Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009.

Thesis (Ph. D.)--University of Virginia, 2007. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Coronary vessel development is a crucial part of heart development requiring the interplay of the epicardial, myocardial and endocardial layers of the heart for proper formation. Coronary vascularization is regulated by a host of transcription factors further regulated by chromatin remodeling enzymes, including Histone Deacetylases (HDACs). To investigate the functions of HDACs in coronary vascular development, we have deleted Hdac3 in endocardial cells using Cre LoxP technology. Endocardial cell-­‐specific deletion of Hdac3 results in aberrant coronary vessel formation and complete postnatal lethality. We have thus shown that Hdac3 is a critical regulator of the coronary vascular development pathway.

Hox genes encode a conserved family of homeodomain containing transcription factors essential for metazoan development. The establishment of overlapping Hox expression domains specifies tissue identities along the anterior-posterior axis during early embryogenesis and is regulated by chromatin architecture and retinoic acid (RA). Here we present the role nucleosome positioning plays in hox activation during embryogenesis. Using four stages of early embryo development, we map nucleosome positions at 37 zebrafish hox promoters. We find nucleosome arrangement to be progressive, taking place over several stages independent of RA. This progressive change in nucleosome arrangement on invariant sequence suggests that trans-factors play an important role in organizing nucleosomes. To further test the role of trans-factors, we created hoxb1b and hoxb1a mutants to determine if the loss of either protein effected nucleosome positions at the promoter of a known target, hoxb1a. Characterization of these mutations identified hindbrain segmentation defects similar to targeted deletions of mouse orthologs Hoxa1 and Hoxb1 and zebrafish hoxb1b and hoxb1a morpholino (MO) loss-of-function experiments. However, we also identified differences in hindbrain segmentation as well as phenotypes in facial motor neuron migration and reticulospinal neuron formation not previously observed in the MO experiments. Finally, we find that nucleosomes at the hoxb1a promoter are positioned differently in hoxb1b-/- embryos compared to wild-type. Together, our data provides new insight into the roles of hoxb1b and hoxb1a in zebrafish hindbrain segmentation and reticulospinal neuron formation and indicates that nucleosome positioning at hox promoters is dynamic, depending on sequence specific factors such as Hox proteins.

The following work examines the mechanisms by which Tip60-p400 chromatin remodeling complex regulates gene expression in embryonic stem cells (ESCs). Tip60-p400 complex has distinct functions in undifferentiated and differentiated cells. While Tip60-p400 is often associated with gene activation in differentiated cells, its most prominent function in ESCs is to repress differentiation-related genes. I show that Tip60-p400 interacts with Hdac6 and other proteins to form a unique form of the complex in ESCs. Tip60-Hdac6 interaction is stem cell specific and is necessary for Tip60-p400 mediated gene regulation, indicating that Tip60- p400 function is controlled in part through the regulation of Hdac6 during development. Furthermore, I find that Hdac6 is required for the binding of Tip60- p400 to many of its target genes, indicating Hdac6 is necessary for the unique function of Tip60-p400 in ESCs. In addition to accessory proteins like Hdac6, Tip60-p400 also interacts with thousands of coding and noncoding RNAs in ESCs. I show that R-loops, DNA-RNA hybrids formed during transcription of many genes, are important for regulation of chromatin binding by at least two chromatin regulators (Tip60-p400 and PRC2). This finding suggests that transcripts produced by many genes in ESC may serve as a signal to modulate binding of chromatin regulators. However, R-loops might also function to regulate chromatin architecture in differentiated cells as well. Future studies based on this work will be necessary to understand the full repertoire of cell types and chromatin regulators regulated by these structures.

Skeletal muscle is a complicated and heterogeneous striated muscle tissue that serves critical mechanical and metabolic functions in the organism. The process of generating skeletal muscle, myogenesis, is elaborately coordinated by members of the protein kinase family, which transmit diverse signals initiated by extracellular stimuli to myogenic transcriptional hierarchy in muscle cells. Mitogen-activated protein kinases (MAPKs) including p38 MAPK, c-Jun N terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) are components of serine/threonine protein kinase cascades that play important roles in skeletal muscle differentiation. The exploration of MAPK upstream kinases identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4), a serine/threonine protein kinase that modulates p38 MAPK, JNK and ERK activities in multiple cell lines. Our lab further discovered that Map4k4 regulates peroxisome proliferator-activated receptor γ (PPARγ) translation in cultured adipocytes through inactivating mammalian target of rapamycin (mTOR), which controls skeletal muscle differentiation and hypotrophy in kinase-dependent and -independent manners. These findings suggest potential involvement of Map4k4 in skeletal myogenesis. Therefore, for the first part of my thesis, I characterize the role of Map4k4 in skeletal muscle differentiation in cultured muscle cells. Here I show that Map4k4 functions as a myogenic suppressor mainly at the early stage of skeletal myogenesis with a moderate effect on myoblast fusion during late-stage muscle differentiation. In agreement, Map4k4 expression and protein kinase activity are declined with myogenic differentiation. The inhibitory effect of Map4k4 on skeletal myogenesis requires its kinase activity. Surprisingly, none of the identified Map4k4 downstream effectors including p38 MAPK, JNK and ERK is involved in the Map4k4-mediated myogenic differentiation. Instead, expression of myogenic regulatory factor Myf5, a positive mediator of skeletal muscle differentiation is transiently regulated by Map4k4 to partially control skeletal myogenesis. Mechanisms by which Map4k4 modulates Myf5 amount have yet to be determined. In the second part of my thesis, I assess the relationship between Map4k4 and IGF-mediated signaling pathways. Although siRNA-mediated silencing of Map4k4 results in markedly enhanced myotube formation that is identical to the IGF-induced muscle hypertrophic phenotype, and Map4k4 regulates IGF/Akt signaling downstream effector mTOR in cultured adipocytes, Map4k4 appears not to be involved in the IGF-mediated ERK1/2 signaling axis and the IGF-mediated Akt signaling axis in C2C12 myoblasts. Furthermore, Map4k4 does not affect endogenous Akt signaling or mTOR activity during C2C12 myogenic differentiation. The results presented here not only identify Map4k4 as a novel suppressor of skeletal muscle differentiation, but also add to our knowledge of Map4k4 action on multiple signaling pathways in muscle cells during skeletal myogenesis. The effects that Map4k4 exerts on myoblast differentiation, fusion and Myf5 expression implicate Map4k4 as a potential drug target for muscle mass growth, skeletal muscle regeneration and muscular dystrophy.