Linker Histone H1.2 cooperates with Cul4A and PAF1 to drive H4K31 ubiquitylation-mediated transactivation

Abstract

Increasing evidence suggests that linker histone H1 can influence distinct cellular processes by acting as a gene-specific regulator. However, the mechanistic basis underlying such H1 specificity and whether H1 acts in concert with other chromatin-altering activities remain unclear. Here, we show that one of the H1 subtypes, H1.2, stably interacts with Cul4A E3 ubiquitin ligase and PAF1 elongation complexes and that such interaction potentiates target gene transcription via induction of H4K31 ubiquitylation, H3K4me3, and H3K79me2. H1.2, Cul4A, and PAF1 are functionally cooperative because their individual knockdown results in the loss of the corresponding histone marks and the deficiency of target gene transcription. H1.2 interacts with the serine 2-phosphorylated form of RNAPII, and we argue that it recruits the Cul4A and PAF1 complexes to target genes by bridging the interaction between the Cul4A and PAF1 complexes. These data define an expanded role for H1 in regulating gene transcription and illustrate its dependence on the elongation competence of RNAPII.

Figure 1. Identification of the Cul4A and PAF1 complexes as interaction partners of linker histone H1.2

(A) The six human H1 subtypes and their associated factors were isolated from nuclear extracts of HeLa S3 cells stably expressing Flag-HA-H1 subtypes through sequential immunoaffinity chromatographies. Three independent affinity purifications from HeLa nuclear extracts expressing Flag-HA-H1 subtypes were used for the MudPIT mass spectrometry assay. Among the reproducible and significant (p-value < 0.001) proteins identified in all three analyses, all subunits of both Cul4A and PAF1 complexes were detected by multiple peptides. The table summarizes the peptide count and the amino acid coverage of Cul4A and PAF1 components co-purified with H1 subtypes. (B) The purified samples shown in (A) were resolved on 4–20% SDS-PAGE, and the presence of the Cul4A and PAF1 complexes was confirmed by immunoblot analysis (C) The reconstituted Cul4A-DDB1-ROC1 (CDR) E3 ligase complex was incubated with glutathione-Sepharose beads containing GST alone, GST-H1.2 full length (FL), GST-H1.2 N-terminal tail (NT), GST-H1.2 globular domain (GD) and GST-H1.2 C-terminal tail (CT). The bound proteins were analyzed by immunoblotting with the indicated antibodies. Input corresponds to 10% of the CDR complex used in the binding reactions. (D) GST alone (lane 2) or GST-H1.2 (lane 3), immobilized on glutathione-Sepharose beads, was incubated with recombinant Cul4A, DDB1 and ROC1. After washing with washing buffer, the bound proteins were immunoblotted with anti-Flag antibody. Ten percent of the input proteins were examined by immunoblotting (lane 1). (E) For the pull-down experiments, the purified PAF1 complex was incubated with GST or the indicated GST-H1.2 fusions and subjected to immunoblotting after extensive washing. Input lane represents 10% of the PAF1 complex used in the binding reactions. (F) GST pull-down assays were conducted as described in (D), but using Flag-tagged subunits of the PAF1 complex that were individually expressed and purified from Sf9 cells. Binding of each protein was analyzed by immunoblotting. Lane 1 represents 10% of the input. See also Figure S1.

Figure 2. Requirements of H1.2 and WDR5 for Cul4A-mediated H4 ubiquitylation

(A) The purified H1.2-associated factors were assayed for in vitro ubiquitin ligase activity using individual core histones in the presence of E1, E2 and His-ubiquitin. Reactions were separated on 15% SDS-PAGE and analyzed by immunoblotting with anti-His antibody. (B) In vitro ubiquitylation assays were performed as in (A), but using HeLa H1-depleted oligonucleosomes as substrates. (C) Mock- or H1.2-depleted 293T cells were transfected with expression vectors for Flag-core histones and/or Cul4A. 48 h post-transfection, whole cell extracts were prepared and immunoprecipitated with anti-Flag antibody. Levels and the monoubiquitylation status of ectopic histones were determined by immunoblotting with anti-Flag antibody. The mobility-shifted bands correspond to monoubiquitylated histones. (D) The CDR, CDRV and CDRW complexes containing neddylated Cul4A were assayed for ubiquitin ligase activity using HeLa H1-depleted oligonucleosomes as substrates. (E) Mock-, VprBP-, or WDR5-depleted cells were transfected with expression vectors for Flag-H4 and/or Myc-Cul4A. Flag-H4 proteins were immunoprecipitated and analyzed by immunoblotting with anti-Flag antibody. (F) Following the expression of Flag-wild type and mutant H4 and/or Cul4A in 293T cells, the ubiquitylation of ectopic H4 proteins was monitored by immunoblotting as in (C). (G) Cell lysates from 293T cells transfected with Flag-H4 were immunoprecipitated with anti-Flag antibody and resolved in SDS–PAGE. The band corresponding to ubiquitylated H4 was excised and analyzed by LC–MS/MS mass spectrometry. The MS/MS spectrum shows that the Lys 31 residue is ubiquitylated in the peptide 24-DNIQGITKPAIR-35. (H) The CDRW complex containing un-neddylated or neddylated Cul4A was assayed for ubiquitin ligase activity using wild type or K31-mutated H4. (I) Mock- or WDR5-depleted cells were transfected with expression vectors for Cul4A and wild type or mutant H4. Ectopic H4 proteins were immunoprecipitated from whole cell lysates, and their ubiquitylation was probed with anti-Flag antibody. See also Figure S2.

Figure 3. Regulation of Cul4A activity by PAF1

(A) HeLa H1-depleted nucleosomes were incubated with neddylated CDRW and/or PAF1 complexes in the presence of E1, E2 and Flag-ubiquitin for in vitro ubiquitylation assays. The ubiquitylated histones were detected by immunoblotting with anti-Flag antibody. (B) Mock- or PAF1-depleted 293T cells were transfected with expression vectors encoding Flag-H4 and/or Cul4A for 48 h. Flag-H4 was purified and analyzed by immunoblotting with anti-Flag antibody. (C) Reconstituted PAF1 complex was incubated with GST-CDRW complex or GST control. GST fusion proteins were precipitated and subjected to immunoblot analysis to detect the subunits of the PAF1 complex. Input corresponds to 10% of the materials used in the binding reactions. (D) GST pull-down assays were performed using components of the PAF1 complex with GST or GST-CDRW complex. After extensive washing, bound proteins were detected by immunoblotting with anti-Flag antibody. Lane 1 represents 10% of the input. (E) The indicated components of the CDRW complex were individually incubated with Flag-PAF1 complex that was immobilized on M2 agarose. Bound proteins were analyzed by immunoblotting. A non-specific band in the Cul4A immunoblot is indicated by an asterisk. Lane 1 represents 10% of the input. See also Figure S3.

Figure 4. Dependence of H3K4 and K3K79 methylation on H4K31 ubiquitylation

(A) 293T cells were transfected with plasmids coding for HA-ubiqutin and Flag-wild type or K31-mutated H4 in the presence or absence of Cul4A for 48 h. After formaldehyde-crosslinking, mononucleosomes were prepared and subjected to immunoprecipitations using anti-Flag antibody. The levels of histone modifications in the isolated nucleosomes were analyzed by immunoblotting. (B) Mock-, H1.2- or PAF1-depleted 293T cells were transfected with expression vectors for HA-ubiquitin and Flag-H4 and/or Cul4A. Mononucleosomes were prepared as in (A), and histone modifications were analyzed by immunoblotting.

Figure 5. Coordinated actions of H1.2, Cul4A and PAF1 in target gene transcription

(A) 293T cells were depleted of H1.2, Cul4A or PAF1, and subjected to microarray analysis. Venn diagrams show the overlapping target genes of H1.2, Cul4A and PAF1. See also Table S1. (B) To validate the gene expression array data, the mRNA levels of the nine down-regulated and one unaffected genes in H1.2/Cul4A/PAF1-depleted cells were quantified by qRT-PCR using primers listed in Supplemental Experimental Procedures. The rescue effects of H1.2/Cul4A/PAF1 expression in H1.2/Cul4A/PAF1-depleted cells were also analyzed as indicated. The values are expressed as fold changes from the mRNA levels in undepleted cells. Results represent the means ± S.D. of three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Statistical tests were performed using one-way ANOVA followed by Bonferroni’s post hoc test. (C) Chromatin immunoprecipitation (ChIP) experiments were performed in 293T cells depleted of H1.2, Cul4A or PAF1 using the indicated antibodies. Precipitation efficiencies relative to non-enriched input samples were determined for seven locations across the human HoxA2 region by quantitative PCR (qPCR) with primers depicted at the bottom and listed in Supplemental Experimental Procedures. Percentage input is determined as the amount of immunoprecipitated DNA relative to input DNA. Nonspecific background signals obtained from a control rabbit IgG are shown by dotted lines, and error bars represent the standard deviation obtained from three independent experiments. See also Figure S4.

Figure 6. Recruitments of H1.2, Cul4A and PAF1 at HoxA2 locus via RNAPII pSer2

293T cells were treated with DMSO or flavopiridol (200 nM) for 3 h, and ChIP assays of the HoxA2 locus were performed using indicated antibodies. ChIP-enriched DNA was quantified by qPCR using the primers indicated at the bottom. The dotted lines represent the signal from negative control rabbit IgG. The data are the means of three independent experiments ± S.D. See also Figure S5.

Figure 7. Selective recognition of RNAPII pSer2 by H1.2

(A) Cell extracts were prepared from DMSO-treated or flavopiridol-treated cells and incubated with GST (lanes 2, 4, 7 and 8), GST-H1.2 (lanes 3 and 5), GST-CDRW (lanes 8 and 10), Flag peptide (lanes 12 and 14) or Flag-PAF1 complex (lanes 13 and 15) immobilized on beads. After extensive washing, RNAPII binding was analyzed by immunoblot with anti-RNAPII antibody. Lanes 1, 6 and 11 represent 10% of the input. (B) The biotinylated CTD heptapeptide repeats that were unmodified or phosphorylated at either position Ser2 or Ser5 were immobilized onto streptavidin-agarose beads and incubated with Flag-H1.2. H1.2 binding to the CTD peptides was determined by immunoblotting with anti-Flag antibody. Input corresponds to 10% of H1.2 used in the binding reactions. (C) Unmodified or phosphorylated CTD peptides were immobilized and incubated with the indicated H1.2 domains, and then pull-down assays were preformed as in (B). Lane 1 represents 10% of H1.2 domains used in the binding reactions. (D) Cell extracts were prepared from DMSO- or flavopiridol-treated cells as in (A), and immunoprecipitated with anti-H1.2 antibody. The amount and phosphorylation level of RNAPII pulled down were examined by immunoblotting. (E) The Ser2 phosphorylated CTD peptides were immobilized and incubated with the Cul4A and PAF1 complexes in the presence or absence of H1.2, and then pull-down assays were performed as in (B). (F) Model for the cooperative role of H1.2, Cul4A and PAF1. Our studies present evidence for H1.2 action targeting post-initiation steps, in which H1.2 selectively recognizes RNAPII CTD Ser2 phosphorylation and brings the Cul4A and PAF1 complexes to target genes. The recruitment and activity of the Cul4A and PAF1 complexes, in turn, stimulate H4K31 ubiquitylation, H3K4me3 and H3K79me2, thereby leading to more productive elongation phase of transcription. Therefore, selective tethering of H1.2 to target loci via CTD Ser2 phosphorylation is essential for the Cul4A and PAF1 complexes to maintain an active state of gene transcription.