Histone Acetyltransferases p300 and CBP Coordinate Distinct Chromatin Remodeling Programs in Vascular Smooth Muscle Plasticity

Abstract

Background: Vascular smooth muscle cell (VSMC) phenotypic switching contributes to cardiovascular diseases. Epigenetic regulation is emerging as a key regulatory mechanism, with the methylcytosine dioxygenase TET2 acting as a master regulator of smooth muscle cell phenotype. The histone acetyl-transferases p300 and CREB-binding protein (CBP) are highly homologous and often considered to be interchangeable, and their roles in smooth muscle cell phenotypic regulation are not known.

Methods: We assessed the roles of p300 and CBP in human VSMC with knockdown, in inducible smooth muscle-specific knockout mice (inducible knockout [iKO]; p300^iKO or CBP^iKO), and in samples of human intimal hyperplasia.

Results: P300, CBP, and histone acetylation were differently regulated in VSMCs undergoing phenotypic switching and in vessel remodeling after vascular injury. Medial p300 expression and activity were repressed by injury, but CBP and histone acetylation were induced in neointima. Knockdown experiments revealed opposing effects of p300 and CBP in the VSMC phenotype: p300 promoted contractile protein expression and inhibited migration, but CBP inhibited contractile genes and enhanced migration. p300^iKO mice exhibited severe intimal hyperplasia after arterial injury compared with controls, whereas CBP^iKO mice were entirely protected. In normal aorta, p300^iKO reduced, but CBP^iKO enhanced, contractile protein expression and contractility compared with controls. Mechanistically, we found that these histone acetyl-transferases oppositely regulate histone acetylation, DNA hydroxymethylation, and PolII (RNA polymerase II) binding to promoters of differentiation-specific contractile genes. Our data indicate that p300 and TET2 function together, because p300 was required for TET2-dependent hydroxymethylation of contractile promoters, and TET2 was required for p300-dependent acetylation of these loci. TET2 coimmunoprecipitated with p300, and this interaction was enhanced by rapamycin but repressed by platelet-derived growth factor (PDGF) treatment, with p300 promoting TET2 protein stability. CBP did not associate with TET2, but instead facilitated recruitment of histone deacetylases (HDAC2, HDAC5) to contractile protein promoters. Furthermore, CBP inhibited TET2 mRNA levels. Immunostaining of cardiac allograft vasculopathy samples revealed that p300 expression is repressed but CBP is induced in human intimal hyperplasia.

Conclusions: This work reveals that p300 and CBP serve nonredundant and opposing functions in VSMC phenotypic switching and coordinately regulate chromatin modifications through distinct functional interactions with TET2 or HDACs. Targeting specific histone acetyl-transferases may hold therapeutic promise for cardiovascular diseases.

Keywords: acetylation; chromatin remodeling; epigenetics; histone acetyltransferases; hyperplasia; smooth muscle cells.

Figure 1:. Differential roles for p300 and CBP in SMC phenotypic modulation

A, B) qPCR analysis of contractile genes in hCASMCs 48 hours after p300 or CBP siRNA knockdown or scrambled siRNA control n=4–6 independent experiments. Student’s t test was performed C-F) Western blot analysis of contractile and synthetic proteins in hCASMCs after p300 or CBP siRNA knockdown or scrambled siRNA control after 48 hours n=4–6 independent experiments. G) Photographs of hCASMCs at indicated time points following scratch wound healing assay. White lines indicate wound front. H) Quantitation of wound front area in 3 random fields were measured over time from each condition, (n=4 independent experiments). Data are expressed as mean±SEM Student’s t test: *P<0.05, **P<0.01, ***P<0.001.

Figure 2:. VSMC p300 and CBP oppositely regulate vessel contractility

A, B) qPCR for indicated contractile protein, p300, or CBP mRNA expression from aorta (after adventitia removed) in p300^iKO and CBP^iKO compared to Control mice. C, D) Maximal tension generated in response to phenylephrine (PE) and KCl in aortic rings from p300^iKO or E, F) CBP^iKO mice compared to controls Data are mean±SEM, n=4–5 mice per group, 3 to 4 aortic rings per mouse. Student’s t test was performed: **P<0.01, ***P<0.001.

Figure 3:. Opposing roles for VSMC p300 and CBP in intimal hyperplasia

A) Elastic Van Gieson staining of tissue sections from or p300^iKO or CBP ^iKO or Control mice 21 days after femoral artery wire injury. Quantification of intima: medial ratio of injured femoral arteries (n=4). Immunostaining for B) Ki67, E) H3K27Ac, H) H3K9Ac in cross-sections of injured arteries from p300^iKO, CBP^iKO, or Control mice 21 days after femoral artery injury. All images are co-stained with DAPI. White dashed line denotes outermost edge of neointima, yellow dashed lines (internal and external elastic laminae) define the media. C, D) Quantification of Ki67 staining in the C) neointimal and D) medial layers was done by counting the total number of Ki67/ DAPI positive cells divided by the number of DAPI positive cells. F, G) Neointimal and G) medial quantification of H3K27Ac staining was done by counting the total number of H3K27Ac/ DAPI positive cells divided by the number of DAPI positive cells. I, J) Quantification of H3K9Ac in the I) neointimal and J) medial layer was done by counting the total number of H3K9Ac/ DAPI positive cells divided by the number of DAPI positive cells. Scale bar, 30 μm. Data are expressed as mean±SEM. One-way ANOVA with Bonferroni’s post hoc test was used to compare differences in knockout mice to controls. *P<0.05, **P<0.01, ***P<0.001.

Figure 4:. p300 and CPB mediate distinct patterns of histone acetylation and RNA Polymerase II recruitment at contractile and synthetic gene promoters.

ChIP q-PCR assays for H3K9Ac, H3K27Ac, and POLII in hCASMCs 48 hours after, p300 or CBP or scrambled siRNA control knockdown in hCASMCs. ChIP assay with antibody to A) H3K9Ac or C) H3K27Ac marks at CArG-containing regulatory regions of contractile gene promoters of ACTA2, CNN1, MYH11 and LMOD1 and C) H3K27Ac at KLF4 promoter after p300 knockdown. ChIP-qPCR assays with antibody to B) H3K9Ac or D) H3K27Ac marks at CArG-containing regulatory regions of contractile gene promoters or D) H3K27Ac at KLF4 promoter after CBP knockdown. E-F) ChIP-qPCR assays with antibody to RNA Polymerase II (PolII) after E) p300 or F) CBP knockdown at CArG-containing regulatory regions of contractile gene promoters. A-F) Negative control IgG immunoprecipitations were amplified with indicated primer sets. Data are presented as mean relative enrichment over input ± SEM from n=5–6 independent experiments. Student’s t test: *P<0.05, **P<0.01, ***P<0.001.

Figure 5:. p300 and CBP differentially interact with and regulate TET2

A, B) ChIP-qPCR assays with antibody to 5hmC at CArG-containing regulatory regions of the CNN1 and MYH11 promoters after 48-hour siRNA knockdown of p300 or CBP or scrambled siRNA control in hCASMC following treatment with 50 nM rapamycin or 10 ng/ml PDGF-BB for 48 hours. Negative control IgG immunoprecipitations were amplified with MYH11 primers C) hCASMC were lysed after 48-hour treatment with 50 nM rapamycin or 10 ng/ml PDGF-BB treatment. Equal amounts of cell lysates from each sample were immunoprecipitated with anti-p300 antibody or negative control IgG followed by western bot analysis for p300 or TET2. 10% of the sample was taken prior to immunoprecipitation and these “Input” samples were analyze for p300, TET2, and GAPDH expression by western blotting. D) After 48 hours siRNA knockdown of p300, CBP, or scrambled siRNA control, hCASMC were treated with 200 μg/mL cycloheximide and harvested at indicated time points for western blot analysis with antibodies against TET2 or β-actin (control). Protein levels were quantitated and TET2 levels normalized to β-actin were plotted to determine TET2 protein half-life Nonlinear regression analysis using one phase decay determined that the calculated half-lives were significantly different with p300 knockdown. E, F) RNA was isolated from aorta of p300^iKO, CBP ^iKO, or Control mice after the adventitia was removed, and qPCR performed for TET2 and normalized to 18S ribosomal RNA. Student’s t test was done for panels D-F or Two-way ANOVA with post hoc Sidak multiple comparisons testing analysis for panels A, B) revealed significant differences between conditions as indicated by asterisks. Data are expressed as mean±SEM from n=5–6 independent experiments. *P<0.05, **P<0.01, ***P<0.001.

Figure 6:. Reciprocal regulation of HATs by TET2

A) Western blot and quantitations (normalized to GAPDH) of contractile proteins, p300, CBP, and TET2 after 48 hours of siRNA mediated TET2 or scrambled siRNA control knockdown in hCASMCs. B) qPCR for contractile genes, p300, CBP, and TET2 after 48 hours of TET2 siRNA knockdown or scrambled siRNA control in hCASMC. C) ChIP-qPCR assay for H3K9Ac or D) H3K27Ac after 48 hours of TET2 siRNA knockdown or scrambled siRNA control in hCASMC. C-D) Negative control IgG immunoprecipitations were amplified with indicated primers. Data are expressed as mean±SEM from n=5–6 independent experiments. Student’s t tests: *P<0.05, **P<0.01, ***P<0.001.

Figure 7:. CBP does not affect HDACs expression but recruits HDACs to contractile gene promoters.

A) mRNA expression (qPCR) for indicated HDACs after siRNA mediated knockdown of CBP compared to control siRNA. B) Western blot analysis of indicated HDACs after siRNA mediated knockdown of CBP compared to control siRNA n. C-D) ChIP qPCR assay with antibodies to HDAC2 and HDAC5 after 48 hours of CBP siRNA knockdown or siRNA control in hCASMC. Negative control IgG immunoprecipitations were amplified with indicated primers. Data are expressed as mean±SEM from n=5–6 independent experiments. Student’s t tests: *P<0.05, **P<0.01.

Figure 8:. P300 and CBP are oppositely regulated in human intimal hyperplasia and model of the mechanistic roles of p300 and CBP in VSMCs.

A) Formalin-fixed artery tissue was obtained from similarly aged patients who had received heart transplants (CAV, n=7–8) or who died of noncardiovascular diseases (non-transplant Control coronary arteries n=7–8). Sections of were immunostained for p300 or CBP and smooth muscle α-actin (ACTA2), with DAPI nuclear counterstain. Representative images shown. Student’s t test was performed. Data are expressed as mean±SEM *P<0.05, **P<0.01. B) Proposed model of SMC phenotypic modulation control by p300 and CBP. Vascular injury releases growth factors including PDGF-BB that can activate the mTORC1 pathway and promote VSMC dedifferentiation to the synthetic phenotype. Rapamycin, an mTORC1 inhibitor, induces VSMC differentiation. We show that Rapamycin increases p300 and TET2 expression and p300 and TET2 association. p300 increases H3K9Ac, H3K27Ac as well as POLII binding at contractile promoters and decreases H3K27Ac at synthetic gene promoters, promoting an open conformation in contractile genes and closed chromatin in synthetic genes. CBP recruits HDACs at contractile gene promoters and decreases H3K9Ac, H3K27Ac as well as POLII binding at contractile genes but increases H3K27Ac at synthetic genes.