Recruitment of Serum Response Factor and Hyperacetylation of Histones at Smooth Muscle–Specific Regulatory Regions During Differentiation of a Novel P19-Derived In Vitro Smooth Muscle Differentiation System
Abstract—Little is known regarding transcriptional regulatory mechanisms that control the sequential and coordinate expression of genes during smooth muscle cell (SMC) differentiation. To facilitate mechanistic studies of SMC differentiation, we established a novel P19-derived clonal cell line (designated A404) harboring a smooth muscle (SM) α-actin promoter/intron-driven puromycin resistance gene. Retinoic acid plus puromycin treatment stimulated rapid differentiation of multipotential A404 cells into SMCs that expressed multiple SMC differentiation marker genes, including the definitive SM-lineage marker SM myosin heavy chain. Using this system, we demonstrated that various transcription factors were upregulated coincidentally with the expression of SMC differentiation marker genes. Of interest, the expression of serum response factor (SRF), whose function is critical for SMC-specific transcription, was high in undifferentiated A404 cells, and it did not increase over the course of differentiation. However, chromatin immunoprecipitation analyses showed that SRF did not bind the target sites of endogenous SMC marker genes in chromatin in undifferentiated cells, but it did in differentiated A404 cells, and it was associated with hyperacetylation of histones H3 and H4. The present studies define a novel cell system for studies of transcriptional regulation during the early stages of SMC differentiation, and using this system, we obtained evidence for the involvement of chromatin remodeling and selective recruitment of SRF to CArG elements in the induction of cell-selective marker genes during SMC differentiation.
Smooth muscle cells (SMCs) play pivotal roles in vascular development and in the formation, maturation, and regression of vascular diseases such as atherosclerosis.1 However, the mechanisms that control differentiation and dedifferentiation of SMCs remain largely unknown. One of the biggest technical difficulties in studies of SMC differentiation has been the lack of a good in vitro differentiation system equivalent to C2C12 and 10T1/2 cells used for studying early stages of skeletal muscle differentiation. In addition, SMCs are not terminally differentiated, and once cultured, they rapidly modulate their differentiated phenotype, thus perturbing the transcriptional regulation of many genes, including the smooth muscle–myosin heavy chain (SM-MHC) gene, that serve as markers of the differentiated state of SMC. It is also evident that cultured SMCs cannot be used for studies of cell-fate determination and initial differentiation of SMCs. To overcome these problems, several groups including ours have developed in vitro culture systems in which multipotent cells, including mouse embryonal carcinoma cells (P19), neural crest stem cells (Monc-1), mouse embryonic stem cells, mouse embryonic 10T1/2 cells, and chicken proepicardial cells, are induced to differentiate into SMCs.2 3 4 5
P19 cells have been successfully used for studies of differentiation mechanisms of neurons, glial cells, and cardiac and skeletal muscle cells.6 Recently, we and others showed that P19s were able to differentiate into SMCs with retinoic acid (RA) treatment.7 8 9 These studies clearly demonstrated that P19s were capable of differentiating into SMCs. However, the low efficacy of SMC differentiation of P19 cells represents a major limitation of this system. That is, the original P19 SMC lineage system described involved conversion of a relatively small fraction of cells to SMCs, and it required the use of dilutional cloning techniques to derive pure populations of SMCs necessary for biochemical and molecular analyses.7 As such, it is impossible to study early inductive and differentiation mechanisms with the original P19 system. The goals of the present studies were to develop a high-efficacy system of SMC differentiation derived from P19 cells and to then use this system to study molecular mechanisms that control the early induction of SMC differentiation marker genes.
Materials and Methods
SM-Specific Promoter-Puromycin Resistance Gene Constructs and Selection of Stable Lines
P19 cells were obtained from American Type Culture Collection (CRL-1825). Cells were maintained in α–minimum essential medium (α-MEM, Sigma, M0644) supplemented with 7.5% fetal bovine serum, 200 μg/mL l-glutamine, and penicillin/streptomycin (Life Technologies). The culture methods for SMC differentiation are outlined in Figure 1A⇓. Cells were plated in a 10-cm dish in α-MEM containing 7.5% FBS and 1 μmol/L all trans-RA on day 0. On day 3, RA was removed from the culture medium. On day 4, cells were trypsinized, plated in two 10-cm dishes, and incubated under the presence of 0.5 μg/mL puromycin (Clontech) for either 2 or 5 days.
Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR)
RNA was purified from A404 and P19 cells, the whole aorta, SMC layers of the stomach and bladder, left and right ventricles of the heart, a portion of the liver, and a portion of the cerebrum. Quantitative multiplex PCR was performed with a gene-specific primer set and a QuantumRNA 18S internal standard primer set (Ambion) in a single tube.
Western Blotting and Immunocytochemistry
Western blotting analysis and immunocytochemical staining were performed as previously described.10
Chromatin Immunoprecipitation (ChIP) Assay
A 10-cm dish of subconfluent undifferentiated and differentiated A404 cells (day 7) were treated with formaldehyde as described previously.11 Chromatin samples were immunoprecipitated with anti–serum response factor (SRF), anti-acetylated histone H3, and anti-acetylated H4 antibodies. Immunoprecipitated chromatin samples were reverse–cross-linked, purified, and subjected to PCR analysis.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Isolation of a Puromycin-Selectable P19-Derived Clonal Cell Line That Showed High-Efficacy Formation of a SMC Lineage
To circumvent low efficacy of SMC differentiation of P19 cells, we isolated P19 clones that could be selected by puromycin for SMC lineages. P19 cells were cotransfected with either a −2560 to 2784 SM α-actin promoter/puromycin-N-acetyltransferase (SMA-PAC) or a −4200 to 11600 SM-MHC promoter/PAC (MHC-PAC), and a cytomegalovirus promoter–driven hygromycin gene. Subsequently, cells were treated with hygromycin to select stable transformants. Thirteen and 35 random colonies were isolated from cells transfected with the SMA-PAC and MHC-PAC constructs, respectively. Genomic PCR was performed to determine if there was integration of the PAC genes. Clones containing PAC genes were then further tested for their ability to differentiate into SMCs. Ten SMA-PAC clones and 28 MHC-PAC clones were treated with RA and then treated with puromycin. Two SMA-PAC clones and 4 MHC-PAC clones survived the puromycin selection. These clones showed much higher levels of SM α-actin and SM-MHC expression than RA treated parental P19 cells. Of these, one line designated A404 exhibited extremely rapid and efficacious induction of virtually all known SMC markers, including SM-MHC, SM α-actin, SM22α, SM calponin, and smoothelin (Figure 1B⇑). Because of the exceptionally high propensity for SMC differentiation, A404 cells were used for further studies.
Multipotential A404 Cells Derived From P19 Cells Showed Highly Efficient Conversion Into SMCs When Treated With RA
Undifferentiated A404 cells grew exponentially and had a spindle shape similar to a subpopulation of parental P19 cells. Expression of SM α-actin and SM-MHC was not detected in undifferentiated A404 cells (Figure 1B⇑). The culture methods used for inducing SMC differentiation are outlined in Figure 1A⇑. Cells were treated with 1 μmol/L RA for 3 days and then cultured in the standard medium for 1 day without RA. On day 4, the majority of these cells expressed SM α-actin and SM-MHC (Figure 1B⇑, lane 5, and Figure 2⇓). A minor population of cells was neuron-like (see Figure 2⇓). Of particular note, expression of all the SM marker genes analyzed was much higher than that of parental P19 cells treated with RA (Figure 1B⇑, lane 5 versus 13).
By treating cells with puromycin at a concentration that could eliminate all undifferentiated A404 cells in 2 days, the expression of SM marker genes was further increased (Figure 1B⇑, lanes 6 through 9). SM-MHC protein was also abundantly expressed in puromycin-treated cells, whereas it was not detected in undifferentiated cells (Figure 3⇓). Although both SM1 and SM2 were detected by using RT-PCR, SM2 was not detected by Western blotting analyses.
To assess the efficiency and efficacy of SMC differentiation, we performed immunocytochemical analyses using anti-SM α-actin, anti–SM-MHC, and anti–neuron-specific tubulin (TUJ1) antibodies. Undifferentiated cells were not stained with these antibodies (Figures 2A⇓, 2D⇓, and 2G⇓). By 4 days after RA treatment, >80% of the cells were SMC-like and stained positive for α-actin (Figure 2B⇓). Most SMC-like cells were also stained positively with SM-MHC antibody (Figure 2E⇓). Approximately 5% to 10% of the cells stained positively with a neuron-specific TUJ1 antibody (Figure 2H⇓). After 2 days of puromycin treatment, the fraction of neuron-like cells was decreased, and very few cells (<0.1%) tested positive for TUJ1 (Figure 2I⇓). All other cells (>90%) were SMC-like and stained positive for both SM α-actin and SM-MHC (Figures 2C⇓ and 2F⇓). Five days of puromycin treatment virtually eliminated all neuronal cells based on staining with the TUJ1 antibody (data not shown). These data indicate that treatment with puromycin enriched SM α-actin–positive cells. Consistent with this, the expression level of a basic helix-loop-helix transcription factor, NeuroD, and a microtubule-associated protein, MAP2C, declined during puromycin treatment (Figure 4A⇓, lanes 5 through 9), whereas in puromycin-nontreated cells, expression of these neuronal markers was sustained (lane 8 versus 10).
To eliminate the possibility that the A404 line consisted of 2 subpopulations that could only differentiate into either SM or neuronal lineages, we isolated 11 subclones from A404 cells by dilutional cloning. All 11 clones were able to differentiate into SMCs and neurons on RA treatment. These results clearly eliminate the possibility that the A404 cell line consists of 2 subsets of cells that have the ability to differentiate into only SMCs or neuronal cells. Rather, results show that A404 cells are capable of differentiating into multiple cell lineages.
The SM α-actin promoter/intron regulatory sequence is activated in developing striated muscle cells in mouse embryos during development.12 As such, it is possible that puromycin selection of RA-treated A404 cells might result in the selection of cardiac and/or skeletal myocytes. However, consistent with previous studies of McBurney et al13 that showed very low efficacy of induction of skeletal or cardiac lineages in RA-treated P19 cells, very weak expression of cardiac α-actin was observed in RA-treated A404 cells on day 4 (Figure 4B⇑, lane 2). Moreover, cardiac α-actin expression was decreased by puromycin selection (lanes 3 and 4). No expression of cardiac α-MHC, skeletal α-actin, and a cardiomyocyte-specific homeobox gene Nkx2-5 was detected by using RT-PCR analyses (Figure 4B⇑ and data not shown). These data indicate that very few cells differentiated into cardiac muscle lineages by RA treatment and that puromycin treatment did not enrich for cardiomyocytes within this cell system.
Various Transcription Factors Implicated in Control of SMC Differentiation Are Induced by RA in A404 Cells
A number of cis elements have been identified as important for the control of SMC-specific genes.14 However, relatively little is known regarding transcription factors that regulate the expression of these genes particularly during the early stages of formation of SMC lineages from multipotential cells. To begin elucidating the circuitry of transcription factors that induce SMC marker genes during the early stages of SMC differentiation and to test the potential utility of A404 cells for studies of transcriptional regulation of SMC marker genes, we analyzed a catalog of transcription factors implicated in the control of SMC marker genes15 16 (Figure 5⇓). Various transcription factors were found to be differentially regulated during SMC differentiation of A404 cells. For example, a Krüppel-like zinc finger transcription factor BTEB2 (KLF5) that we and others found was important for the transcriptional control of SMC marker genes, including SM22α,15 16 was induced on day 1. BTEB2 expression was also detected in SM tissues including the stomach and bladder. We also found that GATA6 was induced on day 1 in A404 cells, and it remained elevated throughout the course of SMC differentiation. In contrast, GATA4 and GATA5 were expressed only transiently at early time points. Although these initial results are descriptive, they demonstrate that various transcription factors implicated in the control of SMC differentiation are induced in the early stages of differentiation of A404 cells and most importantly before detectable upregulation of SMC differentiation markers. As such, the RA-treated A404 cell system described herein should have utility for studies of the transcriptional regulatory circuits that control cell specification and gene expression during the early stages of SMC differentiation.
Whereas SRF Was Abundantly Expressed in Multipotential A404 Cells, Only Cells That Undergo RA-Stimulated SMC Differentiation Showed SRF Binding to CArG-Containing SMC Genes Within Chromatin
SRF-binding sites or CArG elements are crucial for the transcription of virtually all SMC differentiation marker genes characterized to date, including SM-MHC and SM α-actin.1 17 In chicken proepicardial cells, Landerholm et al5 found that SRF was markedly upregulated during SMC differentiation in vitro and that inhibition of SRF function resulted in a reduction in the expression of SMC differentiation marker genes. Similarly, the expression of SRF and its binding to CArG elements coincide with the upregulation of SM γ-actin during chicken gizzard development.18 These results and observations that SRF is highly expressed in developing muscle cells suggest that the high-level expression of SRF may contribute to SMC-selective transcriptional control. However, we found that SRF expression was not increased during differentiation of A404 cells into SMCs, but rather it was abundantly expressed in both undifferentiated and differentiated A404 cells (Figure 5⇑). An alternative possibility is that the activity of SRF may be regulated at the translational and/or post-translational levels. To test if the CArG-binding activity of SRF was increased in association with SMC differentiation, we performed electrophoretic mobility shift assays (EMSAs) using nuclear extracts prepared from undifferentiated and differentiated A404 cells. No increases in SRF binding activity were observed between nuclear extracts derived from undifferentiated versus differentiated A404 cells (Figure 6⇓) despite the fact that the differentiated cells showed marked increases in the expression of multiple CArG-dependent SMC differentiation marker genes.
We hypothesized that although SRF was abundantly expressed and was active in binding to CArG elements in vitro, SRF might not be able to bind CArG elements of the endogenous SMC differentiation marker genes because of the closed state of nucleosomal target sites. To directly test this hypothesis, we performed ChIP assays, which detect binding of transcription factors to target sites in chromatin in living cells. Undifferentiated and differentiated A404 cells were treated with formaldehyde, and cross-linked chromatin was subjected to chromatin immunoprecipitation with anti-SRF antibody. Neither SM α-actin nor SM-MHC CArG regions were amplified from anti-SRF chromatin immunoprecipitates derived from undifferentiated A404 cells (Figure 7⇓, lane 2), whereas the c-fos promoter, which has been shown to be constitutively occupied by SRF in cells,19 was highly enriched in the anti-SRF chromatin immunoprecipitates from undifferentiated A404 cells (Figure 7E⇓, lane 2). In contrast, both α-actin and SM-MHC CArG regions were enriched in immunoprecipitates from differentiated A404 cell samples (lane 5). The enrichment of SM-MHC CArG regions in differentiated A404 cells was highly selective in that we saw no enrichment of these regions in immunoprecipitates from differentiated L6 rat skeletal muscle cells.11 However, in separate studies we found that the CArG region of the skeletal actin promoter was bound by SRF within chromatin in L6 skeletal muscle cells.11 The SM-MHC proximal promoter region that contains a TATA-box and transcription start site but not a CArG element showed no amplification (Figure 7C⇓). Likewise, the amylase gene, which is not CArG-dependent, showed no amplification (Figure 7G⇓). Results of these ChIP assays thus provide clear evidence showing that differentiation of multipotential A404 cells into SMCs is associated with increased SRF binding to the SM-MHC and SM α-actin CArG elements within intact chromatin in the absence of any detectable change in SRF expression or binding activity as measured by using EMSAs.
We next tested whether activation of the endogenous SMC marker genes might involve chromatin remodeling. It has been extensively documented that acetylation of histones H3 and H4 play a central role in chromatin remodeling.20 We thus also performed ChIP analyses with anti-acetylated histone H3 and H4 antibodies. Results showed that acetylation of histone H4 was increased in differentiated A404 cells compared with undifferentiated cells at CArG-containing regulatory regions of the SM α-actin and SM-MHC genes (Figure 7A⇑, lane 14 versus 17). This increase was seen in the CArG regions within the 5′-flanking region of the SM α-actin gene as well as within the 5′-flanking and first intronic regions of the SM-MHC gene. No increase in acetylation of H4 was observed in skeletal actin or amylase genes. Interestingly, the SM-MHC 5′-flanking CArG region also showed hyperacetylation of histone H3, but this was not observed at the α-actin 5′-CArG region or SM-MHC intronic CArG region. The SM-MHC transcription start site showed hyperacetylation of both histones H3 and H4. These results provide evidence for differential hyperacetylation of histones at the regulatory regions of SMC differentiation marker genes. Moreover, taken together with results of ChIP assays, these findings suggest that the induction of CArG-containing SMC differentiation marker genes such as the SM α-actin and SM-MHC genes during early SMC differentiation may be regulated, at least in part, by changes in chromatin structure mediated by histone acetylation. To our knowledge, these results are the first to provide evidence for a role of chromatin remodeling in control of SMC differentiation.
Establishment of a Highly Efficient In Vitro SMC Differentiation System
Multipotential P19 cells have potential utility for various studies of SMC biology because of their ability to differentiate into SMCs after RA treatment. However, their utility for many studies, particularly investigations of early differentiation steps, has been greatly compromised by the low frequency of differentiation of wild-type P19 cells into SMC lineages. In the present studies, we have isolated a derivative of multipotential P19 cells that showed extremely high efficacy of SMC differentiation. Unlike parental P19 cells or other P19 derivatives, the great majority of A404 cells underwent differentiation into SMCs within 4 days of RA treatment. Indeed, based on immunocytochemical analyses, >80% of the total A404 cell population stained positively for SM α-actin by 4 days after RA treatment. A stably integrated SM α-actin promoter–puromycin gene permitted further enrichment of SMCs, and after 2 to 5 days of treatment with puromycin, >90% of cells stained positively for both SM α-actin and the definitive SMC lineage marker SM-MHC. The high efficacy of SMC differentiation observed with A404 cells is in marked contrast with that seen with parental P19 cells where <1% to 5% of cells were estimated to differentiate into SMCs within 4 days (R.S. Blank and G.K. Owens, unpublished observations, 1995). Indeed, in the present studies, SM α-actin expression was barely detectable in RA-treated P19 cells at 4 days after treatment by using RT-PCR analyses (Figure 1B⇑, lane 13). To circumvent this limitation, we previously used dilutional cloning steps that took several months to isolate SMC-like cell lineages.7 To improve the efficacy of SMC differentiation, Suzuki et al8 showed that inhibition of a transcription factor Brn-2 resulted in a higher abundance in SMCs in a P19-derived clonal line compared with wild-type P19 cells when these cells were treated with RA. Although it is difficult to directly compare the efficacy of SMC differentiation between the Brn-2–blocked clone and A404 cells because of significant differences in culture methods used to induce SMC differentiation, expression of SM α-actin and SM-MHC was observed only in later stages culture (day 8 to 20) in the studies by Suzuki et al,8 whereas these markers are readily detectable within 4 days in RA-treated A404 cells.
The results showing the very high efficacy of SMC differentiation of A404 cells compared with parental P19 cells suggest that A404 cells are different from parental P19 cells at least in terms of the induction of SMC-specific genes. The precise mechanisms that resulted in the unique properties of A404 cells are not clear. However, the fact that we obtained multiple P19-derived lines that showed a higher propensity for SMC differentiation indicated that A404 cells are not an outlier clone resulting from spurious activation or inactivation of a gene (or genes) as a result of random insertion of the puromycin resistance constructs. Rather, results suggest that these P19 derivative lines (including A404) may represent a subpopulation of parental P19 cells that have undergone certain as yet undefined determination events that result in their exhibiting a very high propensity for RA-induction of SMC lineages. Irrespective of the precise origins of A404 cells, our observations that RA-treated A404 cells coordinately expressed virtually all widely accepted SMC marker genes, and that this was preceded by the induction of a number of transcription factors implicated in control of SMC differentiation, strongly suggest that these cells retain transcriptional regulatory mechanisms that control SMC differentiation, and that they will be a valuable model system for studying early stages of SMC differentiation. However, it is important to note that there are a number of unresolved issues with respect to these P19-derived cell lines. For example, it is unclear to what extent these cells provide a general model for studying differentiation control processes present in different SMC subtypes, which is an important consideration given recent evidence from our laboratory showing differential transcriptional control of the SM-MHC gene in various SMC subtypes in transgenic mice.11 Further studies are also needed to identify differences in gene expression patterns between A404 (and similar lines) versus parental P19 cells as a means of determining mechanisms responsible for the unique ability of these cells to undergo rapid and coordinate induction of SMC marker genes when treated with RA. However, as illustrated by our use of A404 cells to identify novel mechanisms that regulate SRF binding to the CArG elements within endogenous SMC genes (see below), these cells should have considerable utility for advancing our knowledge of molecular mechanisms that control early stages of SMC differentiation.
Chromatin Remodeling and Recruitment of SRF to the CArG-Containing Regulatory Regions
Although there is extensive evidence showing that SRF is required for expression of SMC-specific genes, SRF is ubiquitously expressed in a variety of cell-types and is necessary for expression of various nonmuscle genes. Thus, a critical question in the field has been to determine how ubiquitously expressed factors such as SRF can regulate SMC-specific transcription. Using ChIP methods, we demonstrated that SRF bound the SMC-specific target regions in chromatin in differentiated cells, whereas it did not bind these target regions in undifferentiated cells despite its abundant expression. These results imply that mechanisms permitting SMC-specific recruitment of SRF to the target sites in chromatin are involved in the initiation of transcription of SMC-specific genes during the early stages of SMC differentiation. We also demonstrated that histones at the CArG containing regulatory regions were hyperacetylated in differentiated cells. Importantly, the skeletal α-actin CArG region was not bound by SRF and was not associated with histone hyperacetylation in differentiated A404 cells, suggesting that SMC-specific regulatory regions were selectively remodeled during A404 cell differentiation. Furthermore, we recently found that the SM-MHC CArG regions were not bound by SRF in non-SM cells including L6 skeletal myocytes and Rat1 fibroblasts, but were bound by SRF in cultured SMCs.11 These data indicate that closed chromatin structure may inhibit SRF from access to the CArG-containing SMC-specific regulatory regions in undifferentiated cells and non-SM cells. Taken together, results support a model in which the induction of SMC-specific genes during the early stages of SMC differentiation is mediated in part by chromatin remodeling and access of transcription factors such as SRF to their cis-binding regions. However, much additional work will be required to determine whether this selective chromatin remodeling is involved in the induction of SMC-specific genes during differentiation of various SMC-subtypes in vivo.
Although it is generally believed that changes in chromatin structure play a central role in cell lineage–specific gene activation during development,21 little is known regarding molecular mechanisms that regulate chromatin structure during differentiation of mammalian cells. Gerber et al22 recently demonstrated that MyoD was able to bind regulatory regions of skeletal muscle–specific genes, which were silent in undifferentiated 10T1/2 cells and induced remodeling of the chromatin structure. Based on these data and data of a few other model systems, it has been suggested that during development sequence-specific transcription factors bind to silent nucleosomal target sites, recruit histone modifying enzymes such as histone acetyl transferases, and promote cell type–selective chromatin remodeling.20 Results of the present studies imply that unidentified sequence-specific transcription factors might bind to the closed state of nucleosomal target sites and initiate opening of SMC-specific transcriptional regulatory regions, thereby permitting other transcription factors including SRF to access target sites within these regions during early stages of SMC differentiation. Alternatively, SRF might be activated by post-translational modifications such as phosphorylation on RA treatment, and activated SRF might then bind the nucleosomal closed CArG elements and cause chromatin remodeling. However, we did not observe increased CArG binding activity in nuclear extracts prepared from differentiated A404 cells in EMSAs, although it is possible that such activation might not be detected in in vitro binding assays by using short oligonucleotide probes. This latter model would also not explain how SRF could selectively bind CArG elements found within SMC-specific genes as opposed to ubiquitously expressed genes like c-fos that are CArG-dependent.
In summary, results of the present studies provide novel evidence for involvement of chromatin remodeling in the induction of SMC-specific genes during SMC differentiation of multipotential P19-derived A404 cells. Moreover, results demonstrate the utility of this novel in vitro SMC differentiation system for studies of mechanisms that control SMC differentiation marker genes within chromatin during the early stages of SMC differentiation.>
This study was supported by grants from the National Institutes of Health (RO1HL57353, RO1HL38854, and PO1HL19242 to G.K.O.) and by a fellowship grant from the Virginia Affiliate of the American Heart Association (VA-F98255V to I.M.). We gratefully acknowledge the expert technical assistance of Diane Raines.
Original received February 14, 2001; revision received April 10, 2001; accepted April 11, 2001.
- © 2001 American Heart Association, Inc.
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