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.

Disrupting the DREAM transcriptional repressor complex induces apolipoprotein overexpression and systemic amyloidosis in mice.

DREAM (Dp, Rb-like, E2F, and MuvB) is a transcriptional repressor complex that regulates cell proliferation, and its loss causes neonatal lethality in mice. To investigate DREAM function in adult mice, we used an assembly-defective p107 protein and conditional deletion of its redundant family member p130. In the absence of DREAM assembly, mice displayed shortened survival characterized by systemic amyloidosis but no evidence of excessive cellular proliferation. Amyloid deposits were found in the heart, liver, spleen, and kidneys but not the brain or bone marrow. Using laser-capture microdissection followed by mass spectrometry, we identified apolipoproteins as the most abundant components of amyloids. Intriguingly, apoA-IV was the most detected amyloidogenic protein in amyloid deposits, suggesting apoA-IV amyloidosis (AApoAIV). AApoAIV is a recently described form, whereby WT apoA-IV has been shown to predominate in amyloid plaques. We determined by ChIP that DREAM directly regulated Apoa4 and that the histone variant H2AZ was reduced from the Apoa4 gene body in DREAM's absence, leading to overexpression. Collectively, we describe a mechanism by which epigenetic misregulation causes apolipoprotein overexpression and amyloidosis, potentially explaining the origins of nongenetic amyloid subtypes.

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.

Isoform-specific promotion of breast cancer tumorigenicity by TBX3 involves induction of angiogenesis.

TBX3 is a member of the highly conserved family of T-box transcription factors involved in embryogenesis, organogenesis and tumor progression. While the functional role of TBX3 in tumorigenesis has been widely studied, less is known about the specific functions of the different isoforms (TBX3iso1 and TBX3iso2) which differ in their DNA-binding domain. We therefore sought to investigate the functional consequence of this highly conserved splice event as it relates to TBX3-induced tumorigenesis. By utilizing a nude mouse xenograft model, we have identified differential tumorigenic potential between TBX3 isoforms, with TBX3iso1 overexpression more commonly associated with invasive carcinoma and high tumor vascularity. Transcriptional analysis of signaling pathways altered by TBX3iso1 and TBX3iso2 overexpression revealed significant differences in angiogenesis-related genes. Importantly, osteopontin (OPN), a cancer-associated secreted phosphoprotein, was significantly up-regulated with TBX3iso1 (but not TBX3iso2) overexpression. This pattern was observed across three non/weakly-tumorigenic breast cancer cell lines (21PT, 21NT, and MCF7). Up-regulation of OPN in TBX3iso1 overexpressing cells was associated with induction of hyaluronan synthase 2 (HAS2) expression and increased retention of hyaluronan in pericellular matrices. These transcriptional changes were accompanied by the ability to induce endothelial cell vascular channel formation by conditioned media in vitro, which could be inhibited through addition of an OPN neutralizing antibody. Within the TCGA breast cancer cohort, we identified an 8.1-fold higher TBX3iso1 to TBX3iso2 transcript ratio in tumors relative to control, and this ratio was positively associated with high-tumor grade and an aggressive molecular subtype. Collectively, the described changes involving TBX3iso1-dependent promotion of angiogenesis may thus serve as an adaptive mechanism within breast cancer cells, potentially explaining differences in tumor formation rates between TBX3 isoforms in vivo. This study is the first of its kind to report significant functional differences between the two TBX3 isoforms, both in vitro and in vivo.

Mapping Retinoic Acid-Dependant 5mC Derivatives in Mouse Embryonic Fibroblasts.

Methylase-assisted bisulfite sequencing (MAB-seq) is a derivatization technique to evaluate the presence of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) at base-pair resolution. Although MAB-seq was originally designed to study these metabolites under steady-state conditions, we have developed an alternative protocol to evaluate the dynamics of 5-fC/5-caC accumulation in response to agonists, such as all-trans retinoic acid (ATRA). In addition, this protocol utilizes a lower quantity of the M.SssI enzyme without compromising methylation efficiency and requires less bench time. Herein, we describe the use of MAB-seq assay to evaluate the generation of 5-fC/5-caC in response to ATRA in mouse embryonic fibroblasts, using the hypermethylated in cancer 1 (Hic1) locus as a model system.

TBX3 promotes progression of pre-invasive breast cancer cells by inducing EMT and directly up-regulating SLUG.

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high-resolution, isoform-specific context, we conducted genome-wide chromatin-immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial-mesenchymal transition (EMT)-related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3-induced migration and invasion. Assessing TBX3 by immunohistochemistry in early-stage (stage 0 and stage I) breast cancers revealed high expression in low-grade lesions. Within a second independent early-stage non-high-grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early-stage pre-invasive breast cancer to invasive carcinoma through the low-grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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.

Regulation of B cell linker protein transcription by PU.1 and Spi-B in murine B cell acute lymphoblastic leukemia.

B cell acute lymphoblastic leukemia (B-ALL) is frequently associated with mutations or chromosomal translocations of genes encoding transcription factors. Conditional deletion of genes encoding the E26-transformation-specific transcription factors, PU.1 and Spi-B, in B cells (ΔPB mice) leads to B-ALL in mice at 100% incidence rate and with a median survival of 21 wk. We hypothesized that PU.1 and Spi-B may redundantly activate transcription of genes encoding tumor suppressors in the B cell lineage. Characterization of aging ΔPB mice showed that leukemia cells expressing IL-7R were found in enlarged thymuses. IL-7R-expressing B-ALL cells grew in culture in response to IL-7 and could be maintained as cell lines. Cultured ΔPB cells expressed reduced levels of B cell linker protein (BLNK), a known tumor suppressor gene, compared with controls. The Blnk promoter contained a predicted PU.1 and/or Spi-B binding site that was required for promoter activity and occupied by PU.1 and/or Spi-B as determined by chromatin immunoprecipitation. Restoration of BLNK expression in cultured ΔPB cells opposed IL-7-dependent proliferation and induced early apoptosis. We conclude that the tumor suppressor BLNK is a target of transcriptional activation by PU.1 and Spi-B in the B cell lineage.

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.

Regulation of follicular B cell differentiation by the related E26 transformation-specific transcription factors PU.1, Spi-B, and Spi-C.

Splenic B-2 cells can be divided into two major subsets: follicular (FO) and marginal zone (MZ) B cells. FO and MZ B cells are generated from immature transitional B cells. Few transcription factors have been identified that regulate FO B cell differentiation. The highly related proteins PU.1, Spi-B, and Spi-C are transcription factors of the E26-transformation-specific family and are important for B cell differentiation and function. To determine whether these proteins play a role in the differentiation of FO B cells, we performed a detailed analysis of splenic B cells in mice with inactivating mutations in the genes encoding PU.1 (Sfpi1) or Spi-B (Spib). Sfpi1(+/-) Spib(-/-) (PUB) mice had a 9-fold reduction in the frequency of CD23(+) FO B cells compared with that of wild-type mice. In contrast, PUB mice had a 2-fold increase in the frequency of MZ B cells that was confirmed by immunofluorescence staining. Expression of Spi-C in Eµ-Spi-C transgenic PUB mice partially rescued frequencies of CD23(+) B cells. Gene expression analysis, in vitro reporter assays, and chromatin immunoprecipitation experiments showed that transcription of the Fcer2a gene encoding CD23 is activated by PU.1, Spi-B, and Spi-C. These results demonstrate that FO B cell differentiation is regulated by the E26-transformation-specific transcription factors PU.1, Spi-B, and Spi-C.

Polycomb-like 2 associates with PRC2 and regulates transcriptional networks during mouse embryonic stem cell self-renewal and differentiation.

Polycomb group (PcG) proteins are conserved epigenetic transcriptional repressors that control numerous developmental gene expression programs and have recently been implicated in modulating embryonic stem cell (ESC) fate. We identified the PcG protein PCL2 (polycomb-like 2) in a genome-wide screen for regulators of self-renewal and pluripotency and predicted that it would play an important role in mouse ESC-fate determination. Using multiple biochemical strategies, we provide evidence that PCL2 is a Polycomb Repressive Complex 2 (PRC2)-associated protein in mouse ESCs. Knockdown of Pcl2 in ESCs resulted in heightened self-renewal characteristics, defects in differentiation, and altered patterns of histone methylation. Integration of global gene expression and promoter occupancy analyses allowed us to identify PCL2 and PRC2 transcriptional targets and draft regulatory networks. We describe the role of PCL2 in both modulating transcription of ESC self-renewal genes in undifferentiated ESCs as well as developmental regulators during early commitment and differentiation.

Opposing regulatory roles of phosphorylation and acetylation in DNA mispair processing by thymine DNA glycosylase.

CpG dinucleotides are mutational hotspots associated with cancer and genetic diseases. Thymine DNA glycosylase (TDG) plays an integral role in CpG maintenance by excising mispaired thymine and uracil in a CpG context and also participates in transcriptional regulation via gene-specific CpG demethylation and functional interactions with the transcription machinery. Here, we report that protein kinase C alpha (PKCalpha) interacts with TDG and phosphorylates amino-terminal serine residues adjacent to lysines acetylated by CREB-binding protein (CBP) and p300 (CBP/p300). We establish that acetylation and phosphorylation are mutually exclusive, and their interplay dramatically alters the DNA mispair-processing functions of TDG. Remarkably, acetylation of the amino-terminal region abrogates high-affinity DNA binding and selectively prevents processing of G:T mispairs. In contrast, phosphorylation does not markedly alter DNA interactions, but may preserve G:T processing in vivo by preventing CBP-mediated acetylation. Mutational analysis suggests that the acetyl-acceptor lysines are not directly involved in contacting DNA, but may constitute a conformationally sensitive interface that modulates DNA interactions. These findings reveal opposing roles of CBP/p300 and PKCalpha in regulating the DNA repair functions of TDG and suggest that the interplay of these modifications in vivo may be critically important in the maintenance of CpG dinucleotides and epigenetic regulation.

Biochemical analysis of arginine methylation in transcription.

Protein arginine methylation has emerged as an important mechanism for regulating the functions of proteins involved in diverse aspects of gene regulation such as transcriptional activation and repression, mRNA processing and nuclear-cytoplasmic shuttling. This modification is catalyzed by the PRMT family of enzymes which utilize intracellular S-adenosyl methionine as a cofactor to dimethylate-specific arginines found within many target proteins.The establishment of in vitro biochemical assays as well as the development of modification-specific antibodies, and more recently mass spectrometry, have increased our understanding of the mechanism of catalysis of the PRMT family of enzymes. In the following discussion, we present some of the more commonly used in vivo and in vitro techniques which can be utilized to study the mechanism of arginine methylation and its role in transcription.

Transcriptional control by adenovirus E1A conserved region 3 via p300/CBP.

The human adenovirus type 5 (HAdV-5) E1A 13S oncoprotein is a potent regulator of gene expression and is used extensively as a model for transcriptional activation. It possesses two independent transcriptional activation domains located in the N-terminus/conserved region (CR) 1 and CR3. The protein acetyltransferase p300 was previously identified by its association with the N-terminus/CR1 portion of E1A and this association is required for oncogenic transformation by E1A. We report here that transcriptional activation by 13S E1A is inhibited by co-expression of sub-stoichiometric amounts of the smaller 12S E1A isoform, which lacks CR3. Transcriptional inhibition by E1A 12S maps to the N-terminus and correlates with the ability to bind p300/CBP, suggesting that E1A 12S is sequestering this limiting factor from 13S E1A. This is supported by the observation that the repressive effect of E1A 12S is reversed by expression of exogenous p300 or CBP, but not by a CBP mutant lacking actyltransferase activity. Furthermore, we show that transcriptional activation by 13S E1A is greatly reduced by siRNA knockdown of p300 and that CR3 binds p300 independently of the well-characterized N-terminal/CR1-binding site. Importantly, CR3 is also required to recruit p300 to the adenovirus E4 promoter during infection. These results identify a new functionally significant interaction between E1A CR3 and the p300/CBP acetyltransferases, expanding our understanding of the mechanism by which this potent transcriptional activator functions.

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.

Identification of a second CtBP binding site in adenovirus type 5 E1A conserved region 3.

C-terminal binding protein (CtBP) binds to adenovirus early region 1A (AdE1A) through a highly conserved PXDLS motif close to the C terminus. We now have demonstrated that CtBP1 also interacts directly with the transcriptional activation domain (conserved region 3 [CR3]) of adenovirus type 5 E1A (Ad5E1A) and requires the integrity of the entire CR3 region for optimal binding. The interaction appears to be at least partially mediated through a sequence ((161)RRNTGDP(167)) very similar to a recently characterized novel CtBP binding motif in ZNF217 as well as other regions of CR3. Using reporter assays, we further demonstrated that CtBP1 represses Ad5E1A CR3-dependent transcriptional activation. Ad5E1A also appears to be recruited to the E-cadherin promoter through its interaction with CtBP. Significantly, Ad5E1A, CtBP1, and ZNF217 form a stable complex which requires CR3 and the PLDLS motif. It has been shown that Ad513SE1A, containing the CR3 region, is able to overcome the transcriptional repressor activity of a ZNF217 polypeptide fragment in a GAL4 reporter assay through recruitment of CtBP1. These results suggest a hitherto-unsuspected complexity in the association of Ad5E1A with CtBP, with the interaction resulting in transcriptional activation by recruitment of CR3-bound factors to CtBP1-containing complexes.