Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 232-250).

Thesis--University of Oklahoma. / Bibliography: leaves 90-93.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph.D.)--University of Wisconsin--Madison, 2006 / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 126-139).

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 160-169). Also available on the Internet.

The XIST gene is implicated in X-chromosome inactivation, yet the RNA contains no apparent open reading frame. An accumulation of XIST RNA is observed near its site of transcription, the inactive X chromosome (Xi). A series of molecular cytogenetic studies comparing properties of XIST RNA to other protein coding RNAs, support a critical distinction for XIST RNA; XIST RNA does not concentrate at Xi simply because it is transcribed and processed there. Most notably, morphometric and 3-D analysis reveals that XIST RNA and Xi are coincident in 2-D and 3-D space; hence the XIST RNA essentially paints Xi. Several results indicate that the XIST RNA accumulation has two components, a minor one associated with transcription and processing, and a spliced major component, which stably associates with Xi. Upon transcriptional inhibition the major spliced component remains in the nucleus and often encircles the extra-prominent heterochromatic Barr body. The continually transcribed XIST gene and its poly-adenylated RNA consistently localize to a nuclear region devoid of splicing factor/poly A RNA rich domains. XIST RNA remains with the nuclear matrix fraction after removal of chromosomal DNA. XIST RNA is released from its association with Xi during mitosis, but shows a unique highly particulate distribution. Collective results indicate that XIST RNA may be an architectural element of the interphase chromosome territory, possibly a component of non-chromatin nuclear structure that specifically associates with Xi. XIST RNA is a novel nuclear RNA which potentially provides a specific precedent for RNA involvement in nuclear structure and cis-limited gene regulation via higher-order chromatin packaging.

Synthesis of histone proteins occurs largely during the S phase of the cell cycle and coincides with DNA replication to provide adequate amounts of histones necessary to properly package newly replicated DNA. Controlling transcription from cell cycle dependent and proliferation specific genes, including histone H4, is an important level of regulation in the overall governance of the cell growth process. Coordination of histone gene transcription results from the cumulative effects of cell signaling pathways, dynamic chromatin structure and multiple transcription factor interactions. The research of this dissertation focused on the characterization and identification of transcription factors interacting on the human histone H4 gene FO108. I also focused on the elucidation of regulatory elements within the histone coding region. Our results suggest a possible mechanism by which a transcription factor facilitates reorganization of histone gene chromatin structure. The histone promoter region between -418 nt and -215 nt, Site III, was previously identified as both a positive and negative cis-regulatory element for transcription. Results of in vitroanalyses presented in this dissertation identified multiple transcription factors interacting at Site III. These factors include H4UA-1/YY1, AP-2, AP-2 like factor and distal factor (NF-1 like factor). Transient transfection experiments show that Site III does not confer significant influence on transcription; however, there may exist a physiological role for Site III which would not be detected in these assay systems. We analyzed the histone H4 gene sequences for additional transcription factor binding motifs and identified several putative YY1 binding sites. Using electrophoretic mobility shift assays (EMSA), we found that Site IV, Site I and two elements within the histone H4 coding region are capable of interacting with YY1. In transient transfection experiments using reporter constructs containing either Site III or one of the coding region elements as potential promoter regulatory elements, and an expression vector encoding YY1, we observed levels of expression up to 2.7 fold higher than from the reporters lacking these elements. Therefore, YY1 appears to interact at multiple regulatory sites of the histone gene and can influence transcription through these elements. Prior to this study, the role of the coding region in histone gene expression was not known. To determine if the coding region is involved in regulating transcription, I constructed and tested a series of heterologous reporter constructs containing various sequences of the histone coding region. Results from these experiments demonstrated that the histone coding region contains three repressor elements. Extensive in vitro analysis indicated that the three repressor elements interact with the repressor CDP/cut. Further analysis showed that CDP/cut interactions with the repressor elements are cell cycle regulated and proliferation specific. CDP/cut interactions increase during the cell cycle when histone transcription decreases. These observations are consistent with the hypothesis that CDP/cutis a cell cycle regulated repressor factor which influences transcription of the histone H4 gene as such. The proximal promoter region of the histone H4 gene between -70 nt and +190 nt is devoid of normal nucleosome structure. This same region contains multiple CDP/cut binding sites. We hypothesized that CDP/cut is involved with chromatin remodeling of the histone gene. DNase I footprinting and EMSA results show purified recombinant CDP/cut interacts specifically with the histone promoter reconstituted into nucleosome cores. Thus, CDP/cutmay facilitate the organization of chromatin of the histone gene. In conclusion, the research presented in this dissertation supports the hypothesis that expression from the human histone H4 gene FO108 is regulated by multiple cis-regulatory elements which interact with several proteins. CDP/cut interacts with Site II, the three repressor elements in the histone coding region and at Distal Site I. YY1 interacts at Site IV, Site III, Site I, and twice in the coding region. ATF/CREB interacts with Site IV and Site I. Distal factor interacts with Site III and within the histone coding region. IRF 2 interacts with Site II and Distal Site I. Thus, histone gene expression is probably regulated by transcription factors CDP/cut, YY1, IRF 2 and ATF/CREB interacting with multiple regulatory elements dispersed throughout its promoter and the coding region. Cell cycle regulation of these transcription factors may contribute to cell cycle dependent expression of the histone gene.

Proliferation and differentiation are essential processes for the growth and development of higher eukaryotic organisms. Regulation of gene expression is essential for control of cell division and differentiation. Normal eukaryotic cells have a limited proliferative capacity, and ultimately undergo cellular senescence and apoptosis. Terminal differentiation of cells is associated with loss of proliferative capacity and acquisition of specialized functions. Proliferation and differentiation are processes required for the creation and maintenance of diverse tissues both during embryonic development and postnatal life. The cell cycle is the process by which cells reproduce, and requires duplication and segregation of hereditary material. Loss of cell cycle control leads to genetic instability and cancer. Expression of replication-dependent histone genes is tightly coupled to DNA synthesis, thus making histone genes a good model for studying cell cycle regulation. The HiNF-D complex interacts with all five classes (H1, H2A, H2B, H3 and H4) of histone genes in a cell cycle-dependent manner. The CCAAT displacement protein (CDP)/Cux and the tumor suppressor pRB are key components of the HiNF-D complex. However, the molecular interactions that enable CDP/Cux and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters are poorly understood. Transient transfection assays show that CDP/Cux represses the histone H4 promoter and that the pRB large pocket domain functions with CDP/Cux as a co-repressor. Direct interaction between CDP/Cux C-terminus and the pRB pocket domain was observed in GST pull-down assays. Furthermore, co-immunoprecipitation assays and immunofluorescence microscopy established that CDP/Cux and pRB form complexes in vivo and associate in situ. pRB interaction and co-repression with CDP/Cux is independent of pRB phosphosphorylation sites, as revealed by GST pull-down assays and transient transfection assays using a series of pRB mutant proteins. Thus, several converging lines of evidence indicate that complexes between CDP/Cux and pRB repress cell cycle-regulated histone gene promoters. CDP/Cux is regulated by phosphorylation and acetylation at the C-terminus, which contains two repressor domains and interacts with histone deacetylase HDAC1. In vivo function of the CDP/Cux C-terminus in development and gene regulation was assessed in genetically targeted mice (Cutl1tm2Ejn, referred to as Cutl1ΔC). The mice express a mutant CDP/Cux protein with a deletion of the C-terminus including the homeodomain. Indirect immunofluorescence microscopy showed that the mutant protein exhibited significantly reduced nuclear localization in comparison to the wildtype protein. Consistent with these data, DNA binding activity of HiNF-D was lost in nuclear extracts derived from mouse embryonic fibroblasts (MEFs) or adult tissues of homozygous mutant (Cutl1 ΔC -/-) mice, indicating the functional loss of CDP/Cux in the nucleus. No significant difference in growth characteristics or total histone H4 mRNA levels was observed between wildtype and Cutl1 ΔC -/- MEFs in culture. However, the histone H4.1 (murine FO108) gene containing CDP/Cux binding sites have reduced expression levels in homozygous mutant MEFs. Stringent control of growth and differentiation appears to be compromised in vivo. Homozygous mutant mice exhibit stunted growth (20-50% weight reduction), a high postnatal death rate of 60-70%, sparse abnormal coat hair and severely reduced fertility. Hair follicle deformities and severely diminished fertility in Cutl1 ΔC -/- mice suggest that CDP/Cux is required for normal development of dermal tissues and reproductive functions. Together the data presented in this dissertation provide new insight into the in vivo functions of CDP/Cux in the regulation of histone gene expression, growth control and differentiation.

Human immunodeficiency virus type 1 (HIV-1) infections have different patterns of expression in different T-cell lines. HIV-1 encodes regulatory as well as structural genes. The role of HIV-1 regulatory gene expression in determining different patterns of infection was explored in four T-cell lines: C8166, H9, A3.01, and Jurkat. The hypothesis being tested was that differences in the expression of regulatory genes would determine differences in the kinetics of infection. To study patterns of regulatory and structural gene expression, RNA was isolated from cultures infected with HIV-1-NL4-3 (NL4-3). During the early and acute phases of infection, the absolute amounts of viral RNA differed in the four T-cell lines. However, the relative proportions of messages for regulatory and structural genes were similar. Thus, differences in the kinetics of infection in C8166, H9, A3.01 and Jurkat cells were not determined by differences in the relative levels of expression of regulatory and structural genes. Analyses of RNA samples from the chronic phase of infection revealed the consistent appearance of novel RNase sensitive sites in H9 and Jurkat cultures. These marked the emergence of viral variants with high ability to establish chronic virus producers. These variants were specifically selected in the chronic phase since they did not undergo selection during serial passage of the virus through the lytic phase of infection. Sequence analysis of the region with the novel RNase sensitive sites revealed the co-mapping of nucleotide changes with each of the novel sites. Most of these differences represented a sense mutation in tat and the abrogation of the initiator methionine of vpu. However, the selected mutations in tat and vpuwere not sufficient, by themselves, to affect the ability of NL4-3 to establish chronic virus producers (Chapters I and II). Further studies on the roles of viral sequences in the chronic phase of infection were undertaken using constructed viruses. Two molecularly cloned viruses, NL4-3 and HIV-1-HXB-2 (HXB-2), were used as parents. NL4-3 has a low ability to establish chronic virus producers. In contrast, HXB-2 has a high ability to establish chronic virus producers. NL4-3 encodes all known HIV-1 genes, whereas HXB-2 is defective for three auxiliary genes: vpr, vpu, and nef. In addition, both viruses differ at other positions throughout the genome. The first series of constructed viruses tested whether differences in auxiliary gene expression determined differences in the ability of NL4-3 and HXB-2 to establish chronic virus producers. NL4-3 mutants containing all possible combinations of the three defective genes in HXB-2 were constructed. Analysis of the ability of these mutants to establish chronic virus producers revealed that vpr and neflimit the ability of NL4-3 to establish chronic virus producers. This was shown by viruses with defects in both of these genes having high ability to establish chronic virus producers (Chapter III). The second series of constructs tested for the roles of non-auxiliary as well as auxiliary gene sequences on chronic virus production by creating recombinants between NL4-3 and HXB-2. Tests of these recombinants revealed that a gag, pol, vif, and vpr fragment could affect the ability of fragments containing defective auxiliary genes to establish chronic virus producers. Taken together, these results indicate that vpr, nef, and 5' internal sequences play important roles in determining the ability to establish chronic virus producers (Chapter IV).