Regulation of smooth muscle-specific transcription by serum response factor and formin homology domain containing protein 1

The regulation of vascular smooth muscle cell (SMC) differentiation is important during vasculogenesis, angiogenesis, and cardiovascular diseases, such as atherosclerosis and restenosis. Previous studies have shown that SMC differentiation marker gene expression is regulated by serum response factor (SRF) and the myocardin family of SRF co-factors (myocardin and the myocardin-related factors, MRTF-A and MRTF-B). A major goal of the current studies was to identify post-translational modifications of SRF that regulate SMC-specific gene expression. By screening phosphorylation deficient and mimetic mutations in SRF -/- ES cells, I identified T159 as a phosphorylation site that significantly inhibits SMC-specific gene expression. In vitro and in vivo labeling studies demonstrated that T159 was phosphorylated by protein kinase PKA, and results from gel shift and chromatin immunoprecipitation assays demonstrated that T159 phosphorylation inhibited SRF binding to the CArG elements present within the promoters of the SMC-specific genes. Based upon the identification of Ubc9 in a yeast-two-hybrid screen for SRF binding proteins, I also tested the role of sumoylation on SRF activity. In vitro sumoylation assays identified K147 as the major SRF sumoylation site, but a sumoylation deficient K147R mutation had no effect on SRF-dependent SMC-specific gene expression. Our lab has also demonstrated that MRTF nuclear localization and activity is regulated by changes in actin dynamics, and a second goal was to determine whether the diaphanous formin, FHOD1, played a significant role in this process. Using RNAi techniques I demonstrated that FHOD1 was important for SMC differentiation marker gene expression in 10T1/2 and that over expression of a constitutively active version of FHOD1 strongly up-regulated SMC-specific promoter activity. Additional studies showed that phosphorylation of FHOD1 in the diaphanous auto-regulatory domain may contribute to FHOD1 activation and that FHOD1-mediated actin polymerization in the nucleus may be important for FHOD1's effects on MRTF activation. Taken together, my results indicate that PKA-mediated phosphorylation of SRF and FHOD1-mediated actin polymerization regulate SMC-transcription providing two novel signaling mechanisms for the control of SMC phenotype. Future experiments extending these findings should lead to a better understanding of SMC's role in cardiovascular disease and to targets for treating these conditions

The 16p11.2 homologs fam57ba and doc2a generate certain brain and body phenotypes

Deletion of the 16p11.2 CNV affects 25 core genes and is associated with multiple symptoms affecting brain and body, including seizures, hyperactivity,macrocephaly, and obesity. Available data suggest thatmost symptoms are controlled by haploinsufficiency of two or more 16p11.2 genes. To identify interacting 16p11.2 genes, we used a pairwise partial loss of function antisense screen for embryonic brainmorphology, using the accessible zebrafish model. fam57ba, encoding a ceramide synthase, was identified as interacting with the doc2a gene, encoding a calcium-sensitive exocytosis regulator, a genetic interaction not previously described. Using genetic mutants, we demonstrated that doc2a+/-fam57ba+/-double heterozygotes show hyperactivity and increased seizure susceptibility relative to wild-type or single doc2a-/-or fam57ba-/-mutants. Additionally, doc2a+/-fam57ba+/-double heterozygotes demonstrate the increased body length and head size. Single doc2a+/-and fam57ba+/-heterozygotes do not show a body size increase; however, fam57ba-/-homozygous mutants show a strongly increased head size and body length, suggesting a greater contribution from fam57ba to the haploinsufficient interaction between doc2a and fam57ba. The doc2a+/-fam57ba+/-interaction has not been reported before, nor has any 16p11.2 gene previously been linked to increased body size. These findings demonstrate that one pair of 16p11.2 homologs can regulate both brain and body phenotypes that are reflective of those in people with 16p11.2 deletion. Together, these findings suggest that dysregulation of ceramide pathways and calcium sensitive exocytosis underlies seizures and large body size associated with 16p11.2 homologs in zebrafish. The data inform consideration of mechanisms underlying human 16p11.2 deletion symptoms

Regulation of smooth muscle -specific transcription by serum response factor and formin homology domain containing protein 1

The regulation of vascular smooth muscle cell (SMC) differentiation is important during vasculogenesis, angiogenesis, and cardiovascular diseases, such as atherosclerosis and restenosis. Previous studies have shown that SMC differentiation marker gene expression is regulated by serum response factor (SRF) and the myocardin family of SRF co-factors (myocardin and the myocardin-related factors, MRTF-A and MRTF-B). A major goal of the current studies was to identify post-translational modifications of SRF that regulate SMC-specific gene expression. By screening phosphorylation deficient and mimetic mutations in SRF -/- ES cells, I identified T159 as a phosphorylation site that significantly inhibits SMC-specific gene expression. In vitro and in vivo labeling studies demonstrated that T159 was phosphorylated by protein kinase PKA, and results from gel shift and chromatin immunoprecipitation assays demonstrated that T159 phosphorylation inhibited SRF binding to the CArG elements present within the promoters of the SMC-specific genes. Based upon the identification of Ubc9 in a yeast-two-hybrid screen for SRF binding proteins, I also tested the role of sumoylation on SRF activity. In vitro sumoylation assays identified K147 as the major SRF sumoylation site, but a sumoylation deficient K147R mutation had no effect on SRF-dependent SMC-specific gene expression. Our lab has also demonstrated that MRTF nuclear localization and activity is regulated by changes in actin dynamics, and a second goal was to determine whether the diaphanous formin, FHOD1, played a significant role in this process. Using RNAi techniques I demonstrated that FHOD1 was important for SMC differentiation marker gene expression in 10T1/2 and that over expression of a constitutively active version of FHOD1 strongly up-regulated SMC-specific promoter activity. Additional studies showed that phosphorylation of FHOD1 in the diaphanous auto-regulatory domain may contribute to FHOD1 activation and that FHOD1-mediated actin polymerization in the nucleus may be important for FHOD1's effects on MRTF activation. Taken together, my results indicate that PKA-mediated phosphorylation of SRF and FHOD1-mediated actin polymerization regulate SMC-transcription providing two novel signaling mechanisms for the control of SMC phenotype. Future experiments extending these findings should lead to a better understanding of SMC's role in cardiovascular disease and to targets for treating these conditions

\u3ci\u3eDrosophila\u3c/i\u3e Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu