Regulation of an inducible promoter by an HP1beta-HP1gamma switch

Abstract

The mammalian heterochromatin protein 1 (HP1) family of proteins was recently shown to be involved in transient repression of inducible promoters. One of these promoters is the HIV1 long terminal repeat, which, during viral latency, recruits a non-processive RNA polymerase II (RNAPII) that synthesizes a short regulatory transcript. Here, we have used this promoter to examine the interplay of HP1alpha, HP1beta and HP1gamma with RNAPII. We find that, in the absence of stimulation, HP1beta is present on the promoter together with the non-processive RNAPII and functions as a negative regulator. On activation, HP1beta bound to methylated H3K9 is rapidly released concurrent with histone H3 phospho-acetylation, and is replaced by HP1gamma. This isoform localizes to the promoter but also inside the coding region, together with the processive RNAPII. Our data show that HP1 recruitment-release is a sequential mechanism that is precisely regulated and highly dependent on transcription.

Figure 1

Heterochromatin protein 1 inactivation modifies HIV1 transcription. (A,B) Schematic representation of HIV1 constructions. (C,D) J-Lat A1 cells were transfected with the indicated siRNAs. After 2 days, messenger RNA and protein levels were determined by RT-qPCR (C) or western blot (D), respectively. (E,F) At 48 h after siRNA transfection in the indicated cell lines, transcript levels were quantified by RT-qPCR. (G,H) Cells were transfected with the indicated siRNAs and treated or not treated with 10 nM PMA. For J-Lat A1 cells, percentage of GFP-positive cells was scored by fluorescence-activated cell sorting (G). For HeLa LTR-Luc cells, luciferase activity was measured (H). CycloB, cyclophilin B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HP1, heterochromatin protein 1; LTR, long terminal repeat; PMA, phorbol-myristate acetate; RT-qPCR, real-time quantitative PCR; siRNA, short interfering RNA.

Figure 2

Activation-dependent recruitment and release of heterochromatin 1 proteins on the HIV1 long terminal repeat. After 2 h of PMA treatment of J-Lat A1 cells, chromatin immunoprecipitation experiments were performed with antibodies specific for HP1α, HP1β and HP1γ (A), or histone H3 tail modifications (B). Enrichment in HP1 LTR chromatin was measured by quantitative PCR using primers spanning the TS region (see Fig 3C). Values are averages from three independent experiments. ac, acetylated; dimet, dimethylated; H, histone; HP1, heterochromatin protein 1; K, lysine; LTR, long terminal repeat; PMA, phorbol-myristate acetate; pS, phospho-Serine; TS, transcription start.

Figure 3

Heterochromatin protein 1 factors colocalize with RNA polymerase II on HIV1 chromatin. After 2 h of PMA treatment of J-Lat A1 cells, chromatin immunoprecipitation experiments were performed with antibodies specific for HP1β, HP1γ or RNAPII amino terminus. Enrichments after immunoprecipitation from (A) unstimulated or (B) PMA-treated cells were measured using quantitative PCR. (C) Schematic representation of PCR amplicons on the HIV1 construct. HP1, heterochromatin protein 1; LTR, long terminal repeat; PMA, phorbol-myristate acetate; RNAP, RNA polymerase II.

Figure 4

HP1β and HP1γ recruitment to the HIV1 long terminal repeat is dependent on the RNA polymerase II. (A) ChIP–reChIP was performed on J-Lat A1 cells treated with PMA for 2 h or untreated. Chromatin was immunoprecipitated first with HP1 or control antibodies and then with anti-RNAPII. (B) Flag-HP1β and Flag-HP1γ were immunoprecipitated (IP) from HeLa S3 cells stably expressing these proteins. Hypo- and hyperphosphorylated forms of RNAPII (IIa and IIo, respectively) were then detected by western bolt with antibodies against RNAPII CTD either non-phosphorylated (8WG16) or phosphorylated on serine 2 or on serine 5 (H5 or H14, respectively), or with an anti-Flag antibody. (C) Pulldown of HA-HP1β with GST-RNAPII CTD expressed in Escherichia coli. (D) J-Lat A1 cells were treated with PMA and actinomycin D as indicated. ChIP experiments were performed using the indicated antibodies. In (A) and (D), chromatin enrichment after immunoprecipitation was measured by using quantitative PCR with primers spanning the transcription start region. Data shown are averaged from two independent experiments. ChIP, chromatin immunoprecipitation; CTD, carboxy-terminal domain; GST, glutathione S-transferase; HA, haemagglutinin; HP1, heterochromatin protein 1; PMA, phorbol-myristate acetate; RNAPII, RNA polymerase II.