Miano Lab
Smooth muscle cell (SMC) differentiation is defined as the expression of a transcriptome encoding for a battery of contractile/cytoskeletal proteins that coordinates the contractile phenotype of this cell type (A). In several pathological conditions, such as atherosclerosis or restenosis following balloon angioplasty (B), this differentiated phenotype is subverted to a tumor-like, de-differentiated state (C). This implies that SMC of the vessel wall can undergo profound changes in the expression of genes that delimit its normal function. Thus, contractile/cytoskeletal genes such as SM22a and the smooth muscle isoforms of myosin heavy chain, alpha actin, and calponin are all compromised in the setting of vascular disease. The pathological changes in the vessel wall, as well as changes in SMC gene expression, may be attenuated with a variety of drugs. In the Miano Lab, retinoids have been under study for a number of years as a potential therapeutic intervention for vascular diseases. Retinoids induce a number of genes through ligand-activated nuclear receptors. In a screen for retinoid-inducible genes, we discovered a number of genes whose expression is compromised in the setting of vascular disease. These genes as well as some of the aforementioned contractile/cytoskeletal genes are under intense study to elucidate their transcriptional regulation through promoterology (the study of gene promoters and regulatory sequences controlling gene expression).
The Miano Lab Research Pipeline therefore begins with some model of human pathology. While we study primarily atherosclerosis and restenosis (B), we are also interested in other diseases where vascular SMC changes occur such as Alzheimer’s Disease (not shown). Gene promoters identified in various screens are then assayed in vitro and ultimately in transgenic animal models such as mice and zebrafish. Our transgenic mouse models of SMC gene promoters encompass conventional transgenic approaches using the bacterial lacZ reporter gene or large genomic sequences contained in bacterial artificial chromosomes. Panel D shows an embryo carrying the SM22alpha promoter linked to a nuclear lacZ reporter gene. This reporter affords exquisite cellular resolution as shown in panel E. Promoters regulating genes such as SM22alpha have been exploited in tissue-specific knockout studies. Recently, we have turned to Danio rerio (zebrafish) as a tractable model system to assess mammalian SMC-related gene promoters (F). In this manner, we can rapidly appraise the regulation of SMC genes in this versatile vertebrate system for eventual validation in the more labor-intensive mouse model. Defining the activity of SMC gene promoters in vivo provides an opportunity to define DNA sequence elements that are critical for such regulation. We use a variety of bioinformatic tools to define evolutionarily-conserved DNA sequences that may be critical for SMC gene activity. For example, VISTA plots are generated through a process known as comparative genomics (G). The completion of numerous genomes has greatly facilitated this analysis. Thus, the largely pink band across the top of panel G reflects the high conservation of DNA sequence between human and chimp. This conservation progressively decreases as homologous sequences from increasingly divergent animals are evaluated (descending traces in panel G). Regulatory element discovery (the SMC ‘regulome’) is thus a major goal in the Miano Lab. One regulatory element we are particularly interested in is the CArG element (H), whose consensus sequence is CC(A or T)6GG. The CArG element binds serum response factor (SRF) which “toggles” between disparate gene programs based on its association with a variety of cofactors. One such cofactor we have been studying is called myocardin (I). Together SRF-myocardin coordinate a SMC differentiation program of gene expression. Thus, we hypothesize that SRF-myocardin activity is altered in vascular diseases leading to a compromise in expression of many CArG-containing genes such as those encoding for contractile/cytoskeletal proteins. The pipeline of discovery is repeated therefore with the evaluation of new genes and complexes (e.g., SRF-myocardin) in the setting of vascular diseases, followed by analysis of their promoters in mice and fish to define DNA sequences important for regulation etc.