PRISM/PRDM6, a transcriptional repressor that promotes the proliferative gene program in smooth muscle cells

Abstract

Smooth muscle cells (SMCs) display remarkable phenotypic diversity and plasticity and can readily switch between proliferative and differentiated states in response to extracellular cues. In an effort to identify novel transcriptional regulators of smooth muscle phenotypes, we compared the gene expression profiles of arterial and venous SMCs by microarray-based transcriptional profiling. Among numerous genes displaying distinct expression patterns in these two SMC types, we discovered an expressed sequence tag encoding a previously uncharacterized zinc finger protein belonging to the PRDM (PRDI-BF1 and RIZ homology domain) family of chromatin-remodeling proteins and named it PRISM (PR domain in smooth muscle). PRISM is expressed in a variety of smooth muscle-containing tissues and displays especially robust expression in the cardiac outflow tract and descending aorta during embryogenesis. PRISM is localized to the nucleus and contains an amino-terminal PR domain and four Krüppel-like zinc fingers at the carboxy terminus. We show that PRISM acts as a transcriptional repressor by interacting with class I histone deacetylases and the G9a histone methyltransferase, thereby identifying PRISM as a novel SMC-restricted epigenetic regulator. Overexpression of PRISM in cultured primary SMCs induces genes associated with the proliferative smooth muscle phenotype while repressing regulators of differentiation, including myocardin and GATA-6. Conversely, small interfering RNA-mediated knockdown of PRISM slows cell growth and induces myocardin, GATA-6, and markers of SMC differentiation. We conclude that PRISM acts as a novel epigenetic regulator of SMC phenotypic plasticity by suppressing differentiation and maintaining the proliferative potential of vascular SMCs.

FIG. 1.

Deduced amino acid sequence and homology of PRISM with other PR proteins. (A and B) Schematic and deduced amino acid sequence of PRISM. The PR domain is shown in blue and the Krüppel-like zinc fingers (Zn) are shown in red. The amino acid positions of the deletion constructs are indicated. (C) Comparison of PRISM with other proteins from the PR family showing homology through the PR/SET domain.

FIG. 2.

Tissue distribution of PRISM. (A) RT-PCR analysis of PRISM. RT-PCR was performed using RNA isolated from adult mouse tissues as described in Materials and Methods. (B) Northern blot analysis of PRISM. Mouse multiple-tissue Northern blots (Ambion) were probed with a radiolabeled PRISM cDNA fragment. Expression of PRISM in whole E14.0 embryo, adult lung, ovary, heart, brain, and thymus is shown. (C) Expression of human PRISM. A human cardiovascular blot (Clontech) was probed with mouse PRISM. Cardiovascular expression of PRISM is specific to the aorta. PRISM expression was detected in sagittal and transverse sections of E11.5 (D), E13.5 (E), and E15.5 (F, G, H, J, and K) mouse embryos. (I) Transverse section of E18.5 mouse embryo exposed to PRISM riboprobes. Antisense probes corresponding to unique PRISM sequences were used as described in Materials and Methods. Staining of the second branchial arch artery (baa), aorta (a), outflow tract (ot), trachea (tr), lung (lu), pulmonary trunk (pt), ductus arteriosus (da), bladder (bl), bladder lamina propria (lp), suburethral space (su), and central nervous system (cns) is depicted.

FIG. 3.

Transcriptional repression, nuclear localization, and homodimerization of PRISM. (A) PRISM is a transcriptional repressor. Results for cotransfections of a 5× GAL4-UAS luciferase reporter with the GAL4 DBD alone or with 50, 100, and 150 ng of GAL4-PRISM fusions are shown. All luciferase data are normalized to protein content following cell lysis. (B) Nuclear localization of PRISM. Immunocytochemistry of FLAG-PRISM-transfected COS-7 cells. FLAG-PRISM is directed to the nucleus by the carboxy-terminal Zn fingers. (C) PRISM forms homodimers. Immunoprecipitation (IP) experiments of epitope-tagged full-length PRISM and PRISM deletions in cotransfected COS-7 cells. Input controls are shown below the immunoprecipitation panels. IB, immunoblotting. The amino acid positions of the deletion constructs are indicated.

FIG. 4.

Interaction of PRISM with chromatin-remodeling enzymes. (A) Coimmunoprecipitation assays of PRISM and HP1-β. COS-7 cells were transfected either with HP1-β alone or in combination with full-length p300 and deletions of PRISM. Lysates were immunoprecipitated (IP) with anti-myc antibodies, and immunoblots (IB) were performed with anti-FLAG as described in Materials and Methods. (B through D) Coimmunoprecipitations of PRISM and HDAC1, HDAC2, and HDAC3, respectively. COS-7 lysates from class I HDACs alone or from HDACs plus PRISM transfections were incubated with anti-myc (PRISM) and subjected to immunoblot analysis using anti-FLAG (HDACs) antibodies. Input controls for each experiment are shown below. (E) Coimmunoprecipitation experiment using full-length p300 and deletions of PRISM. Lysates were immunoprecipitated with anti-HA (p300), and blots were probed with anti-FLAG (PRISM). Input controls for each experiment are shown below the immunoprecipitation panels. The amino acid positions of the deletion constructs are indicated.

FIG. 5.

PRISM contains two repressive domains and recruits methyltransferase activity. (A) The PR and Zn finger domains of PRISM repress transcription. Cotransfections of the 5× GAL4-UAS luciferase reporter and either the GAL4 DBD alone (pM) or GAL4-PRISM (100 ng) constructs are shown. All data are normalized to protein content as described above. (B) Histone deacetylase inhibitors do not abrogate PRISM-mediated repression. COS-7 cells were cotransfected with 5× GAL4-UAS and either the GAL4 DBD alone or GAL4-PRISM (100 ng) and treated with 100 nM TSA for 24 h prior to harvest. (C) Immunoprecipitations (IP) of PRISM with G9a. Cotransfected lysates were incubated with anti-HA (G9a) antibody followed by immunoblotting (IB) with anti-FLAG (PRISM). (D) PRISM associates with HMT activity. Results from histone methylation assays from transfected cell lysates are shown. Cells were transfected with PRISM, G9a, or both and subjected to immunoprecipitation using the designated antibody and subsequent methyltransferase assays. All samples containing PRISM were immunoprecipitated with anti-FLAG (PRISM) antibodies. The amino acid positions of the deletion constructs are indicated.

FIG. 6.

PRISM regulates the expression of SM proliferative and contractile genes. (A and B) Induction of proliferative SM genes in primary SMCs overexpressing PRISM. (A) Microarray data are presented as increases or decreases compared to levels for SMCs infected with empty adenovirus. (B) Representative RT-PCR products and concomitant real-time RT-PCR values validating the overexpression data obtained as shown in panel A. (C) A growth curve following PRISM knockdown in primary SMCs is depicted. Cells from all time points were harvested in triplicate and subjected to colorimetric analyses using WST-1 reagent as described in Materials and Methods (P = 0.03 at 48 h). (D) Representative RT-PCR products following PRISM knockdown are depicted. The SM-MHC panel was generated from RNA harvested 48 h posttransfection. All other panels were from samples harvested at 24 h.

FIG. 7.

Summary of PRISM domains. A schematic of PRISM, including the amino acid positions of the deletion constructs and their activities, is shown. Repr, transcriptional repression; C, cytoplasmic; N, nuclear.