Lineage-determining transcription factor-driven promoters regulate cell type-specific macrophage gene expression.

Mammalian promoters consist of multifarious elements, which make them unique and support the selection of the proper transcript variants required under diverse conditions in distinct cell types. However, their direct DNA-transcription factor (TF) interactions are mostly unidentified. Murine bone marrow-derived macrophages (BMDMs) are a widely used model for studying gene expression regulation. Thus, this model serves as a rich source of various next-generation sequencing data sets, including a large number of TF cistromes. By processing and integrating the available cistromic, epigenomic and transcriptomic data from BMDMs, we characterized the macrophage-specific direct DNA-TF interactions, with a particular emphasis on those specific for promoters. Whilst active promoters are enriched for certain types of typically methylatable elements, more than half of them contain non-methylatable and prototypically promoter-distal elements. In addition, circa 14% of promoters-including that of Csf1r-are composed exclusively of 'distal' elements that provide cell type-specific gene regulation by specialized TFs. Similar to CG-rich promoters, these also contain methylatable CG sites that are demethylated in a significant portion and show high polymerase activity. We conclude that this unusual class of promoters regulates cell type-specific gene expression in macrophages, and such a mechanism might exist in other cell types too.

Fat Distribution in Women Is Associated With Depot-Specific Transcriptomic Signatures and Chromatin Structure.

BACKGROUND: Preferential accumulation of fat in the upper body (apple shape) is associated with higher risk of developing metabolic syndrome relative to lower body fat (pear shape). We previously discovered that chromatin openness partially defined the transcriptome of preadipocytes isolated from abdominal and gluteofemoral fat. However, the molecular mechanisms underlying interindividual variation in body shape are unknown. METHODS: Adipocyte fraction was isolated from abdominal and gluteofemoral fat biopsies of premenopausal women (age and body mass index matched) segregated initially only by their waist-to-hip ratio. We evaluated transcriptomic and chromatin accessibility using RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq) along with key clinical parameters. RESULTS: Our data showed that higher lower body fat mass was associated with better lipid profile and free fatty acid decrease after glucose administration. Lipid and glucose metabolic pathways genes were expressed at higher levels in gluteofemoral adipocyte fraction in pears, whereas genes associated with inflammation were higher both in abdominal and gluteofemoral apple adipocyte fraction. Gluteofemoral adipocyte chromatin from pear-shaped women contained a significantly higher number of differentially open ATAC-seq peaks relative to chromatin from the apple-shaped gluteofemoral adipocytes. In contrast, abdominal adipocyte chromatin openness showed few differences between apple- and pear-shaped women. We revealed a correlation between gene transcription and open chromatin at the proximity of the transcriptional start site of some of the differentially expressed genes. CONCLUSIONS: Integration of data from all 3 approaches suggests that chromatin openness partially governs the transcriptome of gluteofemoral adipocytes and may be involved in the early metabolic syndrome predisposition associated with body shape.

Agonist binding directs dynamic competition among nuclear receptors for heterodimerization with retinoid X receptor.

Retinoid X receptor (RXR) plays a pivotal role as a transcriptional regulator and serves as an obligatory heterodimerization partner for at least 20 other nuclear receptors (NRs). Given a potentially limiting/sequestered pool of RXR and simultaneous expression of several RXR partners, we hypothesized that NRs compete for binding to RXR and that this competition is directed by specific agonist treatment. Here, we tested this hypothesis on three NRs: peroxisome proliferator-activated receptor gamma (PPARγ), vitamin D receptor (VDR), and retinoic acid receptor alpha (RARα). The evaluation of competition relied on a nuclear translocation assay applied in a three-color imaging model system by detecting changes in heterodimerization between RXRα and one of its partners (NR1) in the presence of another competing partner (NR2). Our results indicated dynamic competition between the NRs governed by two mechanisms. First, in the absence of agonist treatment, there is a hierarchy of affinities between RXRα and its partners in the following order: RARα > PPARγ > VDR. Second, upon agonist treatment, RXRα favors the liganded partner. We conclude that recruiting RXRα by the liganded NR not only facilitates a stimulus-specific cellular response but also might impede other NR pathways involving RXRα.

Alternatively Constructed Estrogen Receptor Alpha-Driven Super-Enhancers Result in Similar Gene Expression in Breast and Endometrial Cell Lines.

Super-enhancers (SEs) are clusters of highly active enhancers, regulating cell type-specific and disease-related genes, including oncogenes. The individual regulatory regions within SEs might be simultaneously bound by different transcription factors (TFs) and co-regulators, which together establish a chromatin environment conducting to effective transcription. While cells with distinct TF profiles can have different functions, how different cells control overlapping genetic programs remains a question. In this paper, we show that the construction of estrogen receptor alpha-driven SEs is tissue-specific, both collaborating TFs and the active SE components greatly differ between human breast cancer-derived MCF-7 and endometrial cancer-derived Ishikawa cells; nonetheless, SEs common to both cell lines have similar transcriptional outputs. These results delineate that despite the existence of a combinatorial code allowing alternative SE construction, a single master regulator might be able to determine the overall activity of SEs.

Classification of different types of estrogen receptor alpha binding sites in MCF-7 cells.

Estrogen Receptor alpha (ERα) is a ligand-activated transcription factor and it has a prominent role in both physiological and pathological processes of the reproductive system. ERα has been investigated extensively in breast cancer and the MCF-7 breast-cancer-derived cell line is a widely used model for the study of its behavior. In this paper we provide a systematic catalog of the possible scenarios of binding to more than 80,000 ERα transcription factor binding sites based on the mechanism of ERα binding to DNA (upon both vehicle and estradiol (E2) treatment). A key feature of the estrogen-driven genetic programs is the presence or absence of the specific response element referred to as the estrogen response element (ERE). While ERα-driven super-enhancers are key components of estrogen-dependent genetic programs, three additional classes of enhancers could be identified: one with the presence of ERE where the ERα bound to the DNA prior of E2-treatment, one where the E2 was required for ERα binding even in the presence of ERE, and one where the ERα binding is established through the response elements of the collaborating factors. Our results suggest that different scenarios of ERα binding result in different genetic programs.

Differential open chromatin profile and transcriptomic signature define depot-specific human subcutaneous preadipocytes: primary outcomes.

BACKGROUND: Increased lower body fat is associated with reduced cardiometabolic risk. The molecular basis for depot-specific differences in gluteofemoral (GF) compared with abdominal (A) subcutaneous adipocyte function is poorly understood. In the current report, we used a combination of Assay for Transposase-Accessible Chromatin followed by sequencing (ATAC-seq), RNA-seq, and chromatin immunoprecipitation (ChIP)-qPCR analyses that provide evidence that depot-specific gene expression patterns are associated with differential epigenetic chromatin signatures. METHODS: Preadipocytes cultured from A and GF adipose tissue obtained from premenopausal apple-shaped women were used to perform transcriptome analysis by RNA-seq and assess accessible chromatin regions by ATAC-seq. We measured mRNA expression and performed ChIP-qPCR experiments for histone modifications of active (H3K4me3) and repressed chromatin (H3K27me3) regions respectively on the promoter regions of differentially expressed genes. RESULTS: RNA-seq experiments revealed an A-fat and GF-fat selective gene expression signature, with 126 genes upregulated in abdominal preadipocytes and 90 genes upregulated in GF cells. ATAC-seq identified almost 10-times more A-specific chromatin-accessible regions. Using a combined analysis of ATAC-seq and global gene expression data, we identified 74 of the 126 abdominal-specific genes (59%) with A-specific accessible chromatin sites within 200 kb of the transcription start site (TSS), including HOXA3, HOXA5, IL8, IL1b, and IL6. Interestingly, only 14 of the 90 GF-specific genes (15%) had GF-specific accessible chromatin sites within 200 kb of the corresponding TSS, including HOXC13 and HOTAIR, whereas 25 of them (28%) had abdominal-specific accessible chromatin sites. ChIP-qPCR experiments confirmed that the active H3K4me3 chromatin mark was significantly enriched at the promoter regions of HOXA5 and HOXA3 genes in abdominal preadipocytes, while H3K27me3 was less abundant relative to chromatin from GF. This is consistent with their A-fat specific gene expression pattern. Conversely, analysis of the promoter regions of the GF specific HOTAIR and HOXC13 genes exhibited high H3K4me3 and low H3K27me3 levels in GF chromatin compared to A chromatin. CONCLUSIONS: Global transcriptome and open chromatin analyses of depot-specific preadipocytes identified their gene expression signature and differential open chromatin profile. Interestingly, A-fat-specific open chromatin regions can be observed in the proximity of GF-fat genes, but not vice versa. TRIAL REGISTRATION: Clinicaltrials.gov, NCT01745471 . Registered 5 December 2012.

Poly(ADP-ribose) polymerase-2 is a lipid-modulated modulator of muscular lipid homeostasis.

There is a growing body of evidence that poly(ADP-ribose) polymerase-2 (PARP2), although originally described as a DNA repair protein, has a widespread role as a metabolic regulator. We show that the ablation of PARP2 induced characteristic changes in the lipidome. The silencing of PARP2 induced the expression of sterol regulatory element-binding protein-1 and -2 and initiated de novo cholesterol biosynthesis in skeletal muscle. Increased muscular cholesterol was shunted to muscular biosynthesis of dihydrotestosterone, an anabolic steroid. Thus, skeletal muscle fibers in PARP2-/- mice were stronger compared to those of their wild-type littermates. In addition, we detected changes in the dynamics of the cell membrane, suggesting that lipidome changes also affect the biophysical characteristics of the cell membrane. In in silico and wet chemistry studies, we identified lipid species that can decrease the expression of PARP2 and potentially phenocopy the genetic abruption of PARP2, including artificial steroids. In view of these observations, we propose a new role for PARP2 as a lipid-modulated regulator of lipid metabolism.

The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2.

Hepatocyte nuclear factor 4 alpha (HNF4α) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4α is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4α. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4α. However, based on our previous results we hypothesized that HNF4α is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4α in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4α leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4α. Accordingly, we have observed decreasing but not disappearing binding of HNF4α to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4α chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4α-dependent hepatic gene expression.

Inducible super-enhancers are organized based on canonical signal-specific transcription factor binding elements.

Super-enhancers are established through the interactions of several enhancers and a large number of proteins, including transcription factors and co-regulators; however, the formation of these interactions is poorly understood. By re-analysing previously published estrogen receptor alpha (ERα) ChIP-seq data sets derived from the MCF-7 cell line, we observed that in the absence of stimulation, future super-enhancers are represented by one or a few transcription factor binding event(s) and these extraordinary enhancers possess a response element largely specific to the ERα dimer. Upon hormonal stimulation, these primary binding sites are surrounded by a large amount of ERα and the critical components of active enhancers, such as P300 and MED1, and together with neighbouring sites bound by newly recruited ERα, they generate the functional super-enhancers. To further validate the role of canonical elements in super-enhancer formation, we investigated some additional signal-dependent transcription factors, confirming that certain, distinguished binding elements have a general organizer function. These results suggest that certain signal-specific transcription factors guide super-enhancer formation upon binding to strong response elements. These findings may reshape the current understanding of how these regulatory units assemble, highlighting the involvement of DNA elements instead of protein-protein interactions.

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.