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).

Basic helix-loop-helix transcription factors play important roles in pathways that result in the formation of such tissues as skeletal muscle, the nervous system, pancreas and heart. A recently discovered B-cell restricted bHLH transcription factor activated Bcell factor 1 (ABF -1) is suspected to play a role in the regulation of B cell differentiation. ABF -1 was initially characterized as being a transcriptional repressor because it was able to prevent the transactivation abilities of E47 in HeLa cells and the region responsible was hypothesized to reside within the C-terminal portion. This study has further mapped the repression domain to the C-terminal portion bearing the bHLH domain. In addition, we report the discovery of a region in the N-terminus that has a secondary negative effect on the transactivation ability of E2A. To complement these studies, the subcellular localization and nuclear localization signal of GFP-tagged ABF -1 proteins was performed using fluorescent microscopy. These studies suggest that ABF-1 exerts it function through the bHLH domain and the region of the nuclear localization signal lies within amino acids 71-103. Taken together, the ability of the C-terminal end to repress E2A mediated transcription may be answered through the conservation of two amino acids, serine and lysine, located at amino acid positions 124 and 125 in the first helix of the HLH domain. This conservation is seen in known transcriptional repressors, such as ceABF -1, MyoR/musculin and capsulin, but is absent in all other identified bHLH proteins.

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.

Successful reproduction in plants is a highly-regulated process reliant on the integration of both endogenous and external cues. Different accessions of the model plant Arabidopsis thaliana have been collected, including those with a winter annual or rapid-cycling flowering habit. Natural variation and mutant screens have enabled many flowering time genes to be identified. A key regulator of flowering is FLOWERING LOCUS C (FLC). FLC is a repressor of flowering and is regulated by a number of genes, including those in the autonomous and FRIGIDA-mediated pathways. Of particular interest are FRIGIDA (FRI) and FRIGIDA-LIKE 1 (FRL1) and the autonomous pathway members, FCA and FY. FRI and FRL 1 promote FLC expression making them dominant repressors of flowering. FRI is proposed to initiate chromatin remodelling at the FLC locus leading to increased FLC expression. Once elevated, FLC levels are maintained until plants undergo an extended period of cold, therefore flowering occurs in spring. In contrast, FCA and FY promote flowering by repressing FLC expression. FCA has also been identified as a receptor of the plant hormone abscisic acid (ABA). Upon binding to FCA, ABA is proposed to disrupt/inhibit the FCA:FY interaction which results in delayed flowering. To characterise the FCA:ABA interaction and identify the ABA binding site, a number of truncated FCA proteins were utilised. Initially a FCA:FY GST-pulldown was used to identify the ABA binding site. However, when ABA failed to inhibit the FCA:FY interaction a direct binding assay using [�H]-ABA was employed. Another Arabidopsis ABA receptor, G-protein coupled receptor 2 (GCR2), was used as a positive control in these binding assays. Both FCA and GCR2 failed to bind [�H]-ABA suggesting a broader issue with the binding assay. The identification of FCA and GCR2 as ABA receptors can be attributed to the quality of the protein assayed, the sensitivity of the binding assay and the subsequent data analysis. This study resulted in the retraction of the original paper (Razem et at, 2006) reporting FCA as an ABA receptor. To investigate the molecular mechanism by which FRI and FRL1 act as positive regulators of FLC expresion, a biochemical approach was taken. FRI and FRL1 have no known homology to any other protein or domain and the only method for assessing protein function is through plant complementation experiments. In the absence of sequence homology, or a timely functional assay, a classical approach was taken to produce soluble protein for analysis. Truncation of predicted regions of disorder and expression, solubility and stability screens produced soluble protein of reasonable purity. This allowed characterisation of the biochemical properties of FRI and FRL1. Interaction studies between FRI and FRL1, and the zinc finger protein SUPRESSOR OF FRIGIDA 4 (SUF4), were also carried out. Polyclonal antibodies against FRI and FRL1, made during this study, were useful for protein detection in these experiments. The interaction studies, together with plant complementation experiments, suggest that the C-terminus of FRI is essential for protein function, while the N-terminus improves FRI activity. These findings provide a better understanding of how the components of the proposed "FRI-complex" may interact to promote FLC expression.

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.

Successful reproduction in plants is a highly-regulated process reliant on the integration of both endogenous and external cues. Different accessions of the model plant Arabidopsis thaliana have been collected, including those with a winter annual or rapid-cycling flowering habit. Natural variation and mutant screens have enabled many flowering time genes to be identified. A key regulator of flowering is FLOWERING LOCUS C (FLC). FLC is a repressor of flowering and is regulated by a number of genes, including those in the autonomous and FRIGIDA-mediated pathways. Of particular interest are FRIGIDA (FRI) and FRIGIDA-LIKE 1 (FRL1) and the autonomous pathway members, FCA and FY. FRI and FRL 1 promote FLC expression making them dominant repressors of flowering. FRI is proposed to initiate chromatin remodelling at the FLC locus leading to increased FLC expression. Once elevated, FLC levels are maintained until plants undergo an extended period of cold, therefore flowering occurs in spring. In contrast, FCA and FY promote flowering by repressing FLC expression. FCA has also been identified as a receptor of the plant hormone abscisic acid (ABA). Upon binding to FCA, ABA is proposed to disrupt/inhibit the FCA:FY interaction which results in delayed flowering. To characterise the FCA:ABA interaction and identify the ABA binding site, a number of truncated FCA proteins were utilised. Initially a FCA:FY GST-pulldown was used to identify the ABA binding site. However, when ABA failed to inhibit the FCA:FY interaction a direct binding assay using [�H]-ABA was employed. Another Arabidopsis ABA receptor, G-protein coupled receptor 2 (GCR2), was used as a positive control in these binding assays. Both FCA and GCR2 failed to bind [�H]-ABA suggesting a broader issue with the binding assay. The identification of FCA and GCR2 as ABA receptors can be attributed to the quality of the protein assayed, the sensitivity of the binding assay and the subsequent data analysis. This study resulted in the retraction of the original paper (Razem et at, 2006) reporting FCA as an ABA receptor. To investigate the molecular mechanism by which FRI and FRL1 act as positive regulators of FLC expresion, a biochemical approach was taken. FRI and FRL1 have no known homology to any other protein or domain and the only method for assessing protein function is through plant complementation experiments. In the absence of sequence homology, or a timely functional assay, a classical approach was taken to produce soluble protein for analysis. Truncation of predicted regions of disorder and expression, solubility and stability screens produced soluble protein of reasonable purity. This allowed characterisation of the biochemical properties of FRI and FRL1. Interaction studies between FRI and FRL1, and the zinc finger protein SUPRESSOR OF FRIGIDA 4 (SUF4), were also carried out. Polyclonal antibodies against FRI and FRL1, made during this study, were useful for protein detection in these experiments. The interaction studies, together with plant complementation experiments, suggest that the C-terminus of FRI is essential for protein function, while the N-terminus improves FRI activity. These findings provide a better understanding of how the components of the proposed "FRI-complex" may interact to promote FLC expression.