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 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.
Transcriptional repression by nuclear receptors has been correlated to binding of the putative co-repressor, N-CoR. A complex has been identified that contains N-CoR, the Mad presumptive co-repressor ...mSin3, and the histone deacetylase mRPD3, and which is required for both nuclear receptor- and Mad-dependent repression, but not for repression by transcription factors of the ets-domain family. These data predict that the ligand-induced switch of heterodimeric nuclear receptors from repressor to activator functions involves the exchange of complexes containing histone deacetylases with those that have histone acetylase activity.
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.
Different classes of mammalian transcription factors-nuclear receptors, cyclic adenosine 3′,5′-monophosphate-regulated enhancer binding protein (CREB), and signal transducer and activator of ...transcription-1 (STAT-1)-functionally require distinct components of the coactivator complex, including CREB-binding protein (CBP/p300), nuclear receptor coactivators (NCoAs), and p300/CBP-associated factor (p/CAF), based on their platform or assembly properties. Retinoic acid receptor, CREB, and STAT-1 also require different histone acetyltransferase (HAT) activities to activate transcription. Thus, transcription factor-specific differences in configuration and content of the coactivator complex dictate requirements for specific acetyltransferase activities, providing an explanation, at least in part, for the presence of multiple HAT components of the complex.
In this study we examine the mechanisms of dynamic DNA methylation of the p15ink4b tumor suppressor gene. Using conventional ChIP and ChiPseq, we identify the p15ink4b promoter as a target for the ...ZNF217 oncogene, the CoREST complex, and DNMT3A. Treatment of cells with TGF-I2 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-I2-dependent demethylation of p15ink4b. DNA immunoprecipitation of 5mC and 5hmC indicates that 5mC undergoes conversion to 5hmC prior to activation of p15ink4b. Remarkably, overexpression of ZNF217 inhibits active demethylation and expression of the p15ink4b gene by preventing recruitment of SMAD2/3 and TDG. These findings suggest that active demethylation is essential for regulating a subset of TGF-I2-dependent genes. Importantly, disruption of active demethylation by the ZNF217 oncogene may be a paradigm for other oncogenic signals on DNA methylation dynamics.
In this study we examine the mechanisms of dynamic DNA methylation of the p15ink4b tumor suppressor gene. Using conventional ChIP and ChiPseq, we identify the p15ink4b promoter as a target for the ...ZNF217 oncogene, the CoREST complex, and DNMT3A. Treatment of cells with TGF-β 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-β-dependent demethylation of p15ink4b. DNA immunoprecipitation of 5mC and 5hmC indicates that 5mC undergoes conversion to 5hmC prior to activation of p15ink4b. Remarkably, overexpression of ZNF217 inhibits active demethylation and expression of the p15ink4b gene by preventing recruitment of SMAD2/3 and TDG. These findings suggest that active demethylation is essential for regulating a subset of TGF-β-dependent genes. Importantly, disruption of active demethylation by the ZNF217 oncogene may be a paradigm for other oncogenic signals on DNA methylation dynamics.
► p15ink4b gene is a target for ZNF217/CoREST and DNMT3A ► TGF-β triggers active DNA demethylation of p15ink4b ► TGF-β-dependent active demethylation requires TDG or MBD4 ► Overexpressing ZNF217 inhibits active DNA demethylation and expression of p15ink4b
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