An Atlas of Promoter Chromatin Modifications and HiChIP Regulatory Interactions in Human Subcutaneous Adipose-Derived Stem Cells.

The genome of human adipose-derived stem cells (ADSCs) from abdominal and gluteofemoral adipose tissue depots are maintained in depot-specific stable epigenetic conformations that influence cell-autonomous gene expression patterns and drive unique depot-specific functions. The traditional approach to explore tissue-specific transcriptional regulation has been to correlate differential gene expression to the nearest-neighbor linear-distance regulatory region defined by associated chromatin features including open chromatin status, histone modifications, and DNA methylation. This has provided important information; nonetheless, the approach is limited because of the known organization of eukaryotic chromatin into a topologically constrained three-dimensional network. This network positions distal regulatory elements in spatial proximity with gene promoters which are not predictable based on linear genomic distance. In this work, we capture long-range chromatin interactions using HiChIP to identify remote genomic regions that influence the differential regulation of depot-specific genes in ADSCs isolated from different adipose depots. By integrating these data with RNA-seq results and histone modifications identified by ChIP-seq, we uncovered distal regulatory elements that influence depot-specific gene expression in ADSCs. Interestingly, a subset of the HiChIP-defined chromatin loops also provide previously unknown connections between waist-to-hip ratio GWAS variants with genes that are known to significantly influence ADSC differentiation and adipocyte function.

Transcriptional Control of Subcutaneous Adipose Tissue by the Transcription Factor CTCF Modulates Heterogeneity in Fat Distribution in Women.

Determining the mechanism driving body fat distribution will provide insights into obesity-related health risks. We used functional genomics tools to profile the epigenomic landscape to help infer the differential transcriptional potential of apple- and pear-shaped women's subcutaneous adipose-derived stem cells (ADSCs). We found that CCCTC-binding factor (CTCF) expression and its chromatin binding were increased in ADSCs from pear donors compared to those from apple donors. Interestingly, the pear enriched CTCF binding sites were located predominantly at the active transcription start sites (TSSs) of genes with active histone marks and YY1 motifs and were also associated with pear enriched RNAPII binding. In contrast, apple enriched CTCF binding sites were mainly found at intergenic regions and when identified at TSS, they were enriched with the bivalent chromatin signatures. Altogether, we provide evidence that CTCF plays an important role in differential regulation of subcutaneous ADSCs gene expression and may influence the development of apple vs. pear body shape.

Extensive proteome and functional genomic profiling of variability between genetically identical human B-lymphoblastoid cells.

In life-science research isogenic B-lymphoblastoid cell lines (LCLs) are widely known and preferred for their genetic stability - they are often used for studying mutations for example, where genetic stability is crucial. We have shown previously that phenotypic variability can be observed in isogenic B-lymphoblastoid cell lines. Isogenic LCLs present well-defined phenotypic differences on various levels, for example on the gene expression level or the chromatin level. Based on our investigations, the phenotypic variability of the isogenic LCLs is accompanied by certain genetic variation too. We have developed a compendium of LCL datasets that present the phenotypic and genetic variability of five isogenic LCLs from a multiomic perspective. In this paper, we present additional datasets generated with Next Generation Sequencing techniques to provide genomic and transcriptomic profiles (WGS, RNA-seq, single cell RNA-seq), protein-DNA interactions (ChIP-seq), together with mass spectrometry and flow cytometry datasets to monitor the changes in the proteome. We are sharing these datasets with the scientific community according to the FAIR principles for further investigations.

Unique role for lncRNA HOTAIR in defining depot-specific gene expression patterns in human adipose-derived stem cells.

Accumulation of fat above the waist is an important risk factor in developing obesity-related comorbidities independently of BMI or total fat mass. Deciphering the gene regulatory programs of the adipose tissue precursor cells within upper body or abdominal (ABD) and lower body or gluteofemoral (GF) depots is important to understand their differential capacity for lipid accumulation, maturation, and disease risk. Previous studies identified the HOX transcript antisense intergenic RNA (HOTAIR) as a GF-specific lncRNA; however, its role in adipose tissue biology is still unclear. Using three different approaches (silencing of HOTAIR in GF human adipose-derived stem cells [GF hASCs], overexpression of HOTAIR in ABD hASCs, and ChIRP-seq) to localize HOTAIR binding in GF hASC chromatin, we found that HOTAIR binds and modulates expression, both positively and negatively, of genes involved in adipose tissue-specific pathways, including adipogenesis. We further demonstrate a direct interaction between HOTAIR and genes with high RNAPII binding in their gene bodies, especially at their 3' ends or transcription end sites. Computational analysis suggests HOTAIR binds preferentially to the 3' ends of genes containing predicted strong RNA-RNA interactions with HOTAIR. Together, these results reveal a unique function for HOTAIR in hASC depot-specific regulation of gene expression.

The Role of ARID1A in the Nonestrogenic Modulation of IGF-1 Signaling.

Gaining pharmacologic access to the potential of ARID1A, a tumor suppressor protein, to mediate transcriptional control over cancer gene expression is an unresolved challenge. Retinoid X receptor ligands are pleiotropic, incompletely understood tools that regulate breast epithelial cell proliferation and differentiation. We found that low-dose bexarotene (Bex) combined with the nonselective beta-blocker carvedilol (Carv) reduces proliferation of MCF10DCIS.com cells and markedly suppresses ARID1A levels. Similarly, Carv synergized with Bex in MCF-7 cells to suppress cell growth. Chromatin immunoprecipitation sequencing analysis revealed that under nonestrogenic conditions Bex + Carv alters the concerted genomic distribution of the chromatin remodeler ARID1A and acetylated histone H3K27, at sites related to insulin-like growth factor (IGF) signaling. Several distinct sites of ARID1A enrichment were identified in the IGF-1 receptor and IRS1 genes, associated with a suppression of both proteins. The knock-down of ARID1A increased IGF-1R levels, prevented IGF-1R and IRS1 suppression upon Bex + Carv, and stimulated proliferation. In vitro IGF-1 receptor neutralizing antibody suppressed cell growth, while elevated IGF-1R or IRS1 expression was associated with poor survival of patients with ER-negative breast cancer. Our study demonstrates direct impact of ARID1A redistribution on the expression and growth regulation of IGF-1-related genes, induced by repurposed clinical drugs under nonestrogenic conditions. IMPLICATIONS: This study underscores the possibility of the pharmacologic modulation of the ARID1A factor to downregulate protumorigenic IGF-1 activity in patients with postmenopausal breast cancer undergoing aromatase inhibitor treatment.

Transcriptional and DNA Methylation Signatures of Subcutaneous Adipose Tissue and Adipose-Derived Stem Cells in PCOS Women.

Polycystic ovary syndrome (PCOS) is often associated with metabolic syndrome features, including central obesity, suggesting that adipose tissue (AT) is a key organ in PCOS pathobiology. In this study, we compared both abdominal (ABD) and gluteofemoral (GF) subcutaneous AT in women with and without PCOS. ABD and GF subcutaneous ATs from PCOS and BMI/WHR-matched control women were analyzed by RT-qPCR, FACS and histology. ABD and GF adipose-derived stem cell (ASC) transcriptome and methylome were analyzed by RNA-seq and DNA methylation array. Similar to the control group with abdominal obesity, the GF AT of PCOS women showed lower expression of genes involved in lipid accumulation and angiogenesis compared to ABD depot. FACS analysis revealed an increase in preadipocytes number in both AT depots from PCOS. Further pathway analysis of RNA-seq comparisons demonstrated that the ASCs derived from PCOS are pro-inflammatory and exhibit a hypoxic signature in the ABD depot and have lower expression of adipogenic genes in GF depot. We also found a higher CpG methylation level in PCOS compared to control exclusively in GF-ASCs. Our data suggest that ASCs play an important role in the etiology of PCOS, potentially by limiting expansion of the healthy lower-body AT.

DNA Methylation as a Marker of Body Shape in Premenopausal Women.

Preferential accumulation of fat in the gluteo-femoral (GF) depot (pear shape) rather than in the abdominal (A) depot (apple shape), protects against the development of metabolic diseases but the underlying molecular mechanism is still unknown. Recent data, including our work, suggest that differential epigenetic marking is associated with regulation of genes attributed to distinct fat distribution. Here, we aimed to compare the genomic DNA methylation signatures between apple and pear-shaped premenopausal women. To investigate the contribution of upper and lower body fat, we used paired samples of A-FAT and GF-FAT, analyzed on the BeadChip Methylation Array and quantified the differentially methylated sites between the 2 groups of women. We found unique DNA methylation patterns within both fat depots that are significantly different depending on the body fat distribution. Around 60% of the body shape specific DNA methylation sites identified in adipose tissue are maintained ex vivo in cultured preadipocytes. As it has been reported before in other cell types, we found only a hand full of genes showing coordinated differential methylation and expression levels. Finally, we determined that more than 50% of the body shape specific DNA methylation sites could also be detected in whole blood derived DNA. These data reveal a strong DNA methylation program associated with adipose tissue distribution with the possibility that a simple blood test could be used as a predictive diagnostic indicator of young women who are at increased risk for progressing to the apple body shape with a higher risk of developing obesity related complications. Clinical Trial Registration:https://clinicaltrials.gov/ct2/show/NCT02728635 and https://clinicaltrials.gov/ct2/show/NCT02226640, identifiers NCT02728635 and NCT02226640.

NR2F2 Orphan Nuclear Receptor is Involved in Estrogen Receptor Alpha-Mediated Transcriptional Regulation in Luminal A Breast Cancer Cells.

Nuclear Receptor Subfamily 2 Group F Member 2 (NR2F2) is a member of the steroid/thyroid hormone receptor superfamily with a crucial role in organogenesis, angiogenesis, cardiovascular development and tumorigenesis. However, there is limited knowledge about the cistrome and transcriptome of NR2F2 in breast cancer. In this study, we mapped the regulatory mechanism by NR2F2 using functional genomic methods. To investigate the clinical significance of NR2F2 in breast cancer, The Cancer Genome Atlas (TCGA) data were used. These results show that a high NR2F2 is associated with better survival of a specific subset of patients, namely those with luminal A breast cancer. Therefore, genome-wide NR2F2 and estrogen receptor alpha (ERα) binding sites were mapped in luminal A breast cancer cells using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq), revealing that most NR2F2 overlap with ERα that are co-occupied by forkhead box A1 (FOXA1) and GATA binding protein 3 (GATA3) in active enhancer regions. NR2F2 overlaps with highly frequent ERα chromatin interactions, which are essential for the formation of ERα-bound super-enhancers. In the process of the transcriptome profiling of NR2F2-depleted breast cancer cells such differentially expressed genes have been identified that are involved in endocrine therapy resistance and are also ERα target genes. Overall, these findings demonstrate that the NR2F2 nuclear receptor has a key role in ERα-mediated transcription and it can offer a potential therapeutic target in patients with luminal A breast cancer.

COUP-TFII is a modulator of cell-type-specific genetic programs based on genomic localization maps.

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is a member of the steroid/thyroid hormone receptor superfamily, but its ligand has not yet been identified. Little is known about the role of the COUP-TFII nuclear receptor in cancer cells. In this study, we mapped the cistrome of COUP-TFII in three different cancer cells, namely breast cancer cells (MCF-7), myelogenous leukaemia cells (K562) and liver cancer cells (HepG2) using publicly available ChIP-seq data. Our results show that COUP-TFII co-localises with master transcription factors (TFs) in a cell-specific manner such as estrogen receptor alpha in MCF-7, hepatocyte nuclear factor alpha in HepG2, and GATA-binding factor in K562, while the shared, non-specific COUP-TFII binding sites are co-occupied by CTCF. We identified chromatin environments for these COUP-TFII and master TF co-bound sites together with COUP-TFII and CTCF co-bound sites. Our results show that COUP-TFII and master TF co-bound sites are marked with active enhancer specific histone modifications (H3K27ac and H3K4me1), while COUP-TFII and CTCF co-bound sites reveal active promoter specific histone marks (H3K27ac and H3K4me3). These results describe the genomic context and role of COUP-TFII in the cell-type specific transcriptional programs. Furthermore, we report that the VEGFA gene regulated by shared COUP-TFII and CTCF co-bound regulatory elements is involved in long-range looping in a cell-type-independent manner. These findings provide a genomic insight into the regulation and angiogenic role of COUP-TFII.

Development of an antibody control system using phage display.

While chromatin immunoprecipitation has become a widely-used method in the field of transcription regulation studies, serious limitations connected to the complexity and relatively little standardization of the method serve as obstacles for its use in clinical research. In this paper we introduce a method for developing bacteriophage-based controls for the better standardization of the chromatin immunoprecipitation reactions. Random phage display libraries were selected with ChIP-grade antibodies for several rounds and individual monoclonal phages were isolated. These monoclonal phages can be propagated, characterized, capillary sequenced and if needed later cloned from in-silico data. Using such control tools allows for a better characterization of the immunoprecipitation stage needed for further clinical research in the field of chromatin-immunoprecipitation-based studies.

Enhancer mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer.

The degree of intrinsic and interpatient phenotypic heterogeneity and its role in tumor evolution is poorly understood. Phenotypic drifts can be transmitted via inheritable transcriptional programs. Cell-type specific transcription is maintained through the activation of epigenetically defined regulatory regions including promoters and enhancers. Here we have annotated the epigenome of 47 primary and metastatic estrogen-receptor (ERα)-positive breast cancer clinical specimens and inferred phenotypic heterogeneity from the regulatory landscape, identifying key regulatory elements commonly shared across patients. Shared regions contain a unique set of regulatory information including the motif for transcription factor YY1. We identify YY1 as a critical determinant of ERα transcriptional activity promoting tumor growth in most luminal patients. YY1 also contributes to the expression of genes mediating resistance to endocrine treatment. Finally, we used H3K27ac levels at active enhancer elements as a surrogate of intra-tumor phenotypic heterogeneity to track the expansion and contraction of phenotypic subpopulations throughout breast cancer progression. By tracking the clonality of SLC9A3R1-positive cells, a bona fide YY1-ERα-regulated gene, we show that endocrine therapies select for phenotypic clones under-represented at diagnosis. Collectively, our data show that epigenetic mechanisms significantly contribute to phenotypic heterogeneity and evolution in systemically treated breast cancer patients.

Nuclear receptors in transgenerational epigenetic inheritance.

Nuclear Receptors are ligand-activated transcription factors that translate information about the lipid environment into specific genetic programs, a property that renders them good candidates to be mediators of rapid adaptation changes of a species. Lipid-based morphogens, endocrine hormones, fatty acids and xenobiotics might act through this class of transcription factors making them regulators able to fine-tune physiological processes. Here we review the basic concepts and current knowledge on the process whereby small molecules act through nuclear receptors and contribute to transgenerational changes. Several molecules shown to cause transgenerational changes like phthalates, BPA, nicotine, tributylin bind and activate nuclear receptors like ERs, androgen receptors, glucocorticoid receptors or PPARγ. A specific subset of observations involving nuclear receptors has focused on the effects of environmental stress or maternal behaviour on the development of transgenerational traits. While these effects do not involve environmental ligands, they change the expression levels of Estrogen and glucocorticoid receptors of the second generation and consequently initiate an altered genetic program in the second generation. In this review we summarize the available literature about the role of nuclear receptors in transgenerational inheritance.