G9a Selectively Represses a Class of Late-Replicating Genes at the Nuclear Periphery
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G9a selectively represses a class of late-replicating genes at the nuclear periphery Tomoki Yokochia,1, Kristina Poducha, Tyrone Rybaa, Junjie Lua, Ichiro Hiratania, Makoto Tachibanab, Yoichi Shinkaib, and David M. Gilberta,2 aDepartment of Biological Science, Florida State University, Tallahassee, FL 32306; and bExperimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved September 25, 2009 (received for review June 4, 2009) We have investigated the role of the histone methyltransferase G9a ery and that G9a-null ESCs are selectively depleted of the in the establishment of silent nuclear compartments. Following con- H3K9me2 localized at the periphery (13). Chromatin at the nuclear ditional knockout of the G9a methyltransferase in mouse ESCs, 167 periphery also is replicated late during S-phase, and differentiation genes were significantly up-regulated, and no genes were strongly of ESCs leads to changes in the replication timing of large chro- down-regulated. A partially overlapping set of 119 genes were matin domains, accompanied by the movement of those domains up-regulated after differentiation of G9a-depleted cells to neural toward or away from the nuclear periphery and the respective precursors. Promoters of these G9a-repressed genes were AT rich and silencing or activation of genes within those domains (14). To- H3K9me2 enriched but H3K4me3 depleted and were not highly DNA gether, these results suggested the possibility that G9a may help methylated. Representative genes were found to be close to the establish compartments of facultative heterochromatin at the nu- nuclear periphery, which was significantly enriched for G9a-depen- clear periphery. Here, we have investigated this hypothesis using a dent H3K9me2. Strikingly, although 73% of total genes were early conditional-knockout ESC line that allows acute effects of G9a loss replicating, more than 71% of G9a-repressed genes were late repli- to be evaluated within the first several cell cycles following G9a cating, and a strong correlation was found between H3K9me2 and disruption. We find that G9a loss leads to depletion of H3K9me2 late replication. However, G9a loss did not significantly affect sub- at the nuclear periphery and de-repression of a set of genes with nuclear position or replication timing of any non-pericentric regions H3K9me2-enriched promoters. No genes were down-regulated, of the genome, nor did it affect programmed changes in replication indicating that G9a is not required for the activation of transcription timing that accompany differentiation. We conclude that G9a is a in ESCs. An overlapping set of genes was de-repressed by G9a loss gatekeeper for a specific set of genes localized within the late in neural precursor cells (NPCs) derived from these ESCs. Intrigu- replicating nuclear periphery. ingly, the majority of G9a-repressed genes were late replicating, but the loss of G9a had no detectable effect on the replication timing histone methylation ͉ nucleus ͉ replication timing ͉ transcription of these genes or on the changes in replication timing that took place during ESC differenation to NPCs. We conclude that G9a mediates osttranslational modifications of chromatin are central to the dimethylation of H3K9 within late-replicating chromatin at the Pregulation of many chromosomal functions and are intimately nuclear periphery and is required within this genomic context to tied to transcriptional regulation (1). The histone methyltransferase silence a defined set of genes. (HMTase) G9a, in a complex with G9a-like protein (GLP), is Results responsible for methylation of lysine 9 of histone H3 (H3K9me), commonly associated with gene repression (2, 3). Although H3K9 Changes in Histone Methylation Following G9a Knockout. Stable and can be mono-, di-, or trimethylated (-me1, -me2, -me3, respectively), irreversible genetic knockout often leads to compensatory genetic G9a-knockout ESCs have significantly reduced levels of H3K9me2 and epigenetic changes that can confuse interpretation of the (2, 4, 5). H3K9me2 and H3K9me3 create a platform for the binding resulting phenotypes (15). For example, Suv39h1,2-knockout cells of heterochromatin protein 1 (HP1), usually associated with tran- lose H3K9me3 but also gain H3K27me3 in pericentric heterochro- scriptional silencing but sometimes required for transcriptional matin, where it is not seen in wild-type cells (4). This kind of activation (6). G9a also recruits DNA methyltransferases via its adaptation can be largely circumvented through the use of a ankyrin domain and can promote and/or maintain DNA methyl- conditional-knockout, in which cellular responses to the acute loss ation at target sites independent of its HMTase activity (7–10). G9a of the gene product can be monitored. We have constructed a is an essential gene for development; knockout mice die at day 8.5 conditional knockout of G9a in mouse ESC line TT2 (Fig. 1). This (3). Although the precise lethal event during development of cell line has a single copy of the G9a gene flanked by loxP CELL BIOLOGY G9a-null mice is not known, G9a ESCs can differentiate in culture recombination sites and expresses 4-hydroxytamoxifen (OHT)- but fail to methylate the promoter DNA of a set of genes during inducible Cre fusion protein (16). Addition of OHT results in the differentiation; this failure may affect stable silencing of those rapid deletion of G9a (Fig. 1B), a partial reduction in total promoters (9). Despite the importance of G9a to gene expression H3K9me1 and H3K9me3, and a substantial reduction in total and development, the cohort of genes regulated by G9a has not been reported. Moreover, it is not clear whether G9a always Author contributions,: D.M.G. designed research; T.Y., K.P., J.L., and I.H. performed re- functions as a repressor or whether, as occurs at some promoters search; M.T. and Y.S. contributed new reagents/analytic tools; T.Y., K.P., T.R., J.L., and I.H. occupied by HP1 (6), it also can function to activate certain genes. analyzed data; and D.M.G. wrote the paper. In addition to localized effects at specific promoters, G9a has The authors declare no conflict of interest. global effects on chromatin organization. G9a-null cells show a loss This article is a PNAS Direct Submission. of DNA methylation at major satellite DNA and several classes of Data deposition: The data reported in this paper have been deposited in the Gene Expression repetitive and transposable elements (7). In addition, blocks of Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE18082). G9a-dependent H3K9me2 (large, organized chromatin K9 modi- 1Present address: Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo, Chiba fications, LOCKs) have been identified in mouse ESCs that appear 260-8717, Japan. to overlap strongly with chromatin associated with the nuclear 2To whom correspondence should be addressed. E-mail: [email protected]. lamina (11, 12). Interestingly, we previously showed that H3K9me2, This article contains supporting information online at www.pnas.org/cgi/content/full/ but not H3K9me1 or H3K9me3, is enriched in the nuclear periph- 0906142106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906142106 PNAS ͉ November 17, 2009 ͉ vol. 106 ͉ no. 46 ͉ 19363–19368 Downloaded by guest on September 26, 2021 G9a + G9a - A C Mock OHT Days 0 1 2 3 5 7 0 1 2 3 5 7 G9a -Tubulin + OHT Probe H3K9me1 (A) Fig. 1. Construction and characterization of a G9a con- H3K9me2 (A) ditional-knockout mouse ESC line. (A) When TT2G9aflox/⌬ ESCs are treated with OHT, the catalytic center of G9a is H3K9me3 (B) deleted, resulting in loss of a HindIII site that converts the 4.6-kb HindIII fragment on the floxed allele to a 5.8-kb OHT H3K9me1 (C) HindIII fragment on the deleted allele. Also shown is the B - + H3K9me2 (C) position of the probe used for Southern blot confirmation in (B). (B) Southern blot confirming genetic deletion of H3K9me3 (C) G9a 2 days after OHT treatment. The constitutively de- Null (7.3 kb) leted allele produces a 7.3-kb fragment (3, 8). (C) Western Del. (5.8 kb) H3K27me1 (A) blots of whole-cell extracts were performed at daily inter- Flox (4.6 kb) vals after OHT or vehicle-only mock treatment and probed H3K27me2 (A) with antibodies against the indicated proteins or histone H3K27me3 (A) modifications. Histone antibodies are designated (A) for Upstate Biotechnology polyclonal antibodies, (B) for poly- clonal anti-2xH3K9me3 antibodies (4), and (C) for mono- H4K20me3 (A) clonal antibodies (17). H3K9me2 (Fig. 1C), but has no effect on mono-, di- or trimethy- mouse ESCs, and that stable G9a-knockout ESCs preferentially lated H3K27 or H4K20me3. The effects on the different methylated lose peripheral H3K9me2 (13). To determine whether acute G9a forms of H3K9 were similar with either commonly used polyclonal loss also preferentially affects peripheral H3K9me2, we performed antibodies or more recently reported monoclonal antibodies (17) immunofluorescence detection of mono-, di- and trimethyl H3K9 and were similar in a stable G9a-null ESC line. Hence, this before and after the addition of OHT (Fig. 2). H3K9me1 was conditional-knockout cell line provides the opportunity to observe distributed throughout the interior of the nucleus, excluding the the effects of acute G9a loss in the absence of complicating adaptive periphery and clusters of pericentric heterochromatin (chromo- changes. centers), whereas H3K9me3 was localized primarily to the chro- We previously reported that H3K9me2 is enriched at the nuclear mocenters; both these modifications show some reduction in signal periphery in human, mouse, and hamster fibroblasts, as well as in after G9a loss but no change in localization. In contrast, H3K9me2 A Mock OHT DNA H3K9me Merge DNA H3K9me Merge me1 me2 me3 B Mock OHT Fig. 2. G9a knockout leads to the selective loss of Single-Nucleus 30-60 Single-Nucleus 30-60 H3K9me2 at the nuclear periphery.