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Oct1 Is a Switchable, Bipotential Stabilizer of Repressed and Inducible Transcriptional States

2010; Elsevier BV; Volume: 286; Issue: 1 Linguagem: Inglês

10.1074/jbc.m110.174045

1083-351X

Arvind Shakya, Jinsuk Kang, Jeffrey Chumley, Matthew A. Williams, Dean Tantin,

Histone Deacetylase Inhibitors Research

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. 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. IntroductionThe POU 2The abbreviations used are: POUPit-1, Oct1/2, Unc-86MEFmouse embryonic fibroblastNFATnuclear factor of activated T cellsNuRDnucleosome remodeling and histone deacetylationPMAphorbol 12-myristate 13-acetateqRT-PCRquantitative RT-PCRC/EBPβCCAAT-enhancer binding protein-beta. (Pit-1, Oct1/2, Unc-86) transcription factor family includes ∼13 mammalian paralogs as well as representatives from other metazoans (1Herr W. Sturm R.A. Clerc R.G. Corcoran L.M. Baltimore D. Sharp P.A. Ingraham H.A. Rosenfeld M.G. Finney M. Ruvkun G. Genes Dev. 1988; 2: 1513-1516Crossref PubMed Scopus (599) Google Scholar). 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 (2Okita K. Ichisaka T. Yamanaka S. Nature. 2007; 448: 313-317Crossref PubMed Scopus (3497) Google Scholar, 3Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14653) Google Scholar, 4Nakagawa M. Koyanagi M. Tanabe K. Takahashi K. Ichisaka T. Aoi T. Okita K. Mochiduki Y. Takizawa N. Yamanaka S. Nat. Biotechnol. 2008; 26: 101-106Crossref PubMed Scopus (2174) Google Scholar, 5Feng B. Jiang J. Kraus P. Ng J.H. Heng J.C. Chan Y.S. Yaw L.P. Zhang W. Loh Y.H. Han J. Vega V.B. Cacheux-Rataboul V. Lim B. Lufkin T. Ng H.H. Nat. Cell Biol. 2009; 11: 197-203Crossref PubMed Scopus (374) Google Scholar). Oct1/POU2F1 is related to Oct4 and possesses similar in vitro DNA binding specificity (for reviewed, see Ref. 6Kang J. Shakya A. Tantin D. Trends Biochem. Sci. 2009; 34: 491-499Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). As with many transcription factors, these proteins are known to regulate gene expression both positively and negatively (e.g. 7Boyer L.A. Lee T.I. Cole M.F. Johnstone S.E. Levine S.S. Zucker J.P. Guenther M.G. Kumar R.M. Murray H.L. Jenner R.G. Gifford D.K. Melton D.A. Jaenisch R. Young R.A. Cell. 2005; 122: 947-956Abstract Full Text Full Text PDF PubMed Scopus (3459) Google Scholar, 8dela Paz N.G. Simeonidis S. Leo C. Rose D.W. Collins T. J. Biol. Chem. 2007; 282: 8424-8434Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar); 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 (9Shakya A. Cooksey R. Cox J.E. Wang V. McClain D.A. Tantin D. Nat. Cell Biol. 2009; 11: 320-327Crossref PubMed Scopus (80) Google Scholar). One study indicates that Oct1 levels are increased in some human gastric cancers (10Almeida R. Almeida J. Shoshkes M. Mendes N. Mesquita P. Silva E. Van Seuningen I. Reis C.A. Santos-Silva F. David L. J. Pathol. 2005; 207: 396-401Crossref PubMed Scopus (54) Google Scholar). 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 (11Reymann S. Borlak J. BMC Syst. Biol. 2008; 2: 46Crossref PubMed Scopus (47) Google Scholar, 12Ben-Porath I. Thomson M.W. Carey V.J. Ge R. Bell G.W. Regev A. Weinberg R.A. Nat. Genet. 2008; 40: 499-507Crossref PubMed Scopus (1928) Google Scholar, 13Somervaille T.C. Matheny C.J. Spencer G.J. Iwasaki M. Rinn J.L. Witten D.M. Chang H.Y. Shurtleff S.A. Downing J.R. Cleary M.L. Cell Stem Cell. 2009; 4: 129-140Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 14Mattison J. Kool J. Uren A.G. de Ridder J. Wessels L. Jonkers J. Bignell G.R. Butler A. Rust A.G. Brosch M. Wilson C.H. van der Weyden L. Largaespada D.A. Stratton M.R. Futreal P.A. van Lohuizen M. Berns A. Collier L.S. Hubbard T. Adams D.J. Cancer Res. 2010; 70: 883-895Crossref PubMed Scopus (35) Google Scholar), suggesting that Oct1 activity may be deregulated in malignancy. Recent findings showing post-translational regulation of Oct1 support this possibility (15Kang J. Gemberling M. Nakamura M. Whitby F.G. Handa H. Fairbrother W.G. Tantin D. Genes Dev. 2009; 23: 208-222Crossref PubMed Scopus (92) Google Scholar). Although Oct1 has been studied intensively, our current understanding of how it regulates gene transcription is surprisingly limited (see for example, Ref. 16Bertolino E. Singh H. Mol. Cell. 2002; 10: 397-407Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar).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-2) chromatin remodeling complex to the Il2 promoter in naïve CD4 T cells to mediate gene repression. The recruitment is regulated because upon T cell activation Oct1 loses its capacity to associate with NuRD and instead is required for recruitment of Jmjd1a. Phorbol 12-myristate 13-acetate (PMA) treatment is sufficient to switch association from NuRD to Jmjd1a, despite the fact that PMA is insufficient to activate Il2. In resting but previously stimulated T cells, Oct1 is required to maintain Jmjd1a at Il2, remove histone H3K9me2 marks and protect DNA from methylation, promoting the stronger expression associated with secondary stimulation. In DLD-1 colon adenocarcinoma cells, Oct1 bound to the Cdx2 target locus is required for mutually exclusive NuRD and Jmjd1a association. PMA treatment of DLD-1 cells results in reduced Jmjd1a association and represses Cdx2 in a manner requiring Oct1. These results show that Oct1 is a bipotential factor capable of acting through opposing mechanisms to reinforce repressed or inducible states, even at the same target gene.DISCUSSIONHere, we show that Oct1 is a bipotential transcription factor capable of regulated switching between repressive and antirepressive modes. Our data are consistent with the model shown in Fig. 6. Oct1 mediates antirepression at two housekeeping targets (Polr2a and Ahcy) by enabling the recruitment of Jmjd1a and maintaining chromatin free of histone H3K9me2. At the regulated Il2 target locus promoter region, Oct1 mediates recruitment of the NuRD corepressor complex in naïve T cells and recruitment of Jmjd1a in previously activated, resting T cells. When naïve cells are stimulated, Oct1 becomes modified through mechanisms involving MAPK and ERK signaling such that it no longer recruits NuRD and instead, directly or indirectly recruits Jmjd1a. We detected a biochemical interaction between Oct1 and NuRD, but have thus far failed to detect Jmjd1a. Therefore we cannot eliminate the possibility that the association with Jmjd1a is indirect. MAPK signaling is attenuated after the T cell stimulus is removed; however, the antirepressive state is retained even after 8 days in culture without stimulation. Therefore, the modification(s) induced by MAPK signals are either stable, or some other activity (e.g. the induction of Oct1 cofactors) maintains the switched state over longer times. This state allows faster Il2 induction in rested T cells relative to naïve cells and may be constitutive at Polr2a and Ahcy, allowing gene expression to recover more rapidly in wild-type fibroblasts relative to Oct1-deficient fibroblasts following stress exposure.Oct1 also mediates recruitment of NuRD and Jmjd1a and is important for PMA-mediated gene regulation at the Cdx2 Oct1 target locus in DLD-1 cells. Also as with T cells, loss of Oct1 superficially appears to affect Cdx2 expression minimally; however, upon more careful examination Oct1 is critical for correct regulation. Several differences were also noted between the DLD-1 and T cell system. First, PMA mediates transcriptional silencing rather than transcriptional activation. Second, Oct1 is required for regulation by PMA. Third, both NuRD and Jmjd1a were detected in normally cultured cells as measured by ChIP. Sequential ChIP assays failed to detect any co-binding of the two activities, suggesting that DLD-1 cells are composed of a heterogeneous mixture of Oct1 associated with the different activities. Cumulatively, these findings suggest that Oct1 stabilizes both repressed and inducible transcriptional states. This bipotential form of transcription factor regulation has been associated with other transcription factor classes. For example, thyroid hormone receptor can switch from an activator to a repressor of the same targets in a ligand-regulated manner. This switch is mediated by exchange of coactivators and corepressors, including remodeling enzymes such as NuRD (35Xue Y. Wong J. Moreno G.T. Young M.K. Côté J. Wang W. Mol. Cell. 1998; 2: 851-861Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar, 51Collingwood T.N. Urnov F.D. Wolffe A.P. J. Mol. Endocrinol. 1999; 23: 255-275Crossref PubMed Scopus (265) Google Scholar).Chromatin-modifying complexes are central regulators of gene expression (22Jenuwein T. FEBS J. 2006; 273: 3121-3135Crossref PubMed Scopus (203) Google Scholar, 52Denslow S.A. Wade P.A. Oncogene. 2007; 26: 5433-5438Crossref PubMed Scopus (351) Google Scholar, 53Reik W. Nature. 2007; 447: 425-432Crossref PubMed Scopus (1498) Google Scholar), but the mechanisms of targeting are poorly understood. Transcription factors that maintain chromatin with the appropriate epigenetic marks through recruitment of these factors are certain to play important biological functions, for example by maintaining a silent state or priming target genes for later expression in stem/progenitor and memory cells. Our data suggest that Oct1 regulates the expression of its targets, at least in part, through spatial control of chromatin-modifying factors.The absence of Oct1 is associated with abnormal DNA hypomethylation at a CG site proximal to the Il2 transcription start site in naïve T cells, but is also associated with abnormal hypermethylation at that site in previously stimulated, resting T cells. These changes in DNA methylation are likely an indirect readout of Oct1 regulation through NuRD and Jmjd1a. Surprisingly, both in fibroblasts following stress and in resting but previously stimulated T cells Jmjd1a was simultaneously present with H3K9me2 in wild-type cells. These results suggest that Jmjd1a is either not active in these situations (for example due to lack of cofactors) or that an opposing histone methyltransferase activity such as G9a (54Tachibana M. Matsumura Y. Fukuda M. Kimura H. Shinkai Y. EMBO J. 2008; 27: 2681-2690Crossref PubMed Scopus (300) Google Scholar) continually deposits this mark as it is being removed. We favor the latter mechanism because in wild-type T cells, the mark is present but does not accumulate or become expanded such that DNA methylation takes place, but in the absence of Oct1 it does, suggesting a finely regulated balance of activities.The absence of Oct1 in resting T cells leads to poor activation upon restimulation relative to wild-type T cells. These findings have implications for the generation of T cell memory responses and, by extension, the ability to resist infection by certain pathogens. Oct1 is associated with the expression of multiple cytokines, including Il-3, Il-5, Il-8, Il-12/23 (55Pfeuffer I. Klein-Hessling S. Heinfling A. Chuvpilo S. Escher C. Brabletz T. Hentsch B. Schwarzenbach H. Matthias P. Serfling E. J. Immunol. 1994; 153: 5572-5585PubMed Google Scholar, 56Duncliffe K.N. Bert A.G. Vadas M.A. Cockerill P.N. Immunity. 1997; 6: 175-185Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 57Kaushansky K. Shoemaker S.G. O'Rork C.A. McCarty J.M. J. Immunol. 1994; 152: 1812-1820PubMed Google Scholar, 58Cron R.Q. Zhou B. Brunvand M.W. Lewis D.B. Genes Immun. 2001; 2: 464-468Crossref PubMed Scopus (14) Google Scholar, 59Wu G.D. Lai E.J. Huang N. Wen X. J. Biol. Chem. 1997; 272: 2396-2403Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 60Zhou L. Nazarian A.A. Xu J. Tantin D. Corcoran L.M. Smale S.T. Mol. Cell. Biol. 2007; 27: 2698-2712Crossref PubMed Scopus (41) Google Scholar), and more recently Il-4 and Il-13 (61Gervaziev Y.V. Olenina L.V. Krasotkina J.V. Lupatov A.Y. Mazurina S.A. Gervazieva V.B. Int. J. Immunogenet. 2010; 37: 13-20Crossref PubMed Scopus (9) Google Scholar, 62Kiesler P. Shakya A. Tantin D. Vercelli D. Hum. Mol. Genet. 2009; 18: 4513-4520Crossref PubMed Scopus (18) Google Scholar). Oct1 is also associated with other loci of immunological interest (e.g. 63Thévenin C. Lucas B.P. Kozlow E.J. Kehrl J.H. J. Biol. Chem. 1993; 268: 5949-5956Abstract Full Text PDF PubMed Google Scholar, 64Zabel M.D. Wheeler W. Weis J.J. Weis J.H. J. Immunol. 2002; 168: 3341-3350Crossref PubMed Scopus (16) Google Scholar, 65LeBowitz J.H. Kobayashi T. Staudt L. Baltimore D. Sharp P.A. Genes Dev. 1988; 2: 1227-1237Crossref PubMed Scopus (68) Google Scholar). In light of these findings, a reexamination of the role of Oct1 at these targets may shed light on their regulation. For example, a negative activity has been associated with the octamer site in the immunoglobulin heavy chain enhancer in B/T cell hybrids (66Shen L. Lieberman S. Eckhardt L.A. Mol. Cell. Biol. 1993; 13: 3530-3540Crossref PubMed Scopus (18) Google Scholar). It was postulated that a transcriptional repressor occupies this site in non-B cells. Our data suggest that the positive activity in B cells and the negative activity in non-B cells may be one and the same, and involve differential Oct1 cofactor recruitment. Rather than directly promoting expression of the aforementioned target genes, the principal role of Oct1 may be, in one mode, maintenance of repression, and in the opposite mode, prevention of transcriptional repression, i.e. poising for later expression. IntroductionThe POU 2The abbreviations used are: POUPit-1, Oct1/2, Unc-86MEFmouse embryonic fibroblastNFATnuclear factor of activated T cellsNuRDnucleosome remodeling and histone deacetylationPMAphorbol 12-myristate 13-acetateqRT-PCRquantitative RT-PCRC/EBPβCCAAT-enhancer binding protein-beta. (Pit-1, Oct1/2, Unc-86) transcription factor family includes ∼13 mammalian paralogs as well as representatives from other metazoans (1Herr W. Sturm R.A. Clerc R.G. Corcoran L.M. Baltimore D. Sharp P.A. Ingraham H.A. Rosenfeld M.G. Finney M. Ruvkun G. Genes Dev. 1988; 2: 1513-1516Crossref PubMed Scopus (599) Google Scholar). 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 (2Okita K. Ichisaka T. Yamanaka S. Nature. 2007; 448: 313-317Crossref PubMed Scopus (3497) Google Scholar, 3Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14653) Google Scholar, 4Nakagawa M. Koyanagi M. Tanabe K. Takahashi K. Ichisaka T. Aoi T. Okita K. Mochiduki Y. Takizawa N. Yamanaka S. Nat. Biotechnol. 2008; 26: 101-106Crossref PubMed Scopus (2174) Google Scholar, 5Feng B. Jiang J. Kraus P. Ng J.H. Heng J.C. Chan Y.S. Yaw L.P. Zhang W. Loh Y.H. Han J. Vega V.B. Cacheux-Rataboul V. Lim B. Lufkin T. Ng H.H. Nat. Cell Biol. 2009; 11: 197-203Crossref PubMed Scopus (374) Google Scholar). Oct1/POU2F1 is related to Oct4 and possesses similar in vitro DNA binding specificity (for reviewed, see Ref. 6Kang J. Shakya A. Tantin D. Trends Biochem. Sci. 2009; 34: 491-499Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). As with many transcription factors, these proteins are known to regulate gene expression both positively and negatively (e.g. 7Boyer L.A. Lee T.I. Cole M.F. Johnstone S.E. Levine S.S. Zucker J.P. Guenther M.G. Kumar R.M. Murray H.L. Jenner R.G. Gifford D.K. Melton D.A. Jaenisch R. Young R.A. Cell. 2005; 122: 947-956Abstract Full Text Full Text PDF PubMed Scopus (3459) Google Scholar, 8dela Paz N.G. Simeonidis S. Leo C. Rose D.W. Collins T. J. Biol. Chem. 2007; 282: 8424-8434Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar); 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 (9Shakya A. Cooksey R. Cox J.E. Wang V. McClain D.A. Tantin D. Nat. Cell Biol. 2009; 11: 320-327Crossref PubMed Scopus (80) Google Scholar). One study indicates that Oct1 levels are increased in some human gastric cancers (10Almeida R. Almeida J. Shoshkes M. Mendes N. Mesquita P. Silva E. Van Seuningen I. Reis C.A. Santos-Silva F. David L. J. Pathol. 2005; 207: 396-401Crossref PubMed Scopus (54) Google Scholar). 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 (11Reymann S. Borlak J. BMC Syst. Biol. 2008; 2: 46Crossref PubMed Scopus (47) Google Scholar, 12Ben-Porath I. Thomson M.W. Carey V.J. Ge R. Bell G.W. Regev A. Weinberg R.A. Nat. Genet. 2008; 40: 499-507Crossref PubMed Scopus (1928) Google Scholar, 13Somervaille T.C. Matheny C.J. Spencer G.J. Iwasaki M. Rinn J.L. Witten D.M. Chang H.Y. Shurtleff S.A. Downing J.R. Cleary M.L. Cell Stem Cell. 2009; 4: 129-140Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 14Mattison J. Kool J. Uren A.G. de Ridder J. Wessels L. Jonkers J. Bignell G.R. Butler A. Rust A.G. Brosch M. Wilson C.H. van der Weyden L. Largaespada D.A. Stratton M.R. Futreal P.A. van Lohuizen M. Berns A. Collier L.S. Hubbard T. Adams D.J. Cancer Res. 2010; 70: 883-895Crossref PubMed Scopus (35) Google Scholar), suggesting that Oct1 activity may be deregulated in malignancy. Recent findings showing post-translational regulation of Oct1 support this possibility (15Kang J. Gemberling M. Nakamura M. Whitby F.G. Handa H. Fairbrother W.G. Tantin D. Genes Dev. 2009; 23: 208-222Crossref PubMed Scopus (92) Google Scholar). Although Oct1 has been studied intensively, our current understanding of how it regulates gene transcription is surprisingly limited (see for example, Ref. 16Bertolino E. Singh H. Mol. Cell. 2002; 10: 397-407Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar).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-2) chromatin remodeling complex to the Il2 promoter in naïve CD4 T cells to mediate gene repression. The recruitment is regulated because upon T cell activation Oct1 loses its capacity to associate with NuRD and instead is required for recruitment of Jmjd1a. Phorbol 12-myristate 13-acetate (PMA) treatment is sufficient to switch association from NuRD to Jmjd1a, despite the fact that PMA is insufficient to activate Il2. In resting but previously stimulated T cells, Oct1 is required to maintain Jmjd1a at Il2, remove histone H3K9me2 marks and protect DNA from methylation, promoting the stronger expression associated with secondary stimulation. In DLD-1 colon adenocarcinoma cells, Oct1 bound to the Cdx2 target locus is required for mutually exclusive NuRD and Jmjd1a association. PMA treatment of DLD-1 cells results in reduced Jmjd1a association and represses Cdx2 in a manner requiring Oct1. These results show that Oct1 is a bipotential factor capable of acting through opposing mechanisms to reinforce repressed or inducible states, even at the same target gene.