The aim of this study was to gain more information about the interactions between DNA and the histone octamer during the process of transcription. This work used a pUC8 plasmid derivative that contained the core promoter region of the RNA polymerase I of Acanthamoeba castellanii, placed upstream of four repeats of the 5S rDNA nucleosome positioning sequence from the sea urchin, Lytechinus variegatus. The plasmid was reconstituted into chromatin via addition of chicken erythrocyte histone octamers, using polyglutamic acid as a nucleosome assembly factor. The positioning of nucleosomes on the insert was monitored by restriction enzyme digestion. Proper nucleosome positioning was shown to be dependent on the presence of preassembled transcription complexes on the promoter region. The absence of preformed transcription complexes on the promoter region prior to nucleosome reconstitution perturbed the distribution of histone octamers on the repeats of the 5S rDNA. This "mispositioning" effect was related to the location of the RNA polymerase I promoter region upstream of the four repeats of the 5S rDNA fragment. Band shift assays in polyacrylamide gel electrophoresis were used to determine the relative efficiency of nucleosome formation on the promoter-containing fragment, on 5S rDNA and finally on nucleosome core particle DNA. The results indicate that the promoter fragment forms a nucleoprotein complex at lower concentration of histone than the 5S positioning sequence. This complex may not be a nucleosomal structure. The reconstituted plasmid was then used to investigate the transcriptional elongation by RNA polymerase I using the chromatin-like template containing positioned nucleosomes as compared to transcription on improperly positioned nucleosomes and on free DNA. The efficiency of transcription was related to the proper positioning of nucleosomes with regard to the tandemly repeated 208-bp 5S rDNA. The presence of phased nucleosomes in the path of the transcription complex seemed not to inhibit nor to significantly slow down the elongation as compared to free DNA. Furthermore, nucleosome positioning, as assayed by restriction endonuclease digestion, did not change after passage of the polymerase I transcription complex. / Graduation date: 1993

Cytokinins are plant hormones that affect the primary growth of shoots and roots. Application of the cytokinin N6-benzylaminopurine (BAP) to the shoot apical meristem of <i>Arabidopsis thaliana Landsberg erecta </i>(L.) Heynh induces aberrant flower development and a significant genetic response, and some of these phenotypes and expression patterns were carried to the next generation. Analysis of altered transcript levels with Affymetrix GeneChips® indicated significant changes in transcript levels of genes associated with shoot meristem activity, circadian rhythms, cytokinin metabolism, two-component systems, stress and defense responses, auxin regulation, ethylene and salicylic acid biosynthesis, and signal transduction. Specific genes were also mined from the data as potentially responsible for the BAP-induced aberrant floral phenotypes, increased floral organ number, buds in axils of sepals, and mosaic floral organs. Of particular note was a decrease in the transcript levels of CLAVATA1 (CLV1), a gene encoding a receptor kinase involved in organ differentiation and maintenance of shoot and floral meristems. Time course analysis by RT-PCR showed a decline and subsequent recovery of transcript levels of CLV1 and a coincident increase in WUSCHEL (WUS) transcript, consistent with the known suppression of WUS by CLV. WUS encodes a homeodomain protein associated with shoot meristem proliferation. The temporal coincidence of an increased floral organ phenotype with changes in transcript levels of CLV1 and WUS suggests that cytokinins regulate flower development by affecting the activity of genes controlling shoot meristem activity. Aberrant floral phenotypes in subsequent non-treated generations suggest epigenetic inheritance of some BAP-altered transcript patterns. Repressed expression of the majority of significant genes in the untreated T1 population suggests a mechanism of gene silencing, such as methylation, was involved in this epigenetic inheritance. Also, transcript levels of time-keeping genes, including CIRCADIAN CLOCK ASSOCIATED 1 / ELONGATED HYPOCOTYL, and associated genes with oscillating expression patterns, such as COLD-RESPONSIVE, were affected by BAP in treated plants and the subsequent generation, suggesting the capacity of cytokinins to affect the phase of the circadian clock. Hormonal regulation of heritably altered diurnal periodicity and environmental responses may provide a developmental and, therefore, evolutionary advantage to plants.

In vertebrates, the embryonic hindbrain is transiently subdivided along its anterior-posterior (A-P) axis into 8 well defined segments termed rhombomeres (r1-8). Each rhombomere represents a true cellular compartment in transcriptional profile, lineage restriction and neuronal organization. Thus, the vertebrate hindbrain provides a beautiful model for studying mechanisms of anterior-posterior patterning, signal transduction and interpretation, initiation and maintenance of transcriptional profiles, cell sorting and border formation. The Kreisler/MafB gene, which encodes a basic leucine zipper (bZIP) transcription factor that regulates some Hox genes, is one of the first genes to be expressed segmentally in the hindbrain, and is subject to a dynamic and complex regulatory process. However, unlike the Hox genes, Kreisler/MafB is not located within a large cluster of genes and therefore provides a simple system for dissecting the molecular mechanisms involved in hindbrain compartmentalization. In dissecting the mechanisms that govern Kreisler/MafB regulation, we have identified the S5 regulatory element that directs early MafB expression in the future r5-r6 domain. We have found a binding site within S5 that is specific for the Variant Hepatocyte Nuclear Factor 1 (vHNF1) to be essential, but not sufficient for early induction of r5-r6-specific expression. Thus, early inductive events that initiate MafB expression are clearly distinct from later acting ones that modulate its expression levels. Using mouse mutants, we have shown that MafB is dependent on the M33 polycomb protein and other mechanisms of chromatin remodeling. We then utilized transgenic flies and mice as well as binding assays to identify and validate a PcG/trxG response element (PRE), PRE1 which acts to reorganize the surrounding chromatin, regulating S5-dependent expression. To our knowledge, PRE1 is the first validated vertebrate PcG/trxG response element. Thus, PRE1 provides a springboard for further exploration of the mechanisms governing chromatin remodeling.

The first wave of the inducible gene network up-regulated by pathogen-stimulated mononuclear cells encodes a variety of effector proteins with pleitropic biological activities. This class of primary immediate early (IE) genes codes for potent pro-inflammatory cytokines and chemokines that play a prominent role during the manifestation of inflammatory response. In an attempt to better understand induction mechanisms for such genes, I have focused on those coding for human interleukin-1&beta; (IL1B) and tumor necrosis factor &alpha; (TNF), which exhibit both transient IE induction as well as cell-type restriction. Employing a combined approach using cell lines and primary cells, reporter transient transfection, chromatin conformational capture and immunoprecipitation, evaluation of transcript integrity, ectopic expression in a non-competent cell type, and comparison to mouse orthologs, I have determined that a complex array of mechanisms interplay in order to distinctly regulate the Toll-like receptor (TLR) signaling-dependent induction of these two important pro-inflammatory genes whose deregulation provides the etiology for numerous diseases. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), which are the hallmarks of poised IE genes. In contrast, IL1B is stringently regulated by long-distance chromosome gyrations, multistep activation through a unique doubly-paused Pol II which, in association with the monocyte lineage factor Spi1/PU.1 (Spi1), maintains a low TBP and Pol II occupancy prior to activation. Activation and DNA binding of the transcription factors C/EBP&beta; and NF-&kappa;B resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of the positive elongation factor b (P-TEFb). This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Nucleosome occupancy and chromatin modification analyses of the IL1B and TNF promoters, revealed activation-specific changes in chromatin marks that are supportive for nucleosome clearance and formation of nucleosome free regions (NFR). Furthermore, ectopic expression of Spi1, along with a TLR surrogate (over-expressed TNF receptor associated factor 6, TRAF6), in a cell line incompetent for IL1B transcription, is observed to prime the cell's endogenous genome for IL1B induction by appropriately phasing promoter nucleosomes and recruiting paused Pol II in a manner reminiscent of that observed in competent monocytes. Here I report a novel connection between the metabolic state of cells and HIF-1&alpha; in regulating murine Il1b gene expression. With regard to the lipopolysaccharide (LPS) unresponsive state known as endotoxin tolerance, my data revealed that following transient induction, IL1B and TNF remained marked with paused Pol II complexes for up to 24 hours post-stimulation. Upon subsequent LPS exposure, tolerized TNF remained in an unresponsive paused state, while IL1B resumed transcription due to recruitment of positive elongation kinase P-TEFb. Emerging evidence suggests that inflammatory responses of LPS/TLR4 activated macrophages are interconnected with metabolic pathways, resulting in the shift of energy utilization by the cells. Here I report that inhibition of either phosphoinositide 3-kinase (PI3K) or glucose metabolism had a greater affect on the transcriptional response of Il1b than of Tnf. The differences between these two genes, especially for endotoxin tolerance, suggest that il1b may play a distinct role from tnf in chronic inflammation. / Bayer School of Natural and Environmental Sciences; / Biological Sciences / PhD; / Dissertation;

Cytokinins are plant hormones that affect the primary growth of shoots and roots. Application of the cytokinin N6-benzylaminopurine (BAP) to the shoot apical meristem of <i>Arabidopsis thaliana Landsberg erecta </i>(L.) Heynh induces aberrant flower development and a significant genetic response, and some of these phenotypes and expression patterns were carried to the next generation. Analysis of altered transcript levels with Affymetrix GeneChips® indicated significant changes in transcript levels of genes associated with shoot meristem activity, circadian rhythms, cytokinin metabolism, two-component systems, stress and defense responses, auxin regulation, ethylene and salicylic acid biosynthesis, and signal transduction. Specific genes were also mined from the data as potentially responsible for the BAP-induced aberrant floral phenotypes, increased floral organ number, buds in axils of sepals, and mosaic floral organs. Of particular note was a decrease in the transcript levels of CLAVATA1 (CLV1), a gene encoding a receptor kinase involved in organ differentiation and maintenance of shoot and floral meristems. Time course analysis by RT-PCR showed a decline and subsequent recovery of transcript levels of CLV1 and a coincident increase in WUSCHEL (WUS) transcript, consistent with the known suppression of WUS by CLV. WUS encodes a homeodomain protein associated with shoot meristem proliferation. The temporal coincidence of an increased floral organ phenotype with changes in transcript levels of CLV1 and WUS suggests that cytokinins regulate flower development by affecting the activity of genes controlling shoot meristem activity. Aberrant floral phenotypes in subsequent non-treated generations suggest epigenetic inheritance of some BAP-altered transcript patterns. Repressed expression of the majority of significant genes in the untreated T1 population suggests a mechanism of gene silencing, such as methylation, was involved in this epigenetic inheritance. Also, transcript levels of time-keeping genes, including CIRCADIAN CLOCK ASSOCIATED 1 / ELONGATED HYPOCOTYL, and associated genes with oscillating expression patterns, such as COLD-RESPONSIVE, were affected by BAP in treated plants and the subsequent generation, suggesting the capacity of cytokinins to affect the phase of the circadian clock. Hormonal regulation of heritably altered diurnal periodicity and environmental responses may provide a developmental and, therefore, evolutionary advantage to plants.

<p>Cis-elements govern the key step of transcription to regulate gene expression within a cell. Identification of utilized elements within a particular cell line will help further our understanding of individual and cumulative effects of trans-acting factors. These elements can be identified through an assay leveraging the ability of DNaseI to cut DNA that is in an open and accessible state making it hypersensitive to cleavage. Here we develop and explore computational techniques to measure open chromatin from sequencing and microarray data. We are able to identify 94,925 DNaseI hypersensitive sites genome-wide in CD4+ T cells. Interestingly, only 16%-20% of these sites were found in promoters. We also show that these regions are associated with different chromatin modifications. We found that DNaseI data can also be used to identify precise 'footprints' indicating protein-DNA interaction sites. Footprints for specific transcription factors correlate well with ChIP-seq enrichment, reveal distinct conservation patters, and reveal a cell-type specific arrangement of transcriptional regulation. These footprints can be used in addition to or in lieu of ChIP-seq data to better understand genomic regulatory systems.</p> / Dissertation

<p>In <italic>Saccharomyces cerevisiae</italic>, proper maintenance of haploid cell identity requires the SIR complex to mediate the silenced chromatin found at the cryptic mating-type loci, <italic>HML</italic> and <itaic>HMR</italic>. This complex consists of Sir2, a histone deacetylase and the histone binding proteins Sir3 and Sir4. Interestingly, both Sir2 and Sir3 have paralogs from a genome duplication that occurred after the divergence of <italic>Saccharomyces</italic> and <italic>Kluyveromyces species</italic>. The histone deacetylase <italic>HST1</italic> is the paralog of <italic>SIR2</italic> and works with the promoter-specific SUM1 complex to repress sporulation and alpha-specific genes. <italic>ORC1</italic> is the paralog of <italic>SIR3</italic> and is an essential subunit of the Origin Recognition Complex and also recruits SIR proteins to the <italic>HM</italic> loci. I have investigated the functions of these proteins in the non-duplicated species <italic>Kluyveromyces lactis</italic> and compared these functions to those found in <italic>S. cerevisiae</italic>. </p> <p>I have shown that <italic>SIR2</italic> and <italic>HST1</italic> subfunctionalized post-duplication via the duplication, degeneration and complementation mechanism. In <italic>S. cerevisiae</italic>, Sir2 has retained the ability to function like Hst1 when in an <italic>hst1&#916;</italic> strain. I have also shown, with a chimeric Sir2-Hst1 protein, that there are distinct specificity domains for Sir2 interaction with the SIR complex and Hst1 interaction with the SUM1 complex that have diverged between Sir2 and Hst1. Trans-species complementation assays show that the non-duplicated Sir2 from <italic>K. lactis</italic> can interact with both SIR and SUM1 complexes in <italic>S. cerevisiae</italic>.</p> <p>Further analysis into the non-duplicated experimental system of <italic>K. lactis</italic> has revealed that deletion of KlSir2 de-represses the HM loci as well as sporulation and cell-type specific genes. A physical interaction between KlSir2 and the histone binding protein KlSir4 is conserved in <italic>K. lactis</italic>, and both proteins spread across the <italic>HML</italic> locus and associate with telomeres in a manner similar to <italic>S. cerevisiae</italic>. KlSir2 also physically interacts with the DNA-binding protein, KlSum1, to repress sporulation and cell-type specific genes in a promoter-specific manner and recruitment of KlSir2 to these loci is dependent on KlSum1. Surprisingly, deletion of <italic>KlSUM1</italic> also de-represses <italic>HML</italic> and <italic>HMR</italic>, a phenotype not observed in <italic>S. cerevisiae</italic>. I show by chromatin immunoprecipitation that KlSum1 directly regulates the <italic>HM</italic> loci by spreading across these regions in a mechanism that is distinct from its role in repressing sporulation-specific genes. This result indicates that KlSum1 is a key regulator of not only meiotic, but also mating-type transcriptional programming. </p> <p>The <italic>SIR3-ORC1</italic> gene pair has previously been used as an example of neofunctionalization based on accelerated rates of evolution. However, my studies of KlOrc1 show it is distributed across <italic>HML</italic> and associates with Sir2 and Sir4 at telomeres, indicative of it having Sir3-like capabilities to spread across chromatin. This ability of KlOrc1 to spread is distinct from its functions with ORC, and is entirely dependent on its BAH domain. These findings demonstrate that prior to the genome duplication there was a silencing complex that contained both KlSir2 and KlOrc1. In addition to their functions at <italic>HML</italic> and the telomeres, KlOrc1 associates with replication origins and KlSir2 and KlSum1 work in complex to repress sporulation genes in a promoter-specific manner. The multiple functions of both KlOrc1 and KlSir2 in K. lactis indicate that after duplication, these properties were divided among paralogs and subsequently specialized to perform the functions that have been characterized in <italic>S. cerevisiae</italic>.</p> / Dissertation

Mesenchymal stem cells are more and more widely used in tissue engineering due to their pluripotency and no relative ethical problems. Traditional characterization techniques to detect mesenchymal stem cell states include flow cytometry, gene expressing profiling and immunohistochemistry. However, these methods can only provide transient and low level information from current RNA or protein levels about mesenchymal stem cells, which may cause problems when predicting the possible downstream lineages they will commit into. We have developed chromatin immunoprecipitation (ChIP)-based epigenetic technique to detect mesenchymal stem cell states. For the systems we tested, this epigenetic assessor successfully characterized cell state changes and gave similar results obtained from gene expression profiling or protein expression assay. This epigenetic technique can provide information about mesenchymal stem cells states from a more fundamental chromatin level, which is promising for predicting future lineages from current states.

In higher eukaryotes, DNA is packaged with histones and other proteins into chromatin. While this is important in the control of unwanted gene expression, chromatin also serves as a barrier to many vital functions in the cell. Therefore, cells have evolved many different types of chromatin remodeling enzymes to contend with this inhibitory structure and enable gene expression and other functions. The Saccharomyces cerevisiae PHO5 gene is triggered in response to phosphate starvation. In this study, I evaluate the chromatin remodeling requirements of this gene with respect to the multisubunit complexes SWI/SNF and SAGA. I show, for the first time, physical recruitment of SWI/SNF to the PHO5 promoter. I also demonstrate the role of promoter occupancy in influencing requirements for chromatin remodeling enzymes. Further, I describe various interactions between these two complexes at the PHO5 promoter. This study presents evidence for the first instance of excess recruitment of an ATP-dependent remodeler potentially compensating for the lack of a histone acetyltransferase.

The phaseolin (phas) promoter drives the copious production of transcripts encoding the protein phaseolin during seed embryogenesis but is silent in vegetative tissues when a nucleosome is positioned over its three phased TATA boxes. Transition from the inactive state in transgenic Arabidopsis leaves was accomplished by ectopic expression of the transcription factor PvALF (Phaseolus vulgaris ABI3-like factor), and application of abscisic acid (ABA). PvALF belongs to a family of seed-specific transcriptional activators that includes the maize viviparious1 (VP1) and the Arabidopsis abscisic acid-insensitive3 (ABI3) proteins. The major goal of the study is to gain insight to the regulation of seed-specific gene expression in three different aspects. First, since ABI3 (homolog of PvALF) is involved in ABA-mediated expression of several seed-specific protein genes in Arabidopsis, understanding its transcriptional regulation will provide insight to the mechanism by which PvALF expression is controlled. To achieve this, ABI3 promoter deletion analysis using either $-glucuronidase (gus) or green fluorescent protein (gfp) reporter gene fusions have identified various regulatory regions within the ABI3 promoter including two upstream activating sequences and a minimal seed specific expression region. In addition, a 405 bp 5' UTR was shown to play a negative role in ABI3 expression, possibly through post-transcriptional mechanisms. Second, placement of PvALF expression under control of an estradiol-inducible promoter permitted chronological ChIP analysis of changes in histone modifications, notably increased acetylation of H3-K9, as phas chromatin is remodeled (potentiated). A different array of changes (trimethylation of H3-K4) is associated with ABA-mediated activation. In contrast, H3-K14 acetylation decreased upon phas potentiation and increased on activation. Whereas decreases in histone H3 and H4 levels were detected during PvALF-mediated remodeling, slight increases occurred following ABA-mediated activation, suggesting the restoration of histone-phas interactions or the redeposition of histones in the phas chromatin. The observed histone modifications thus provide insight to the factors involved in euchromatinization and activation of a plant gene. Finally, ectopically expressed ABI5 and PvALF renders the activation of phas ABA-independent, suggesting ABI5 acts downstream of ABA during phas activation.