Mechanism of polymerase II transcription repression by the histone variant macroH2A

Abstract

macroH2A (mH2A) is an unusual histone variant consisting of a histone H2A-like domain fused to a large nonhistone region. In this work, we show that histone mH2A represses p300- and Gal4-VP16-dependent polymerase II transcription, and we have dissected the mechanism by which this repression is realized. The repressive effect of mH2A is observed at the level of initiation but not at elongation of transcription, and mH2A interferes with p300-dependent histone acetylation. The nonhistone region of mH2A is responsible for both the repression of initiation of transcription and the inhibition of histone acetylation. In addition, the presence of this domain of mH2A within the nucleosome is able to block nucleosome remodeling and sliding of the histone octamer to neighboring DNA segments by the remodelers SWI/SNF and ACF. These data unambiguously identify mH2A as a strong transcriptional repressor and show that the repressive effect of mH2A is realized on at least two different transcription activation chromatin-dependent pathways: histone acetylation and nucleosome remodeling.

FIG. 1.

macroH2A interferes with both p300- and Gal4-VP16-dependent transcription and histone acetylation. Chromatin was assembled on the pG₅ML array DNA by using recombinant Drosophila Acf1, ISWI, and the nucleosome assembly protein 1, an equimolar mixture of either conventional H2A or variant histone macroH2A, and the three remaining core histones H2B, H3, and H4. (A) DNA supercoiling assay for the assembly of chromatin. The DNA samples were run on 1% agarose gels and stained with ethidium bromide. Lane 1, supercoiled pG₅ML DNA (S); lane 2, topoisomerase I-relaxed pG₅ML DNA (R); lanes 3 and 4 show the plasmid DNA isolated from the chromatin samples assembled with macroH2A or conventional H2A, respectively. “nc” designates nick DNA. (B) Micrococcal nuclease digestion of the assembled macroH2A (lane 2) and conventional H2A (lanes 3 and 4) chromatin samples. The chromatin samples were digested with either 0.2 mU MNase (lanes 2 and 3) or 0.5 mU MNase (lane 4) for 10 min at 22°C, and the DNA was analyzed on 1.2% agarose gels. Lane 1, a 123-bp DNA ladder marker (M). (C) p300- and Gal4-VP16-dependent transcription of conventional H2A (lanes 1 and 2) and histone variant mH2A (lanes 3 and 4) nucleosomal arrays. The arrays were incubated with GAL4-VP16 alone or with both Gal4-VP16 and p300. The results from two independent experiments are shown. (D) HAT assays with nucleosomal arrays assembled with either conventional H2A (lanes 1 and 2) or macroH2A (lanes 3 and 4) nucleosomal arrays. All reaction mixtures contained p300 while Gal4-VP16 was present in mixtures for reactions 2 and 4 only. The positions of the histones are indicated.

FIG. 2.

The presence of mH2A does not affect the binding of Gal4-VP16 to the nucleosome. (A) Schematic of the nucleosomes used in Gal4-VP16 binding studies. A 152-bp DNA fragment, derived from the pG₅ML vector and containing the five Gal4-VP16 sites inserted in the E4 promoter, was PCR amplified and used to reconstitute both conventional and mH2A nucleosomes. The positions of the five Gal4- VP16 binding sites and the nucleosome dyad are designated. nt, nucleotide. (B) Binding of Gal4-VP16 to conventional H2A nucleosomes. Increasing amounts of Gal4-VP16 were added to the solution containing conventional nucleosomes, and Gal4-VP16 binding was assessed by EMSA. The positions of free DNA, nucleosomes (nuc), and Gal4-VP16 nucleosome complexes (cplx.) are designated on the left part of the figures. (C) Data are presented as described for panel B but for macroH2A nucleosomes. (D) Quantification of the data presented in panels B and C. (E) HAT assays with either conventional H2A (lane 2) or macroH2A (lane 4) mononucleosomes. Acetylation of the histone mixtures consisting of conventional histones or containing mH2A is shown in lanes 1 and 3, respectively. All reaction mixtures contained p300 and Gal4-VP16. The positions of the histones are indicated.

FIG. 3.

The NHR of the histone variant macroH2A is involved in both the repression of p300- and Gal4-VP16-dependent transcription and the inhibition of histone acetylation. Chromatin was assembled on the pG₅ML array DNA as described in the legend for Fig. 1 but with the histone-like domain of mH2A (H2A-like) or with the fusion (H2A-NHR) of conventional H2A with the NHR of mH2A. (A) Schematic of the proteins used in the chromatin assembly experiments. (B) 18% SDS electrophoresis of the recombinant conventional core histones, the fusion H2A-NHR, and the H2A-like nucleosomal template. (C) Supercoiling assay for DNA isolated from chromatin assembled with the H2A-like (lane 3) or H2A-NHR (lane 4) nucleosomal template. (D) Micrococcal nuclease analysis of chromatin assembled with either the H2A-like (lanes 1 and 2) or H2A-NHR (lanes 3 and 4) nucleosomal template. The digestion was performed with 0.1 mU MNase (lanes 1 and 3) or with 0.5 mU MNase (lanes 2 and 4) for 10 min at 22°C. DNA was then extracted from the digested samples and analyzed on 1.2% agarose gels. (E) p300- and Gal4-VP16-dependent transcription of H2A-like (lanes 1 and 2) and H2A-NHR (lanes 3 and 4) nucleosomal templates. The results from two independent experiments are shown. (F) HAT assays of H2A-like (lanes 1 and 2) and H2A-NHR (lanes 3 and 4) nucleosomal arrays. Note the complete inhibition of histone acetylation in the H2A-NHR templates.

FIG. 4.

The NHR of mH2A does not affect polymerase II elongation through nucleosomal templates. A 245-bp DNA fragment was used to reconstitute nucleosomes with either conventional H2A or the fusion H2A-NHR. The templates, a mixed population of two positioned nucleosomes, N1 and N2, were then ligated to the elongation Pol II complex immobilized on beads as described previously (21). The Pol II elongation complex was allowed to transcribe the nucleosomal DNA, and the nascent RNA was pulse labeled. The transcription was performed in the presence of either 40 mM, 300 mM, or 1 M KCl, and the labeled RNA was extracted and analyzed. The RNA isolated from the transcription reactions of conventional H2A (lanes 1 to 4), mH2A (lanes 5 to 8), H2A-like (lanes 9 to 12), and H2A-NHR (lanes 13 to 16) nucleosomal templates was analyzed on an 8% denaturing polyacrylamide gel. The transcriptions from preformed stalled elongation complexes are also shown (lanes 1, 5, 9, and 13). M, a marker for the molecular mass of the transcripts.

FIG. 5.

The presence of NHR of mH2A results in alterations in the structure of the H2A-NHR nucleosomes. (A) EMSA of the reconstituted H2A and H2A-NHR nucleosomes (Nuc). (B) 18% SDS-polyacrylamide gel electrophoresis of the histones isolated from H2A (lane 1) and H2A-NHR (lane 2) nucleosomes. The positions of the histones are shown on the left part of the figure. Note that the H2A and H2B Xenopus laevis histones comigrate under the electrophoresis conditions. (C) DNase I footprinting of conventional H2A and H2A-NHR nucleosomes reconstituted on a 152-bp fragment comprising the 5S DNA Xenopus borealis gene. The digestion products were analyzed on an 8% denaturing polyacrylamide gel. The bottom strand of the nucleosomal DNA was P³² labeled. The diamond designates the dyad axis of the nucleosome. Stars indicate the alterations of the H2A-NHR nucleosome DNase I digestion pattern.

FIG. 6.

The NHR of mH2A interferes with SWI/SNF and ACF nucleosome mobilization. Conventional H2A and NHR-H2A nucleosomes were reconstituted by using a 255-bp fragment containing the centrally positioned sequence 601 (19). (A) SWI/SNF mobilization of H2A and NHR-H2A nucleosomes. Both types of nucleosomes were incubated for 45 min at 30°C in the presence of increasing amounts of SWI/SNF and ATP. Mobilization of the histone octamer was revealed by EMSA on 5.5% native polyacrylamide gels. The center- and end-positioned nucleosomes and free DNA are indicated on the left part of the figure. (B) Time course of the SWI/SNF-induced mobilization of conventional H2A and H2A-NHR nucleosomes. The nucleosome solutions were supplemented with ATP and 0.5 μl of SWI/SNF and incubated for the indicated time. The nucleosome mobilization was arrested by apyrase treatment, and the reaction mixtures were stored on ice until they were loaded on the gel. The central- and end-positioned nucleosomes and free DNA are indicated. (C) Mapping of H2A and H2A-NHR nucleosome positions after treatment with SWI/SNF. Both types of particles were incubated for 45 min at 30°C in the presence of increasing amounts of SWI/SNF as indicated. Then, the mobilization reaction was arrested by apyrase treatment, the samples were digested with exonuclease III, and the digestion products were run on an 8% denaturating gel. Stars indicate the radioactively labeled end of the DNA used for the reconstitution. (D) ACF is unable to mobilize H2A-NHR nucleosomes. Conventional H2A and H2A-NHR nucleosomes were reconstituted by using a 241-bp fragment containing the end-positioned sequence 601. Reconstituted conventional H2A and H2A-NHR nucleosomes were incubated for 45 min at 30°C with increasing amounts of ACF in the presence of ATP and run on a 5.5% native acrylamide gel. The center- and end-positioned nucleosomes and free DNA are indicated. nt, nucleotide; M, a marker for the molecular mass of the transcripts.

FIG. 7.

The NHR of mH2A interferes with SWI/SNF nucleosome remodeling. Conventional H2A and H2A-NHR were reconstituted on a radioactively end-labeled 152-bp DNA fragment containing the Xenopus borealis 5S RNA gene. Increasing amounts of SWI/SNF were added to the nucleosome (nuc) solutions, and the remodeling reaction was carried out for 40 min at 30°C. After digestion with DNase I, DNA was extracted and subjected to an 8% sequencing gel. The position of the DNase I cleavage repeat is indicated on the left part of the figure. The DNase I digestion pattern of free DNA is shown in lane 7. The diamond designates the dyad axis of the nucleosome.