CBP/p300 and SIRT1 are involved in transcriptional regulation of S-phase specific histone genes

Abstract

Background: Histones constitute a type of essential nuclear proteins important for chromatin structure and functions. The expression of major histones is strictly confined to the S phase of a cell cycle and tightly coupled to DNA replication.

Methodology/principal findings: With RT-qPCR and ChIP assays, we investigated transcriptional regulation of the S-phase specific histone genes and found that the acetylation level of histones on core histone gene promoters fluctuated during cell cycle in a pattern similar to RNA polymerase II association. Further, we showed that CBP/p300 and SIRT1 were recruited to histone gene promoters in an NPAT-dependent manner, knockdown of which affected histone acetylation on histone gene promoters and histone gene transcription.

Significance: These observations contribute to further understanding of the mechanism by which the expression of canonical histone genes is regulated, and also implicate a link between histone expression and DNA damage repair and cell metabolism.

Figure 1. Histone H3 acetylation and RNA polymerase II association on histone promoter are cell cycle regulated.

HeLa cells were treated with 75 ng/ml of nocodazole for 22 hours to arrest cells in G2/M phase. Then cells were released into cell cycle progression. At 0, 4, 8 or 12 hours after release, cells were harvested for analyses. (A) Synchronization of HeLa cells in different phases of cell cycle. FCAS was carried out to show the position of cells in the cell cycle (upper panel). Cyclin E mRNA level was measured with reverse transcription-quantitative real-time PCR (RT-qPCR; lower panel), n = 3. (B) Cell cycle regulated H2B expression. H2B mRNA level was quantified by RT-qPCR, n = 3. (C) Cell cycle regulated RNA polymerase II association with the H2B promoter. ChIP was performed with normal mouse IgG or anti-RNA polymerase II (anti-Pol II) antibodies. (D) Cell cycle regulated histone H3 acetylation on the H2B promoter. ChIP was performed with either normal rabbit IgG or anti-AcH3 antibodies. qPCR was used to quantify the enrichment of RNA polymerase II (C) or acetylated histone H3 (D) on the H2B promoter region, n = 3.

Figure 2. CBP and p300 are required for histone gene expression and histone acetylation on histone promoters.

In A–C, F and G, HeLa cells were transfected with 100 nM of CBP or p300 specific siRNA with random siRNA as control (Ctrl). For D and E, HeLa cells were transfected with 3 µg/ml of p300-HA encoding plasmids or empty vector CMV. Cells were harvested for Western-Blot, RT-qPCR or ChIP analysis 48 hours post transfection. (A) The specificity and efficiency of the knockdown of CBP and p300. Western-Blot was performed with rabbit anti-p300, mouse anti-CBP or rabbit anti-Sti1 antibody as indicated. Sti1 (Stress-inducible protein 1) level was used as a loading control. (B) Decreased H2B expression in CBP or p300 knockdown cells. n = 5. (C) Decreased H4 expression in CBP or p300 knockdown cells. n = 5. (D) Over-expression of HA-tagged p300. (E) Increased H2B and H4 expression in p300 over-expressing cells. n = 3. (F) Reduced level of acetylated H3, H4 and RNAPII at the H2B promoter by CBP or p300 knockdown. n = 5. (G) Reduced level of acetylated H3, H4 and RNAPII at the H4 promoter by CBP or p300 knockdown. n = 4. (H) The additive effect of double knockdown of CBP and p300. HeLa cells were transfected with 50 nM of CBP and/or p300 specific siRNA; compensating dosage of random siRNA was used to make the concentration of total siRNA at 100 nM, n = 3. (I) Compensation of reduced H2B expression resulted from CBP knockdown by p300 over-expression. Comparison between column 2 and 3 was analyzed with unpaired t test. n = 3.

Figure 3. The acetylation levels of histones at histone gene promoters decrease in cells with p300 knockdown.

(A) p300 downregulation by siRNA resulted in cell accumulation in G1 phase. siRNA transfection was as described in Fig. 2A. (B) Acetylated histone H3 and H4 on the H2B promoter increased in HU treated cells. n = 4. HeLa cells were treated with 2.5 mM of HU for 24 hours before being harvested. HU was dissolved in PBS buffer. (C) Histone H2B promoter acetylation decreased in HU-synchronized cells with p300 knockdown. HeLa cells were transfected with sip300 or control siRNA as indicated. 24 hours after transfection, cells were switched to fresh media with 2.5 mM of HU for another 24 hours. n = 3. (D) Histone H2B mRNA level and histone promoter acetylation level were reduced in p300 knockdown cells in early S phase. Cells synchronized as described in (C) were released into S-phase for 30 minutes before being harvested for FACS, RT-qPCR and ChIP. Comparison of H2B mRNA levels between released control and sip300 groups was analyzed with unpaired t test. n = 4.

Figure 4. CBP and p300 associate with histone promoters in an NPAT-dependent manner.

ChIP assays were performed with rabbit anti-CBP or anti-p300 antibodies. (A) CBP and p300 association with the H2B promoter. n = 5. (B) CBP and p300 association with the H4 promoter. n = 5. (C) Co-immunoprecipitation of CBP with NPAT. Whole cell extracts of HeLa cells transfected with pCMV-NPAT were used to carry out co-immunoprecipitation with rabbit anti-CBP antibodies. Precipitated proteins were detected with Western-blot using mouse anti-CBP antibodies or mouse anti-NPAT antibodies as indicated. (D) The efficacy of NPAT knockdown. Western-Blot was performed with rabbit anti-NPAT or rabbit anti-Sti1 antibody as indicated. Sti1 was used as a loading control. (E) Reduced association of CBP and p300 with the H2B promoter in NPAT knockdown cells. n = 3. (F) Reduced association of CBP and p300 with the H4 promoter in NPAT knockdown cells, n = 3.

Figure 5. Cell cycle regulated association of CBP and NPAT with the H2B promoter.

(A) CBP and NPAT association with histone promoter in different phases of a cell cycle. HeLa cells were synchronized at G2/M, G1, G1/S and mid-S phases as described in Figure 1. ChIP assays were performed with rabbit anti-CBP or rabbit anti-NPAT antibodies with normal rabbit IgG as control. Enrichment of CBP or NPAT on H2B promoter at G1, G1/S or S phase was compared with that of G2/M phase, respectively, n = 5. (B) CBP and NPAT protein levels in different phases of a cell cycle. HeLa cells were synchronized with nocodazole and then released for different time points as indicated. Whole cells extracts were used for Western-Blot analyses. Membranes were blotted with mouse anti-CBP, rabbit anti-NPAT or mouse anti-tubulin. Tubulin was used as loading control.

Figure 6. The HAT activity of CBP/p300 is important for histone gene transcription.

(A) HAT activity of ectopically expressed p300. Plasmids pCMV-p300HA which encodes HA-tagged p300 or pcDNA3.1-p300HAT- which encodes p300 (HAT-) mutant were transfected into HeLa (left panels) or 293T cells (right panels). Western-blot was employed to determine protein expression and the HAT activity. (B) Histone H2B and H4 transcription was enhanced by p300HA but not p300 (HAT-). n = 4. (C) p300 (HAT-) mutant was unable to rescue the down-regulation of H2B transcription resulted from CBP knockdown. HeLa cells were co-transfected with siRNA and plasmid DNA as indicated. 48 hours after transfection, cells were harvested for RT-real time qPCR. Comparison between column 2 and 3 was analyzed with unpaired t test. n = 4.

Figure 7. Involvement of SIRT1 in regulating the expression of S-phase specific histone genes.

(A) H2B expression pattern of HeLa cells released from HU treatment. HeLa cells were synchronized at G1/S border with HU and then released into S phase. Cells were harvested at 2 hour interval and were analyzed with FACS and RT-PCR. 0 hour, G1/S border; 2 hours, early S-phase; 4 and 6 hours, mid-S-phase; 8 and 10 hours, late S-phase; 12 hours, S/G2 border; 16 hour, G2/M phase. (B) Enhanced H2B expression by HDAC inhibitors. HeLa cells synchronized at G1/S transition with HU were released and concomitantly treated with 2 µM TSA or 40 mM NAM for 2 hours. H2B mRNA level at 2 hours post release was set as 1, n = 4. (C) Increased H2B promoter activity as a result of inhibiting SIRT1. n = 3. (D) The efficacy of SIRT1 knockdown. Western-Blot was used to determine the SIRT1 protein level. The level of Sti1 serves as a control. (E) Up-regulated H2B and H4 expression in SIRT1 knockdown cells, n = 3. (F) Repressed H2B expression upon SIRT1 activation. HeLa cells were transfected with SIRT1 specific siRNA for 45 hours and then treated with 20 µM of resveratrol for additional 3 hours, n = 3. RSV, resveratrol, was dissolved in ethanol. Comparison between RSV treated groups with or without siSIRT1 transfection was analyzed with unpaired t test.

Figure 8. SIRT1 associates with histone gene promoters and impacts histone acetylation on target promoters.

(A) SIRT1 associates with histone H2B and H4 promoters. (B) The SIRT1 association with histone promoters requires NPAT. (C) Increased levels of acetylated H3 and H4 on the H2B promoter in SIRT1 knock-down cells. (D) Increased levels of acetylated H3 and H4 on the H4 promoter in SIRT1 knock-down cells. In A–D, n = 3.

Figure 9. Growth phenotypes of HeLa cells knockdown of CBP or SIRT1.

HeLa cells were transfected with 100 nM of different siRNA as indicated. At different time points post transfection, the numbers of viable cells were counted with tryphan blue staining. Random siRNA and siRNA specific for p38/GAPDH or NPAT were controls. Results were mean of triplicates.

Figure 10. A model for the regulation of histone gene transcription.

At G1/S transition of a cell cycle, the cellular NAD+/NADH ratio is proper for the assembly of the OCA-S complex which facilitates the recruitment of NPAT to the histone H2B promoter. NPAT, phosphorylated by cyclin E/cdk2, in turn facilitates the recruitment of CBP/p300 and SIRT1 to histone promoters in a global fashion. CBP/p300, which is also phosphorylated by cyclin E/cdk2, enhances histone acetylation on histone promoter regions hence activating S-phase histone transcription. In late S phase, the CBP/p300 association with histone promoters is decreased, likely accompanied by increased SIRT1 recruitment, which in conjunction with more oxidative cellular redox (higher NAD+ level) stimulates the HDAC activity of SIRT1 specifically targeting histone gene promoters hence accounting for declination of histone expression in a coordinated fashion. This model adds another layer of regulation of redox-modulated histone expression.