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. 2008 Apr 4;283(14):9113-26.
doi: 10.1074/jbc.M708205200. Epub 2008 Feb 7.

Isolation and characterization of a novel H1.2 complex that acts as a repressor of p53-mediated transcription

Kyunghwan Kim  1 , Jongkyu Choi, Kyu Heo, Hyunjung Kim, David Levens, Kimitoshi Kohno, Edward M Johnson, Hugh W Brock, Woojin An
Affiliations

Isolation and characterization of a novel H1.2 complex that acts as a repressor of p53-mediated transcription

Kyunghwan Kim et al. J Biol Chem. .

Abstract

Linker histone H1 has been generally viewed as a global repressor of transcription by preventing the access of transcription factors to sites in chromatin. However, recent studies suggest that H1 can interact with other regulatory factors for its action as a negative modulator of specific genes. To investigate these aspects, we established a human cell line expressing H1.2, one of the H1 subtypes, for the purification of H1-interacting proteins. Our results showed that H1.2 can stably associate with sets of cofactors and ribosomal proteins that can significantly repress p53-dependent, p300-mediated chromatin transcription. This repressive action of H1.2 complex involves direct interaction of H1.2 with p53, which in turn blocks p300-mediated acetylation of chromatin. YB1 and PURalpha, two factors present in the H1.2 complex, together with H1.2 can closely recapitulate the repressive action of the entire H1.2 complex in transcription. Chromatin immunoprecipitation and RNA interference analyses further confirmed that the recruitment of YB1, PURalpha, and H1.2 to the p53 target gene Bax is required for repression of p53-induced transcription. Therefore, these results reveal a previously unrecognized function of H1 as a transcriptional repressor as well as the underlying mechanism involving specific sets of factors in this repression process.

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Figures

FIGURE 1.
FIGURE 1.
Preparation of H1.2 complex and recombinant H1.2. A, schematic diagram for the purification of H1.2 complex from the stable cell line. Numbers indicate the KCl concentration used to purify the individual fractions. Each elution was separated by 4–20% gradient SDS-PAGE and probed with HA and FLAG antibodies as indicated (lane 2). The control preparation with normal HeLa nuclear extract is also included (lane 1). B, subcellular localization of ectopic H1.2. Cytoplasmic and nuclear extracts were prepared as described recently (19) and analyzed by immunoblots with anti-FLAG, anti-lamin A/C, and anti-tubulin antibodies. C, relative levels of ectopic H1.2 versus endogenous H1.2. Nuclear extracts were prepared from control cells (lane 1) and H1.2-expressing cells (lane 2), and Western blot analysis was performed with anti-H1.2 antibody. D, purification of recombinant H1.2. Recombinant H1.2 (rH1.2) was purified as described under “Experimental Procedures.” The purity of the purified H1.2 was confirmed by SDS-PAGE and Coomassie staining analysis. E, mass spectrometric identification of H1.2 complex. After H1.2 complex was fractionated by 4–20% gradient SDS-PAGE, bands were excised and subjected to mass spectrometry analysis as described under “Experimental Procedures.” Identified components of H1.2 complex are indicated on the right. Molecular mass markers are indicated on the left. Lane 1, mock-purified control; lane 2, H1.2 complex. F, immunoblot confirmation of identified factors. Purified H1.2 complex was separated by 4–20% gradient SDS-PAGE, and the presence of selected factors was analyzed with the indicated antibodies. Lane 1, HeLa nuclear extract input; lane 2, mock-purified control; lane 3, H1.2 complex. G, glycerol gradient centrifugation of H1.2 complex. H1.2 complex, purified on phosphocellulose P11 and anti-FLAG antibody affinity columns, was separated by 15–40% glycerol gradient centrifugation as described under “Experimental Procedures.” Fractions were loaded onto a 4–20% SDS-polyacrylamide gel, and proteins were detected by silver staining (uppermost panel) or Western blot (six lower panels). IB, immunoblot; C, cytoplasmic extract; N, nuclear extract; DNA-PK, DNA-dependent protein kinase; com, complex; hnRNP K, heterogeneous nuclear ribonucleoprotein K; f, FLAG.
FIGURE 2.
FIGURE 2.
Interaction of H1.2 with its associated factors. A, purification of GST-H1.2 fusion protein. GST or GST-H1.2 was purified as described under “Experimental Procedures.” The purity of the proteins was analyzed by 15% SDS-PAGE and Coomassie staining analysis. Lane 1, GST; lane 2, GST-H1.2. B, in vitro binding assay of H1.2 and its associated factors. In vitro translated proteins (Xpress-ASXL1, Xpress-β-catenin, Xpress-TGase7, Xpress-CAPERα, Xpress-YB1, and Xpress-WDR5) and recombinant proteins (FLAG-PARP1, HA-FIR, and PURα) were incubated with GST or GST-H1.2 as described under “Experimental Procedures.” After extensive washing, bound proteins were analyzed by immunoblot with anti-Xpress (for ASXL1, β-catenin, TGase7, CAPERα, YB1, and WDR5), anti-HA (for FIR), anti-PARP1, and anti-PURα antibodies. Lane 1, 10% input; lane 2, GST alone; lane 3, GST-H1.2. C, in vivo binding assay of H1.2 and its associated factors. FLAG- or Xpress-tagged H1.2 was transiently transfected with its associated factors (FLAG-, HA-, or Xpress-tagged), and immunoprecipitation was performed as described under “Experimental Procedures.” Lanes 1, 4, 7, and 10, factor-only expression; lanes 2, 5, 8, and 11, H1.2-only expression; lanes 3, 6, 9, and 12, H1.2 and factor co-expression. Asterisks indicate nonspecific bands. D, cellular co-localization of H1.2 with its associated factors. HeLa cells were transfected with expression vectors encoding EGFP-H1.2 and Xpress-ASXL1, FLAG-PARP1, HA-FIR, Xpress-CAPERα, Xpress-YB1, Xpress-PURα, or Xpress-WDR5 as indicated. Cells were fixed with paraformaldehyde and immunostained with anti-FLAG, anti-Xpress, or anti-HA antibody followed by the Cy3-conjugated secondary antibodies. After mounting on glass slides with VECTASHIELD with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories), confocal microscopy was performed as detailed under “EXPERIMENTAL Procedures.” H1.2 is stained green, and its associated factor is stained red. Nucleus is stained blue, and co-localizations of H1.2 and its associated factors are shown in MERGE. The scale bar represents 5 μm. IP, immunoprecipitate; Xp, Xpress.
FIGURE 3.
FIGURE 3.
Repressive effects of H1.2 complex in chromatin acetylation and transcription. A, schematic representation of transcription templates. Arrows indicate the length of DNA to be transcribed. p53 RE, p53 response element. B, schematic summary of transcription and chromatin histone acetyltransferase (HAT) assays. NTPs and PIC indicate nucleotide triphosphates and preinitiation complex, respectively. C, transcription assays with recombinant H1.2 and H1.2 complex. p53ML-S chromatin template (40 ng) and p53ML-L DNA (40 ng) were transcribed with p53 (15 ng), p300 (20 ng), and/or acetyl-CoA (10 μm) as summarized in B and as described recently (29). H1.2 complex (200 and 400 ng) and recombinant H1.2 (40 and 80 ng of H1.2 mixed with 160 and 320 ng of BSA) were used in transcription. Note that results were obtained from three separate transcription experiments as indicated by boxes. Data were quantitated by PhosphorImager and normalized to reactions with p53ML-L DNA and p53 alone (100%). D, chromatin histone acetyltransferase assays with recombinant H1.2 and H1.2 complex. Chromatin template (100 ng) was incubated with recombinant H1.2 (80 ng of H1.2 mixed with 320 ng of BSA) or H1.2 complex (400 ng) in the presence of p53 (30 ng), p300 (40 ng), and 2.5 μm [3H]acetyl-CoA. Txn, relative transcription levels; ND, nondetectable; ACF, ATP-utilizing chromatin assembly and remodeling factor.
FIGURE 4.
FIGURE 4.
Direct interaction of H1.2 with p53. A, p53 interaction with H1.2 in vitro. GST-H1.2 mutants (lanes 1–4) or GST-p53 mutants (lanes 5–7) were analyzed by SDS-PAGE and Coomassie staining analysis. For interaction studies, GST-H1.2 mutants and GST-p53 mutants were incubated with FLAG-tagged p53 and His-tagged H1.2, respectively. After washing, binding of p53 and H1.2 was analyzed by Western blot analysis with anti-FLAG or anti-His antibody. Lane 1, GST-H1.2 full length (FL; amino acids 1–213); lane 2, GST-H1.2 NT (amino acids 1–34); lane 3, GST-H1.2 globular domain (GD; amino acids 35–109); lane 4, GST-H1.2 CT (amino acids 110–213); lane 5, GST-p53 NT (amino acids 1–83); lane 6, GST-p53 DNA binding domain (DBD; amino acids 120–290); lane 7, GST-p53 CT (amino acids 290–393); lanes 8 and 14, 10% input of FLAG-p53 and His-H1.2; lanes 9 and 15, GST control; lanes 10–13, p53 bound to GST-H1.2 mutants; lanes 16–18, H1.2 bound to GST-p53 mutants. B, p53 interaction with H1.2 complex in vitro. GST (lane 1) or GST-p53 full length (lane 2) was incubated with H1.2 complex, and pulldown fractions were analyzed by Western blot analysis using the indicated antibodies. C, p53 interaction with H1.2 in vivo. H1.2 and p53 were expressed in 293T cells and immunoprecipitated using anti-FLAG and anti-Xpress antibodies as indicated (lanes 1–6). Similar experiments were also performed after expression of p53 and H1.2 deletion mutants as described under “Experimental Procedures” (lanes 10–21). Lanes 1 and 4, p53-only expression; lanes 2 and 5, H1.2-only expression; lanes 3 and 6, p53 and H1.2 co-expression; lanes 7–9, expressed H1.2 mutants in whole cell lysates; lanes 10–12, H1.2-only controls; lanes 16–18, p53-only controls; lanes 13–15 and 19–21, H1.2 mutants and p53 mutants co-expressions. The asterisk indicates a nonspecific band containing IgG light chain. D, mutual interaction of endogenous p53 and H1.2. Whole cell extracts from 293T cells were immunoprecipitated with anti-p53 antibody (DO-1) and analyzed by Western blotting with anti-H1.2 and anti-p53 antibodies as indicated. Lane 1, whole cell lysate; lane 2, control IgG; lane 3, anti-p53 precipitates. E, p53 interaction with H1.2 complex in vivo. 293T cells were transfected with FLAG-tagged p53 and Xpress-tagged H1.2-expressing plasmids, and cell lysates were prepared 2 days after transfection. Lysates were immunoprecipitated with anti-FLAG and analyzed by Western blot analysis using the indicated antibodies. Lane 1, whole cell lysate; lane 2, control IgG; lane 3, anti-FLAG precipitates. IP, immunoprecipitate; Xp, Xpress; f, FLAG.
FIGURE 5.
FIGURE 5.
Functional characterization of H1.2, PURα, and YB1. A, analysis of recombinant proteins. Recombinant C-terminal tailless H1.2, PURα, and YB1 were purified as described under “Experimental Procedures.” The purity of the purified proteins was confirmed by SDS-PAGE and Coomassie staining analysis. B, repressive action of H1.2, PURα, and YB1 in p300-mediated, p53-dependent transcription. Chromatin template was transcribed with recombinant H1.2 (50 ng), PURα (100 ng), and/or YB1 (100 ng) as indicated. Final protein concentrations in all reactions were adjusted to be identical by including BSA. C, requirement of H1.2 C terminus for transcriptional repression. Transcription assays were performed as in B but with mutant H1.2 in which the C-terminal tail was deleted. D, effect of H1.2, PURα, and YB1 on p53-dependent transcription in vivo. H1299 cells were transiently transfected with Bax reporter gene together with vectors that express p53, PURα, YB1, and/or wild type/C-terminal tailless H1.2 as indicated. Data are represented as the means ± S.E. of three independent experiments. E, ChIP analysis of Bax promoter. H1299 cells were mock-transfected (lane 2) or transfected with p53, H1.2, PURα, and YB1 (lane 3) and subjected to ChIP analysis with antibodies specific for the indicated proteins. A sample containing 5% total input chromatin was also included for each ChIP assay (lane 1). Similar ChIP experiments on the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) minimal promoter region were also included as a control (lanes 4–6). F, RT-PCR analysis of Bax RNA. H1299 cells were transfected with p53, H1.2, PURα, and/or YB1 as indicated. RT-PCR was performed on total RNA isolated from transfected or mock-transfected cells. RT-PCR of actin RNA was used as a loading control. G, validation of H1.2, PURα, and YB1 knockdown. Cells were stably transfected with shRNA of H1.2, YB1, or PURα, and expression of targeted proteins was checked by Western blot analysis of cell lysates with anti-H1.2, anti-YB1, and anti-PURα antibodies (lanes 2–4). As a control, cells were also transfected with a mock shRNA vector (lane 1). H, up-regulation of Bax gene transcription upon H1.2, PURα, and YB1 knockdown. RNA was extracted from cells transfected with shRNA of H1.2, YB1, or PURα, and relative changes in expression of Bax gene were assessed by real time PCR. Txn, relative transcription levels; RNAi, RNA interference; ND, nondetectable; rH1.2, recombinant H1.2.
FIGURE 6.
FIGURE 6.
Model for the promoter-selective inhibition of p53-dependent transcription by H1 complex. We propose that the H1.2 complex binds to p53 and disrupts p53-mediated recruitment of chromatin-regulating factors to the promoter of target genes. This repressive chromatin state will in turn interfere with the formation of functional preinitiation complexes at the promoter to block gene transcription (see “Discussion” for details). Ac, acetylation; RNAPII, RNA polymerase II.
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