and mechanisms of action of FABP4.Results: The results of the present multicentre study confirmed an association between a decrease in the expression of FABP4 and poor outcomes in GC patients. FABP4 inhibited GC metastasis but did not influence tumour growth in vitro and in vivo. Mechanistically, FABP4 binding with peroxisome proliferator-activated receptor γ (PPAR-γ) facilitated the translocation of PPAR-γ to the nucleus. FABP4 depletion suppressed PPAR-γ-mediated transcription of cell adhesion molecule 3 (CADM3), which preferentially governed GC metastasis. Notably, the PPAR-γ agonist rosiglitazone reversed the metastatic properties of FABP4-deficient GC cells in vitro and demonstrated viable therapeutic potential in multiple mouse models. For GC patients with diabetes, low FABP4 portends better prognosis than high FABP4 after receipt of rosiglitazone treatment. Additionally, chromatin inaccessibility induced by HDAC1 reduced FABP4 expression at the epigenetic level.Conclusions: Our findings suggest that chromatin inaccessibility orchestrates a reduction in FABP4 expression, which inhibits CADM3 transcription via PPAR-γ, thereby resulting in GC metastasis. The antidiabetic drug rosiglitazone restores PPAR-γ/CADM3 activation in FABP4-deficient GC and thus has promising therapeutic potential.

Kidney cell type-specific changes in the chromatin and transcriptome landscapes following epithelial Hdac1 and Hdac2 knockdown.

Recent studies have identified at least 20 different kidney cell types based upon chromatin structure and gene expression. Histone deacetylases (HDACs) are epigenetic transcriptional repressors via deacetylation of histone lysines resulting in inaccessible chromatin. We reported that kidney epithelial HDAC1 and HDAC2 activity is critical for maintaining a healthy kidney and preventing fluid-electrolyte abnormalities. However, to what extent does Hdac1/Hdac2 knockdown affect chromatin structure and subsequent transcript expression in the kidney? To answer this question, we used single nucleus assay for transposase-accessible chromatin-sequencing (snATAC-seq) and snRNA-seq to profile kidney nuclei from male and female, control, and littermate kidney epithelial Hdac1/Hdac2 knockdown mice. Hdac1/Hdac2 knockdown resulted in significant changes in the chromatin structure predominantly within the promoter region of gene loci involved in fluid-electrolyte balance such as the aquaporins, with both increased and decreased accessibility captured. Moreover, Hdac1/Hdac2 knockdown resulted different gene loci being accessible with a corresponding increased transcript number in the kidney, but among all mice only 24%-30% of chromatin accessibility agreed with transcript expression (e.g., open chromatin and increased transcript). To conclude, although chromatin structure does affect transcription, ∼70% of the differentially expressed genes cannot be explained by changes in chromatin accessibility and HDAC1/HDAC2 had a minimal effect on these global patterns. Yet, the genes that are targets of HDAC1 and HDAC2 are critically important for maintaining kidney function.

Chromatin accessibility regulates chemotherapy-induced dormancy and reactivation

Cisplatin-based chemotherapy remains the standard care for non-small cell lung cancer (NSCLC) patients. Relapse after chemotherapy-induced dormancy affects the overall survival of patients. The evolution of cancer cells under chemotherapy stress is regulated by transcription factors (TFs) with binding sites initially buried deep within inaccessible chromatin. The transcription machinery and dynamic epigenetic alterations during the process of dormancy-reactivation of lung cancer cells after chemotherapy need to be investigated. Here, we investigated the chromatin accessibility of lung cancer cells after cisplatin treatment, using an assay for transposase-accessible chromatin sequencing (ATAC-seq). We observed that global chromatin accessibility was extensively improved. Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining (TRRUST) v.2 was used to elucidate TF-target interaction during the process of dormancy and reactivation. Enhancer regions and motifs specific to key TFs including JUN, MYC, SMAD3, E2F1, SP1, CTCF, SMAD4, STAT3, NFKB1, and KLF4 were enriched in differential loci ATAC-seq peaks of dormant and reactivated cancer cells induced by chemotherapy. The findings suggest that these key TFs regulated gene expressions during the process of dormancy and reactivation of cancer cells through altering promoter accessibility of target genes. Our study helps advance understanding of how cancer cells adapt to the stress induced by chemotherapy through TF binding motif accessibility.