Showing papers by "Scott J. Bultman published in 2011"

The ect2 rho Guanine nucleotide exchange factor is essential for early mouse development and normal cell cytokinesis and migration.

Abstract: Ect2 is a member of the human Dbl family of guanine nucleotide exchange factors (RhoGEFs) that serve as activators of Rho family small GTPases. Although Ect2 is one of at least 25 RhoGEFs that can activate the RhoA small GTPase, cell culture studies using established cell lines determined that Ect2 is essential for mammalian cell cytokinesis and proliferation. To address the function of Ect2 in normal mammalian development, we performed gene targeting to generate Ect2 knockout mice. The heterozygous Ect2 +/- mice showed normal development and life span, indicating that Ect2 haplodeficiency was not deleterious for development or growth. In contrast, Ect2 -/- embryos were not found at birth or postimplantation stages. Ect2 -/- blastocysts were recovered at embryonic day 3.5 but did not give rise to viable outgrowths in culture, indicating that Ect2 is required for peri-implantation development. To further assess the importance of Ect2 in normal cell physiology, we isolated primary fibroblasts from Ect2 fl/fl embryos (MEFs) and ablated Ect2 using adenoviral delivery of Cre recombinase. We observed a significant increase in multinucleated cells and accumulation of cells in G2/M phase, consistent with a role for Ect2 in cytokinesis. Ect2 deficiency also caused enlargement of the cytoplasm and impaired cell migration. Finally, although Ect2-dependent activation of RhoA has been implicated in cytokinesis, Ect2 can also activate Rac1 and Cdc42 to cause growth transformation. Surprisingly, ectopic expression of constitutively activated RhoA, Rac1, or Cdc42, known substrates of Ect2, failed to phenocopy Ect2 and did not rescue the defect in cytokinesis caused by loss of Ect2. In summary, our results establish the unique role of Ect2 in development and normal cell proliferation.

Reversible epigenetic modifications of the two cardiac Myosin Heavy Chain genes during changes in expression

Abstract: The two genes of the cardiac myosin heavy chain (MHC) locus-alpha-MHC (aMHC) and beta-MHC (bMHC)--are reciprocally regulated in the mouse ventricle during development and in adult conditions such as hypothyroidism and pathological cardiac hypertrophy. Their expressions are under the control of thyroid hormone T3 levels. To gain insights into the epigenetic mechanisms that underlie this inducible and reversible switching of the aMHC and bMHC isoforms, we have investigated the histone modification patterns that occur over the two cardiac MHC promoters during T3-mediated reversible switching of gene expression. Mice fed a diet of propylthiouracil (PTU, an inhibitor of T3 synthesis) for 2 weeks dramatically reduce aMHC mRNA expression and increase bMHC mRNA levels to high levels, while a subsequent withdrawal of PTU diet for 2 weeks completely reverses the T3-mediated changes in MHC expression. Using hearts from mice treated in this way, we carried out chromatin immunoprecipitation-qPCR assays with antibodies against acetylated histone H3 (H3ac) and trimethylated histone (H3K4me3)-two well-documented markers of activation. Our results show that the reexpression of bMHC is associated at the bMHC promoter with increased H3ac but not H3K4me3. In contrast, the silencing of aMHC is associated at its promoter with decreased H3K4me3, but not decreased H3ac. The epigenetic changes at the two MHC promoters are completely reversed when the gene expression returns to initial levels. These data indicate that during reciprocal and inducible gene expression H3ac parallels bMHC isoform expression while H3K4me3 parallels expression of the tightly linked aMHC isoform.