Significance Methylation of cytosine bases in DNA is an epigenetic modification that influences gene expression. TET (Ten-Eleven Translocation) enzymes regulate DNA methylation status and facilitate DNA demethylation by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products in mammalian genomes. Of the three mammalian TET proteins, Tet1 and Tet2 are the major regulators of 5hmC levels in mouse embryonic stem (ES) cells. We show that Tet1 and Tet2 have distinct roles in mouse ES cells: Tet1 primarily regulates 5hmC levels at gene promoters and transcription start sites, whereas Tet2 mainly regulates 5hmC levels in gene bodies and exon boundaries of highly-expressed genes and exons respectively. Our results suggest a complex interplay between the functions of Tet1 and Tet2 proteins in mESC.
Oct1 and Oct4 are homologous transcription factors with similar DNA-binding specificities. Here we show that Oct1 is dynamically phosphorylated in vivo following exposure of cells to oxidative and genotoxic stress. We further show that stress regulates the selectivity of both proteins for specific DNA sequences. Mutation of conserved phosphorylation target DNA-binding domain residues in Oct1, and Oct4 confirms their role in regulating binding selectivity. Using chromatin immunoprecipitation, we show that association of Oct4 and Oct1 with a distinct group of in vivo targets is inducible by stress, and that Oct1 is essential for a normal post-stress transcriptional response. Finally, using an unbiased Oct1 target screen we identify a large number of genes targeted by Oct1 specifically under conditions of stress, and show that several of these inducible Oct1 targets are also inducibly bound by Oct4 in embryonic stem cells following stress exposure. Oct1 and Oct4 (products of the Pou2f1 and Pou5f1 genes) are members of the POU (Pit-1, Oct1/2, Unc-86) domain transcription factor family (Herr et al. 1988;Ryan and Rosenfeld 1997). This family is defined by the presence of a bipartite DNA-binding domain in which two subdomains, covalently connected by a flexible linker, typically recognize DNA through major groove interactions on opposite sides of the helix (Klemm et al. 1994). The classical DNA recognition sequence is known as an octamer motif (59-ATGCAAAT-39, hereafter called a ''simple'' octamer). However, we demonstrated recently that native binding sites for Oct4 frequently exist in complex paired, overlapping, and nonconsensus configurations (Tantin et al. 2008).Oct4 is a master regulator of the stem cell state and has recently been shown to be one of three proteins sufficient to reprogram differentiated adult mouse and human cells to the embryonic stem (ES) cell lineage (Okita et al. 2007;Takahashi et al. 2007;Nakagawa et al. 2008). The biological function of Oct1 is more enigmatic. Oct1 is known to interact with regulatory sites in interleukin, immunoglobulin, and histone genes (Garrity et al. 1994;Zheng et al. 2003;Ushmorov et al. 2004;Murayama et al. 2006). Oct1 also moderately stimulates gene expression reporter constructs linked to target sequences in transient transfection assays (Sive et al. 1986;LeBowitz et al. 1988). However, we showed that Oct1 is nonessential for native H2B, IgH, and Igk expression (V.E. V.E.H. Wang et al. 2004). Oct1-deficient cells appear morphologically normal in light microscopy and divide at normal rates. Oct1-deficient mice die in mid-late gestation (embryonic days 12,5-18.5 [E12.5-E18.5]) (V.E.H. .We determined previously that Oct1 À/À mouse embryonic fibroblasts (MEFs) are hypersensitive to oxidative and genotoxic stress (Tantin et al. 2005). One explanation for this result is that constitutive products of Oct1-mediated transcription participate in stress response pathways. Support for an alternative hypothesis, namely that Oct1 directly senses cellular stress, comes from the f...
Little is known regarding how the Oct1 transcription factor regulates target gene expression. Using murine fibroblasts and two target genes, Polr2a and Ahcy, we show that Oct1 recruits the Jmjd1a/KDM3A lysine demethylase to catalyze the removal of the inhibitory histone H3K9 dimethyl mark and block repression. Using purified murine T cells and the Il2 target locus, and a colon cancer cell line and the Cdx2 target locus, we show that Oct1 recruits the NuRD chromatin-remodeling complex to promote a repressed state, but in a regulated manner can switch to a different capacity and mediate Jmjd1a recruitment to block repression. These findings indicate that Oct1 maintains repression through a mechanism involving NuRD and maintains poised gene expression states through an antirepression mechanism involving Jmjd1a. We propose that, rather than acting as a primary trigger of gene activation or repression, Oct1 is a switchable stabilizer of repressed and inducible states.The POU 2 (Pit-1, Oct1/2, Unc-86) transcription factor family includes ϳ13 mammalian paralogs as well as representatives from other metazoans (1). The best known example, Oct4/POU5F1, regulates embryonic stem (ES) cell identity and is a key factor used to generate induced pluripotent stem cells from somatic cells (2-5). Oct1/POU2F1 is related to Oct4 and possesses similar in vitro DNA binding specificity (for reviewed, see Ref. 6). As with many transcription factors, these proteins are known to regulate gene expression both positively and negatively (e.g. 7, 8); however, their activity has been thought to be determined by gene context and not subject to regulation.Loss of Oct1 inhibits oncogenic transformation in mouse embryonic fibroblasts (MEFs) and tumorigenicity in p53-deficient mice and xenograft assays, while having little effect on cell growth in culture or transformation by serial passage (9). One study indicates that Oct1 levels are increased in some human gastric cancers (10). In contrast, multiple studies have identified coordinate up-regulation of Oct1 target genes in lung and breast adenocarcinomas, leukemias, and myeloid leukemia stem cells, without concurrent up-regulation of Oct1 itself (11)(12)(13)(14), suggesting that Oct1 activity may be deregulated in malignancy. Recent findings showing post-translational regulation of Oct1 support this possibility (15). Although Oct1 has been studied intensively, our current understanding of how it regulates gene transcription is surprisingly limited (see for example, Ref. 16).Here, we show using three different systems (fibroblasts, primary T cells, and a colon cancer cell line) that Oct1 is a bipotential and switchable transcriptional regulator. In fibroblasts, Oct1 mediates recruitment of the Jmjd1a histone demethylase to target genes (Polr2a and Ahcy) following oxidative stress exposure. In the absence of Oct1, Jmjd1a fails to be recruited, H3K9me2 levels are elevated, and inappropriate repression is observed. In contrast, Oct1 recruits the nucleosome remodeling and histone deacetylation (NuRD/Mi...
Dioxygenases of the TET (Ten-Eleven Translocation) family produce oxidized methylcytosines, intermediates in DNA demethylation, as well as new epigenetic marks. Here we show data suggesting that TET proteins maintain the consistency of gene transcription. Embryos lacking Tet1 and Tet3 (Tet1/3 DKO) displayed a strong loss of 5-hydroxymethylcytosine (5hmC) and a concurrent increase in 5-methylcytosine (5mC) at the eight-cell stage. Single cells from eight-cell embryos and individual embryonic day 3.5 blastocysts showed unexpectedly variable gene expression compared with controls, and this variability correlated in blastocysts with variably increased 5mC/5hmC in gene bodies and repetitive elements. Despite the variability, genes encoding regulators of cholesterol biosynthesis were reproducibly down-regulated in Tet1/3 DKO blastocysts, resulting in a characteristic phenotype of holoprosencephaly in the few embryos that survived to later stages. Thus, TET enzymes and DNA cytosine modifications could directly or indirectly modulate transcriptional noise, resulting in the selective susceptibility of certain intracellular pathways to regulation by TET proteins.DNA methylation | cholesterol biosynthesis | TET methylcytosine oxidases | 5-hydroxymethylcytosine | 5hmC
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