Thesis (Ph. D.)--Florida State University, 2003. / Advisor: Dr. Timothy M. Logan, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Oct. 2, 2003). Includes bibliographical references.

Graduation date: 1992

Thesis (Ph.D.)--Kent State University, 2005. / Title from PDF t.p. (viewed June 29, 2007). Advisor: Asch, David K. Keywords: quinic acid gene cluster, neurospora crassa, catabolite repression. Includes bibliographical references (p. 136-153).

A common organizational feature of any nervous system is the existence of groups of neurons that share a set of common traits but that can be further divided into individual neuron types and subtypes. Understanding the mechanistic basis of neuron type and subtype diversification processes will constitute a major step toward understanding brain development and evolution. In this dissertation, I have explored the mechanistic basis for the specification of motor neuron classes in the nematode C. elegans which serves as a paradigm for neuron diversification processes. Cholinergic motor neurons in the C. elegans ventral nerve cord share common traits, but are also comprised of many distinct classes, each characterized by unique patterns of effector gene expression (e.g. motor neuron class-specific ion channels, signaling molecules, and neurotransmitter receptors). Both the common as well as class-specific traits are directly activated by the terminal selector of cholinergic motor neuron identity, the EBF/COE-like transcription factor UNC-3. Via forward genetic screens to identify mutants that are defective in class specification, I have discovered that the diversification of UNC-3/EBF-dependent cholinergic motor neurons is controlled by distinct sets of phylogenetically conserved, motor neuron class-specific transcriptional repressors. One such repressor is in fact a novel gene previously uncharacterized in C. elegans or any nervous systems and is now named bnc-1. By molecularly dissecting the cis-regulatory region of effector genes, I found that the repressor proteins prevent UNC-3/EBF from activating class-specific effector genes in specific motor neuron subsets via discrete binding sites that are adjacent to those of UNC-3/EBF. And by using CRISPR/Cas9-mediated genome engineering to tag repressor proteins with inducible degrons, I demonstrate that these repressors share the important feature of being continuously required throughout the life of the animal to counteract, in a class-specific manner, the function of the UNC-3/EBF terminal selector that is active in all motor neuron classes. I propose that the strategy of antagonizing the activity of broadly acting terminal selectors of neuron identity in a neuron subtype-specific manner may constitute a general principle of neuron subtype diversification.

The progressive loss of developmental potential is a hallmark of all differentiating cells in multicellular organisms. At the chromatin level, this restriction in cell-fate plasticity is established through the silencing of active and poised lineage-specific genes that are incompatible with the terminal fate of the maturing cell type. The effective and stable inhibition of gene expression relies on the coordinated action of transcriptional repressors. These repressors are often transiently expressed only at the time of cell-fate specification and direct lineage decisions by suppressing alternative developmental programs. However, compared to the numerous studies examining the mechanisms by which cell-type specific transcriptional activators program cellular identity, little is currently known regarding how transient repressors execute permanent silencing of gene regulatory networks. To address this question, I have examined the mechanisms through which the transiently expressed transcription factor (TF) Nkx2.2 represses the acquisition of motor neuron (MN) identity in V3 neuronal progenitors. While it is well-established that Nkx2.2 functions as a transcriptional repressor through its interactions with the Groucho (Grg) family of co-repressors, how these interactions manifest in gene silencing has remained unknown. Moreover, the effects of Nkx2.2 occupancy on chromatin modifications have not been determined. In this dissertation, I demonstrate that surprisingly, Nkx2.2 decommissions enhancers of the MN developmental program not through the recruitment of additional co-repressor proteins but rather through the eviction of co-activator complexes. While this displacement is dependent upon an intact Grg-interacting domain, Nkx2.2 binding does not increase Grg enrichment. In addition, extensive profiling of Nkx2.2 genome-wide binding events in neural precursors unexpectedly revealed that Nkx2.2 occupies not only enhancers of MN progenitor genes acutely repressed by Nkx2.2 but also enhancers of genes expressed exclusively in postmitotic MNs, long after Nkx2.2 expression has been down- regulated. In vivo lineage tracing experiments and in vitro genomic analyses demonstrated that Nkx2.2 also functions in a repressive capacity at these poised regulatory regions. Here, Nkx2.2 binding prevents the activation of postmitotic genetic networks through a preferential enlistment of histone deacetylase complex 2 (HDAC2) proteins. However, this binding is not accompanied by the deposition of repressive chromatin modifications, and removal of Nkx2.2 in differentiating V3 neurons leads to the ectopic expression of the postmitotic MN TFs Isl1 and Hb9. Collectively, these studies indicate that transiently expressed repressors may establish gene suppression by counteracting the activities of transcriptional activators, rather than by directly establishing repressive chromatin signatures. As transcriptional reprogramming of differentiated cell linages often fails to adequately silence the expression programs of the starting population, these results may help to inform new methodologies for instructing cell conversions.

Two novel and highly related C₂H₂ zinc finger proteins (CTIP1/BCL11A/EVI9 and CTIP2/BCL11B/Rit1) have been implicated in COUP-TF signaling, etiology of myeloid and lymphoid malignancies, and hematopoietic cell development. However, the precise cellular function(s) and the contribution of these proteins to neoplastic processes and hematopoietic cell development remain unknown. The goal of the studies described herein was to elucidate the molecular mechanisms underlying the transcriptional repression mediated by these proteins to understand their biological properties, and ultimately, their cellular function(s). CTIP proteins repressed transcription of a reporter gene in a TSA-insensitive manner, suggesting that this repression mechanism(s) may not involve TSA-sensitive histone deacetylation catalyzed by member(s) of class I and II HDACs. One possible mechanism is that CTIP proteins may exert ISA-insensitive histone deacetylation catalyzed by TSA-insensitive HDAC(s), such as SIRT1, to repress transcription. In deed, SIRT1 was found to interact with CTIP proteins both in vitro and in mammalian cells, and was recruited to the promoter template in a CTIP-dependent manner. The proline-rich regions of CTIP proteins and the sirtuin homology domain of SIRT1 were found to be essential for mediating CTIPs•SIRT1 interactions. Moreover, column chromatography revealed that SIRT1 and CTIP2 were components of a large complex in Jurkat cell nuclear extracts. Based on the findings that SIRT1 associates with CTIP proteins in mammalian cells, SIRT1 may underlie the transcriptional repression activity of CTIP proteins. The following results support the hypothesis that SIRT1 may underlie the mechanism(s) of CTIP-mediated transcriptional repression. First, CTIP-mediated transcriptional repression was inhibited, at least partially, by nicotinamide, an inhibitor of the NAD⁺-dependent, TSA-insensitive HDACs. Second, the decrease in levels of acetylated histones H3 and/or H4 at the promoter region of a reporter gene was observed upon overexpression of CTIP proteins, and this effect was inhibited, at least partially, by nicotinamide. Third, endogenous SIRT1 was recruited to the promoter template of a reporter gene in mammalian cells upon overexpression of CTIP proteins. Fourth, SIRT1 enhanced the transcriptional repression mediated by CTIP proteins and this enhancement required the catalytic activity of SIRT1. Finally, SIRT1 enhanced the deacetylation of template-associated histones H3 and/or H4 in CTIP-transfected cells. In summary, results described herein strongly suggest that CTIP-mediated transcriptional repression involves the recruitment of SIRT1 to the template, at which the TSA-insensitive, but nicotinamide-sensitive histone deacetylase catalyzes deacetylation of promoter-associated histones H3 and/or H4. These results contribute additional understanding to the molecular mechanisms underlying transcriptional activity of CTIP proteins, which might be helpful for identification and characterization of the target genes under the control of CTIP proteins in cells of hematopoietic system and/or the central nervous system. / Graduation date: 2004

Thesis (M. Sc.)--York University, 2001. / Typescript. Includes bibliographical references (leaves 69-89). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ67745.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Biochemistry." Includes bibliographical references (p. 124-146).