Histone exchange is associated with activator function at transcribed promoters and with repression at histone loci

Abstract

Transcription in eukaryotes correlates with major chromatin changes, including the replacement of old nucleosomal histones by new histones at the promoters of genes. The role of these histone exchange events in transcription remains unclear. In particular, the causal relationship between histone exchange and activator binding, preinitiation complex (PIC) assembly, and/or subsequent transcription remains unclear. Here, we provide evidence that histone exchange at gene promoters is not simply a consequence of PIC assembly or transcription but instead is mediated by activators. We further show that not all activators up-regulate gene expression by inducing histone turnover. Thus, histone exchange does not simply correlate with transcriptional activity, but instead reflects the mode of action of the activator. Last, we show that histone turnover is not only associated with activator function but also plays a role in transcriptional repression at the histone loci.

Fig. 1. Genome-wide histone exchange in the absence of TBP.

(A) Upper left panel: the TBP anchor-away assay. Upon addition of rapamycin (+rap; red dot), TBP fused to the rapamycin-binding domain FRB is rapidly exported out of the nucleus through its interaction with an RP (ribos) bearing the complementary FKBP rapamycin-binding domain [for details, see (18)]. Upper middle panel: experimental approach. Cells from a TBP anchor-away strain carrying a galactose-inducible H3^HA construct were grown in raffinose and arrested in G₁ by alpha factor. Galactose was then added at T0 to induce H3^HA expression, together with rapamycin to deplete TBP^FRB from the nucleus [TBP–anchor away (AA)]. As a control, rapamycin was replaced by glucose to limit galactose induction of H3^HA when TBP remains nuclear (control). The right panel is a Western blot analysis for H3^HA expression under the indicated conditions. Lower panels: TBP promoter occupancy and H3^HA incorporation under control (glucose, green lines) or TBP depletion (rapamycin, red lines) conditions were monitored at the indicated genes and time points (minutes) by quantitative ChIP using antibodies against TBP or HA. Results are expressed as percentage of input DNA recovered. Note that H3^HA incorporation at RPL28 was measured at the 3′ gene end. (B) ChIP-seq analysis of H3^HA incorporation under control (green) or TBP depletion (red) conditions at T30 after galactose induction. Shown is a representative region in chromosome VII (965,000 to 1,015,000). The H3^HA ChIP-seq reads were normalized to histone H3 ChIP-seq reads and are expressed in arbitrary units. Shown is one of two independent experiments. (C) Metagene profiles of H3^HA incorporation and H3 occupancy under control (green) or TBP depletion (red) conditions at SAGA-dependent, TATA-containing genes (left panel) and at TFIID-dependent, TATA-less genes (56). Plots show a 1.5-kb region aligned at the transcription start site (TSS) and average H3^HA or H3 enrichment for the indicated conditions. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 2. Histone exchange at Gal4-regulated genes in the presence or absence of TBP or the activator Gal4.

(A and B) Histone turnover in the absence of TBP. mRNA (upper panel) and H3^HA incorporation (lower panel) levels for the indicated control, and Gal4-regulated GAL1 and GAL7 genes were monitored by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and quantitative ChIP under the same experimental conditions, as in Fig. 1A. Measurements were made just before (T0) and at 60 min (T60) after galactose addition or not. See fig. S5 for experimental scheme. The PYK1 mRNA signals were divided by 30× to facilitate comparison. GAL11 is a lowly transcribed gene that shows no histone exchange (41). Shown in (A) is the sample color code and a Western blot analysis for H3^HA expression. (C) Histone turnover in the absence of Gal4. Cells from a Gal4 anchor-away strain carrying a cadmium-inducible H3^HA allele were grown in raffinose and arrested in G₁ by alpha factor. Cadmium was added at T0 to induce expression of H3^HA. Cells were then maintained under basal (i.e., raffinose) conditions, or galactose was added 15 min later without or with prior treatment with rapamycin to deplete Gal4^FRB from the nucleus. mRNA (upper panel) and H3^HA incorporation (lower panel) levels at the indicated genes were monitored as in (B). On the left is a Western blot analysis for H3^HA expression. See figs. S5 and S6 for experimental diagrams and biological replicates of this and all subsequent figures.

Fig. 3. Histone exchange at Msn2/4-dependent heat shock–inducible genes.

(A and B) Histone turnover in the absence of TBP. mRNA (upper panel) and H3^HA incorporation (lower panel) levels under control or TBP anchor-away conditions were measured, as in Fig. 2, except that, where indicated (induced), transcription of the Msn2/4-dependent HSP12 and CTT1 genes was induced by heat shock at 37°C starting 10 min after galactose induction of H3^HA expression. The HSP12 mRNA signals were divided by 5 to facilitate comparison. See fig. S5 for experimental scheme. Shown in (A) is the sample color code and a Western blot analysis for H3^HA expression. WT, wild type. (C) Histone turnover in the absence of Msn2/4. mRNA levels (upper panel) and H3^HA incorporation (lower panel) were monitored at the same genes in a wild-type strain and in a strain deleted for MSN2 and MSN4 [msn2/4 (Δ)]. H3^HA induction and heat shock were as in (B). The CTT1 mRNA signals were multiplied by 5 to facilitate comparison. The sample color code and a Western blot analysis for H3^HA expression are shown above in (A).

Fig. 4. Histone exchange at the Ace1-dependent, copper-inducible CUP1 gene is mediated by the Ace1 AD.

(A and B) Histone turnover in the absence of TBP. Similar experiment as before except that, where indicated (induced), Ace1 binding to DNA was induced by adding copper to the medium. (C) Histone turnover in the absence of Ace1. Similar experiment but after rapamycin-mediated nuclear depletion of Ace1 instead of TBP. nt, not tested. See fig. S5 for experimental details. (D) Upper: Competition strategy to assess the relative contribution of the Ace1 DBD and AD in histone exchange. See fig. S5 for experimental scheme and text for details. Lower: Western blot analysis for expression of the Ace1 truncations produced as GFP fusions from a galactose-inducible expression vector (28). (E) mRNA levels and H3^HA incorporation at the Ace1-regulated CUP1 and indicated control genes under basal or inducing conditions in control cells (none) and in cells overexpressing the wild-type (WT) Ace1 DBD or a point mutant (mut) with attenuated DNA binding affinity (28).

Fig. 5. Rap1 activates gene transcription without inducing histone exchange.

(A and B) mRNA and H3^HA incorporation levels at the Rap1-regulated RPS13 and RPL30 genes and at the inactive STE3 and active ADH1 control genes under normal conditions or following TBP anchor away. (C) Same but following auxin (IAA)–mediated degradation of Rap1 fused to auxin-inducible degron (AID; Rap1^AID). See fig. S5 for experimental details. (D and E) Activation of CUP1 by the Ace1 AD and Rap1 AD. Cells depleted for endogenous Ace1 (see fig. S3D) were made to express the Ace1 DBD alone (none) or fused to either the Ace1 or Rap1 AD. Gene expression and H3^HA incorporation were monitored, as before.

Fig. 6. HIR-mediated histone incorporation at the NEG-containing histone loci.

(A) Upper panels: H3^HA incorporation under control (green) or TBP depletion (red) conditions at the four histone loci and at the highly transcribed ADH1 gene. Data are presented as in Fig. 1B but using a different scale on the y axis. Lower panels: H3^HA incorporation (black bars) and H3 occupancy (gray bars) in wild-type cells or in cells carrying a deletion (Δ) of the gene encoding the Hpc2 subunit of the HIR complex. See fig. S5 for experimental detail. H3 occupancy levels are relative to those measured within a subtelomeric region on chromosome VI, which was set to 10. Note that the H3 occupancy results for ADH1 are presented on a different scale. Lower right: Western blot analysis of H3^HA expression. (B) Transcriptional repression by artificial recruitment of the HIR complex. Upper left panel: The activity of a CYC1 promoter–driven β-galactosidase reporter gene (CYC1-LacZ) carrying (red) or not (green) four upstream LexA-binding sites was monitored in G₁-arrested cells expressing LexA alone or LexA fused to the Hir2 subunit of the HIR complex (36). See text and fig. S5 for experimental details. Lower panels: H3^HA incorporation within the upstream promoter regions of the indicated genes. Note the different scales on the y axis. b.s., binding sites.