Regulation of Chromatin Accessibility by the Farnesoid X Receptor Is Essential for Circadian and Bile Acid Homeostasis In Vivo.

The Farnesoid X Receptor (FXR) belongs to the nuclear receptor superfamily and is an essential bile acid (BA) receptor that regulates the expression of genes involved in the metabolism of BAs. FXR protects the liver from BA overload, which is a major etiology of hepatocellular carcinoma. Herein, we investigated the changes in gene expression and chromatin accessibility in hepatocytes by performing RNA-seq in combination with the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) using a novel FXR knockout mouse model (Fxrex5Δ: Nr1h4ex5Δ/ex5Δ) generated through CRISPR/Cas9. Consistent with previous Fxr knockout models, we found that Fxrex5Δ mice develop late-onset HCC associated with increased serum and hepatic BAs. FXR deletion was associated with a dramatic loss of chromatin accessibility, primarily at promoter-associated transcription factor binding sites. Importantly, several genes involved in BA biosynthesis and circadian rhythm were downregulated following loss of FXR, also displayed reduced chromatin accessibility at their promoter regions. Altogether, these findings suggest that FXR helps to maintain a transcriptionally active state by regulating chromatin accessibility through its binding and recruitment of transcription factors and coactivators.

The Role of Thymine DNA Glycosylase in Transcription, Active DNA Demethylation, and Cancer.

DNA methylation is an essential covalent modification that is required for growth and development. Once considered to be a relatively stable epigenetic mark, many studies have established that DNA methylation is dynamic. The 5-methylcytosine (5-mC) mark can be removed through active DNA demethylation in which 5-mC is converted to an unmodified cytosine through an oxidative pathway coupled to base excision repair (BER). The BER enzyme Thymine DNA Glycosylase (TDG) plays a key role in active DNA demethylation by excising intermediates of 5-mC generated by this process. TDG acts as a key player in transcriptional regulation through its interactions with various nuclear receptors and transcription factors, in addition to its involvement in classical BER and active DNA demethylation, which serve to protect the stability of the genome and epigenome, respectively. Recent animal studies have identified a connection between the loss of Tdg and the onset of tumorigenesis. In this review, we summarize the recent findings on TDG's function as a transcriptional regulator as well as the physiological relevance of TDG and active DNA demethylation in cancer.

TDG is a novel tumor suppressor of liver malignancies.

In a recent publication, we demonstrated that conditional deletion of the gene encoding thymine DNA glycosylase (TDG) leads to a late onset of hepatocellular carcinoma (HCC). TDG loss causes disruption in active DNA demethylation in the liver and dysregulation of the farnesoid X receptor and small heterodimer partner (FXR-SHP) regulatory cascade. This leads to a loss of bile acid and glucose homeostasis, which predisposes mice to HCC.

Loss of Thymine DNA Glycosylase Causes Dysregulation of Bile Acid Homeostasis and Hepatocellular Carcinoma.

Thymine DNA glycosylase (TDG) is a nuclear receptor coactivator that plays an essential role in the maintenance of epigenetic stability in cells. Here, we demonstrate that the conditional deletion of TDG in adult mice results in a male-predominant onset of hepatocellular carcinoma (HCC). TDG loss leads to a prediabetic state, as well as bile acid (BA) accumulation in the liver and serum of male mice. Consistent with these data, TDG deletion led to dysregulation of the farnesoid X receptor (FXR) and small heterodimer partner (SHP) regulatory cascade in the liver. FXR and SHP are tumor suppressors of HCC and play an essential role in BA and glucose homeostasis. These results indicate that TDG functions as a tumor suppressor of HCC by regulating a transcriptional program that protects against the development of glucose intolerance and BA accumulation in the liver.

Genome-wide analysis reveals a role for TDG in estrogen receptor-mediated enhancer RNA transcription and 3-dimensional reorganization.

BACKGROUND: The estrogen receptor (ER) is a ligand-dependant transcription factor expressed in many breast cancers and is the target of many endocrine-based cancer therapies. Genome-wide studies have shown that the ER binds to gene-specific enhancer regions in response to ß-estradiol (E2) which undergo transcription producing noncoding enhancer RNA (eRNA). While eRNAs are important for transcriptional activation of neighboring genes, the mechanism remains poorly understood. RESULTS: Using ChIP-Seq we generate a global profile of thymine DNA glycosylase (TDG), an ER coactivator that plays an essential role in DNA demethylation, in response to E2 in the MCF7 breast cancer cell line. Remarkably, we found that in response to E2 TDG localized to enhancers which also recruit ERα, RNA Pol II and other coregulators and which are marked by histone modifications indicative of active enhancers. Importantly, depletion of TDG inhibits E2-mediated transcription of eRNAs and transcription of ER-target genes. Functionally, we find that TDG both sensitizes MCF7 cells to tamoxifen-mediated cytostasis and increases migration and invasion of MCF7 cells. CONCLUSIONS: Taken together we find that TDG plays a central role in mediating transcription at a subset of enhancers and governs how MCF7 cells respond to both estrogenic and anti-estrogenic compounds and may be an effective therapeutic target.

Regulation of Active DNA Demethylation through RAR-Mediated Recruitment of a TET/TDG Complex.

Retinoic acid (RA) plays important roles in development, growth, and homeostasis through regulation of the nuclear receptors for RA (RARs). Herein, we identify Hypermethylated in Cancer 1 (Hic1) as an RA-inducible gene. HIC1 encodes a tumor suppressor, which is often silenced by promoter hypermethylation in cancer. Treatment of cells with an RAR agonist causes a rapid recruitment of an RAR/RXR complex consisting of TDG, the lysine acetyltransferase CBP, and TET 1/2 to the Hic1 promoter. Complex binding coincides with a transient accumulation of 5fC/5caC and concomitant upregulation of Hic1 expression, both of which are TDG dependent. Furthermore, conditional deletion of Tdg in vivo is associated with Hic1 silencing and DNA hypermethylation of the Hic1 promoter. These findings suggest that the catalytic and scaffolding activities of TDG are essential for RA-dependent gene expression and provide important insights into the mechanisms underlying targeting of TET-TDG complexes.

TGF-ß-dependent active demethylation and expression of the p15ink4b tumor suppressor are impaired by the ZNF217/CoREST complex.

In this study we examine the mechanisms of dynamic DNA methylation of the p15(ink4b) tumor suppressor gene. Using conventional ChIP and ChiPseq, we identify the p15(ink4b) promoter as a target for the ZNF217 oncogene, the CoREST complex, and DNMT3A. Treatment of cells with TGF-ß triggers active demethylation involving loss of ZNF217/CoREST/DNMT3A and the corecruitment of SMAD2/3, CBP, and the DNA glycosylase TDG. Knockdown of TDG, or its functional homolog MBD4, prevents TGF-ß-dependent demethylation of p15(ink4b). DNA immunoprecipitation of 5mC and 5hmC indicates that 5mC undergoes conversion to 5hmC prior to activation of p15(ink4b). Remarkably, overexpression of ZNF217 inhibits active demethylation and expression of the p15(ink4b) gene by preventing recruitment of SMAD2/3 and TDG. These findings suggest that active demethylation is essential for regulating a subset of TGF-ß-dependent genes. Importantly, disruption of active demethylation by the ZNF217 oncogene may be a paradigm for other oncogenic signals on DNA methylation dynamics.

Regulation of the BRCA1 gene by an SRC3/53BP1 complex.

BACKGROUND: Steroid Receptor coactivator 3(SRC3) is an oncogene and a member of the SRC family of nuclear receptor coactivator proteins that mediate the transcriptional effects of nuclear hormone receptors as well as other transcription factors. RESULTS: We have used protein purification and mass spectrometry to identify the 53BP1 tumour suppressor as a novel SRC3-associated protein. Copurification was demonstrated using multiple antibodies, and was not dependent on DNA damage suggesting that SRC3 is not directly involved in the DNA damage response. However using chromatin immunoprecipitation(ChIP) and siRNA knockdown, we have demonstrated that both SRC3 and 53BP1 co-occupy the same region of the BRCA1 promoter and both are required for BRCA1 expression in HeLa cells. CONCLUSIONS: Our results suggest that both 53BP1 and SRC3 have a common function that converge at the BRCA1 promoter and possibly other genes important for DNA repair and genomic stability.

Genome analysis identifies the p15ink4b tumor suppressor as a direct target of the ZNF217/CoREST complex.

The ZNF217 oncoprotein is a constituent of a core transcriptional complex that includes CoREST, histone deacetylase 1/2, lysine demethylase 1, and the C-terminal binding protein 1/2. We have combined genome-wide expression profiling and chromatin immunoprecipitation with directed selection and ligation (ChIP-DSL) to identify a subset of genes directly regulated by ZNF217. Our results establish p15(ink4b) as a direct target of the ZNF217 complex. Downregulation of ZNF217 in MCF-7 breast cancer cells resulted in a dramatic increase in p15(ink4b) expression and coincided with increases in dimethylation of H3-K4 and, surprisingly, a decrease in K9/K14-H3 acetylation. Stimulation of HaCaT cells with transforming growth factor beta (TGF-beta) resulted in a release of ZNF217 and a concomitant binding of SMAD2 to the proximal promoter, which preceded increases in ink4b protein expression. Furthermore, the changes in chromatin marks at the p15(ink4b) promoter following TGF-beta stimulation were similar to those observed following ZNF217 downregulation. Collectively, these results establish the ZNF217 complex as a novel negative regulator of the p15(ink4b) gene and may constitute an important link between amplification of ZNF217 and the loss of TGF-beta responsiveness in breast cancer.