Dissecting the roles of the histone chaperones reveals the evolutionary conserved mechanism of transcription-coupled deposition of H3.3

Abstract

The mammalian genome encodes multiple variants of histone H3 including H3.1/H3.2 and H3.3. In contrast to H3.1/H3.2, H3.3 is enriched in the actively transcribed euchromatin and the telomeric heterochromatins. However, the mechanism for H3.3 to incorporate into the different domains of chromatin is not known. Here, taking the advantage of well-defined transcription analysis system of yeast, we attempted to understand the molecular mechanism of selective deposition of human H3.3 into actively transcribed genes. We show that there are systemic H3 substrate-selection mechanisms operating even in yeasts, which encode a single type of H3. Yeast HIR complex mediated H3-specific recognition specificity for deposition of H3.3 in the transcribed genes. A critical component of this process was the H3 A-IG code composed of amino acids 87, 89 and 90. The preference toward H3.3 was completely lost when HIR subunits were absent and partially suppressed by human HIRA. Asf1 allows the influx of H3, regardless of H3 type. We propose that H3.3 is introduced into the active euchromatin by targeting the recycling pathway that is mediated by HIRA (or HIR), and this H3-selection mechanism is highly conserved through the evolution. These results also uncover an unexpected role of RI chaperones in evolution of variant H3s.

Figure 1.

Histone H3.3 is enriched in the actively transcribed region. (A) Human H3s (hH3.1 and hH3.3) with a N-terminal 3HA tag were expressed in the wild-type yeast strain (YC73) at similar levels. Immunoblotting analysis was performed with the whole-cell extract prepared from yeasts that harbor the pRS425-pTFA1-HA-hH3.1 or hH3.3. Tfg2, a subunit of transcription factor IIF was used as a loading control. (B) The schematic diagram of the GAL1 promoter-linked YLR454 gene and GAL1 with the PCR primer pairs used for ChIP. The TATA box represents the promoter and the transcription start site (TSS) is indicated by an arrow. (C) Human H3.3 was preferentially incorporated in the actively transcribed region. The target gene was induced by shifting the culture from raffinose- to galactose-containing medium and then subsequently repressed by addition of glucose. ChIP was performed with the 12CA5 antibody using chromatin solution prepared from the raffinose and glucose culture. Each signal was quantified with real-time quantitative PCR and normalized to the intergenic control and the input DNA. The ChIP value obtained in raffinose was arbitrarily set to 1. Raf indicates the HA-H3 level before induction of target genes, while Glc indicates HA-H3 level accumulated through transcription. The data were reported as the mean ± SD from at least three independent experiments. (D) Yeast H3 (yH3), human H3.1 and hH3.3 with an N-terminal 3HA tag were expressed at similar levels. (E) Comparison of incorporation efficiency of different HA-H3s.

Figure 2.

Amino acid residues in the ID region are important for variant-specific H3 deposition. (A) The amino acid alignment showing sequence difference in ID region among yH3, hH3.1 and hH3.3. H3 point mutants used in figure (C) and S2B-D are shown in parallel. (B) The expression levels of the H3s under the TFA1 promoter were similar. (C) Incorporation of HA-tagged H3s was analyzed by ChIP. Data points of the HA-H3 occupancy obtained from the glucose sample were plotted.

Figure 3.

The yeast HIR complex is involved in the selective incorporation of hH3.3 during transcription. (A) The expression levels of hH3.1 and hH3.3 in the wild-type (YC73), hir1Δ (YC199), hir3Δ (YC264) and hpc2Δ (YC265) detected by immunoblotting analysis. (B and C) The selectivity of H3 toward H3.3 is lost in the absence of HIR1. The occupancy of HA-tagged H3s [hH3.1 and hH3.3 in (B) or yH3 and yH3(VM) in (C)] was analyzed by ChIP. The occupancy levels of HA-yH3s in wild-type yeasts are plotted as open circles (C). (D) Human H3.1 incorporation is abnormally increased during transcription in the absence of HIR subunits (hir3Δ, YC264; hpc2Δ, YC265; hir1Δhir2Δ, YC293). HA-hH3.1 incorporation was analyzed within pGAL1-YLR454 by ChIP. Yeast strains were arrested appropriately by α-factor. (E) Deletion of HIR complex subunits results in an overall loss of Hir3 protein level. The Hir3 protein level in the wild-type (YC73), single (hir1Δ, YC199; hir2Δ, YC208; hir3Δ, YC264; hpc2Δ, YC265) or double deletion mutants (hir1Δhir2Δ, YC293; hir1Δhpc2Δ, YC297; hir1Δhir3Δ, YC296) was detected by immunoblotting analysis with an anti-Hir3 antibody.

Figure 4.

Human HIRA functionally complements the yeast HIR phenotype. (A) The schematic diagram of the domain structure of human HIRA. Filled circle, 7 × WD40 repeat (residues 1–356); open oval, B domain (residues 448–471); filled box, Hir2-like domain (residues 763–962). Protein domains were analyzed using InterProScan software (http://www.ebi.ac.uk/InterProScan/). (B) All human HIRA constructs were expressed in hir1Δ. The immunoblotting analysis was performed with the whole cell extract prepared from hir1Δ (YC199) expressing indicated HIRA constructs. The arrows indicate the position of full-length and truncated HIRA proteins. The asterisk indicates a nonspecific band. Protein molecular mass markers are shown. (C) Expression of human HIRA partially suppressed hH3.1 incorporation in hir1Δ. HIRA-FL and the HIRA-N2(1–729) partially suppressed hH3.1 incorporation in hir1Δ. The incorporation of HA-hH3.1 in yeast strains indicated in Figure 4B was analyzed by ChIP. (D) Expression of human HIRA partially suppresses hH3.1 incorporation in hir1Δhir2Δ (YC293). The incorporation of HA-hH3.1 in hir1Δ and hir1Δhir2Δ carrying YEp352GAPII (empty vector) or YEp352GAPII-HIRA-FL(1-1017)-FLAG was analyzed by ChIP.

Figure 5.

The C-terminal HUN domain of Hpc2 is essential for the selective incorporation of hH3.3 during transcription by maintaining the stability of the HIR complex. (A) The schematic diagram of the domain structure of yeast Hpc2 and human UBN1. Boxes indicate three domains (N-terminal CDI, C-terminal CDII and HUN) conserved among Hpc2 homologs of yeast species and higher eukaryotes. The amino acid alignment (bottom) shows sequence similarity of HUN domains between yeast Hpc2 and human UBN1. (B) Human H3.1 and H3.3 incorporations in yeast strains expressing wild-type Hpc2-13Myc (YC298) or Hpc2ΔC-13Myc (YC299) were analyzed by ChIP. (C) Expression of Hpc2-FL-FLAG, Hpc2-ΔN-FLAG and Hpc2-ΔC-FLAG in hpc2Δ (YC265) was confirmed by immunoblotting by using an anti-FLAG antibody. The molecular mass markers are shown. (D) The Hpc2-ΔC lacking C-terminal HUN domain fails to suppress hH3.1 incorporation in hpc2Δ. The incorporation of HA-hH3.1 was analyzed by ChIP. (E) Lack of C-terminal HUN domain of Hpc2 causes a significant decrease in the Hir3 protein level. The Hir3 protein level in wild-type (YFC193) and hpc2Δ (YC302)-expressing Hpc2-FL-FLAG, Hpc2-ΔN-FLAG and Hpc2-ΔC-FLAG was detected by immunoblotting analysis with an anti-Hir3 antibody. (F) Co-immunoprecipitation assay of Hpc2, Hir2 and Hir3. The whole-cell extract from hpc2Δ cells (YC302) expressing indicated proteins was prepared. Co-IP was performed with anti-Flag antibody followed by immunoblotting with anti-Hir3 or anti-CBP (to detect TAP) antibodies.

Figure 6.

The histone chaperone HIR complex and Asf1 cooperate together for selective accumulation of hH3.3 during transcription. (A) Wild-type Asf1 and the Asf1 V94R mutant with a C-terminal 13Myc tag were expressed in asf1Δhir1Δ (YC252). All yeasts expressed HA-H3 at similar levels. (B) ChIP analysis of HA-hH3.1 and hH3.3 incorporation in asf1Δhir1Δ (YC252). The data points of the HA-hH3.1 and hH3.3 occupancy obtained in hir1Δ (glucose sample) from Figure 3B were depicted as open circles and shown for comparison. (C) ChIP analysis of HA-hH3.1 and -hH3.3 incorporation in asf1Δhir1Δ expressing either wild-type or Asf1 (V94R) mutant. (D) ChIP analysis of HA-hH3.3 and HA-hH3.1 incorporation in cells expressing wild-type Asf1 or yAsf1ΔC. (E) A model suggesting that Asf1 and HIRA complex cooperate for selective accumulation of hH3.3 in the active genes during transcription. Asf1 and HIRA complex are required for disassembly and reassembly of nucleosomes on the path of polymerase II, allowing for the incorporation of new histones. HIRA complex normally mediates recycling of histones and deposits old H3 to assemble nucleosomes. In this model, hH3.3, but not hH3.1, efficiently binds to HIRA complex and competes with recycling histones to incorporate into nucleosomes. (F) A potential role of RI histone chaperones in the evolution of histone H3 variants.