This thesis explores how basic region/leucine zipper (bZIP) transcription factors target gene regulatory sequences. The GCN4 bZIP binds to more than one target site [CRE (TGACGTCA) and cognate AP-1 (TGACTCA)] and exhibits flexibility in -helical structure. These observations suggest that the GCN4 bZIP can establish sequence-selective DNA binding at noncognate target sites. Studies on such noncognate but sequence-selective binding can provide insights into how bZIP proteins search for and localize to their cognate target sites. This thesis investigates DNA binding by the GCN4 bZIP and its structural and functional mimic, the wild-type (wt) bZIP, at noncognate gene regulatory sequences C/EBP (TTGCGCAA), E-box (CACGTG), HRE (GCACGTAG), XRE1 (TTGCGTGA), and related DNA sequences. These DNA-binding activities are sequence-selective, as confirmed by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Full- and half-site DNA-binding affinities, determined by EMSA titrations, decrease from cognate to noncognate binding. At noncognate target sites, the bZIP proteins form a dimer of -helices, as indicated by circular dichroism (CD) spectroscopy and EMSA. These results demonstrate that the bZIP proteins can establish noncognate but sequence-selective DNA binding, and suggest such DNA binding potentially contributes to structure preorganization and rapid translocation of the bZIP proteins when they search for their cognate target sites, to which they then bind with high affinity. This thesis also indicates a highly dynamic DNA-binding model for the bZIP proteins to establish strong and sequence-selective DNA binding. The C/EBP site includes two 5H-LR (TTGCG) half-sites, each of which comprises two 4-bp subsites. The in vitro and in silico results together demonstrate that the basic region at 5H-LR recognizes the 4-bp subsites alternately as distinct units, which requires it to translocate between the subsites, potentially by sliding or hopping. Taken as a whole, this thesis provides further insights into how bZIP transcription factors accomplish sequence-selective DNA binding.

This thesis explores how basic region/leucine zipper (bZIP) transcription factors target gene regulatory sequences. The GCN4 bZIP binds to more than one target site [CRE (TGACGTCA) and cognate AP-1 (TGACTCA)] and exhibits flexibility in -helical structure. These observations suggest that the GCN4 bZIP can establish sequence-selective DNA binding at noncognate target sites. Studies on such noncognate but sequence-selective binding can provide insights into how bZIP proteins search for and localize to their cognate target sites. This thesis investigates DNA binding by the GCN4 bZIP and its structural and functional mimic, the wild-type (wt) bZIP, at noncognate gene regulatory sequences C/EBP (TTGCGCAA), E-box (CACGTG), HRE (GCACGTAG), XRE1 (TTGCGTGA), and related DNA sequences. These DNA-binding activities are sequence-selective, as confirmed by DNase I footprinting and electrophoretic mobility shift assay (EMSA). Full- and half-site DNA-binding affinities, determined by EMSA titrations, decrease from cognate to noncognate binding. At noncognate target sites, the bZIP proteins form a dimer of -helices, as indicated by circular dichroism (CD) spectroscopy and EMSA. These results demonstrate that the bZIP proteins can establish noncognate but sequence-selective DNA binding, and suggest such DNA binding potentially contributes to structure preorganization and rapid translocation of the bZIP proteins when they search for their cognate target sites, to which they then bind with high affinity. This thesis also indicates a highly dynamic DNA-binding model for the bZIP proteins to establish strong and sequence-selective DNA binding. The C/EBP site includes two 5H-LR (TTGCG) half-sites, each of which comprises two 4-bp subsites. The in vitro and in silico results together demonstrate that the basic region at 5H-LR recognizes the 4-bp subsites alternately as distinct units, which requires it to translocate between the subsites, potentially by sliding or hopping. Taken as a whole, this thesis provides further insights into how bZIP transcription factors accomplish sequence-selective DNA binding.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22728号 / 医博第4646号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 萩原 正敏, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Induction of C-reactive protein (CRP) synthesis in hepatocytes by cytokines occurs at the transcriptional level. In Hep3B cells, the transcription factors C/EBPβ, STAT3, and Rel p50 have been shown to participate in this process. A C/EBP binding site centered at -53 and an overlapping nonconsensus κB site on the promoter are critical for CRP expression. We have previously found that an oligonucleotide containing a κB site diminished binding of C/EBPβ to the C/EBP site, suggesting that unidentified Rel proteins present in Hep3B nuclei facilitate the formation of C/EBPβ-complexes. The current studies were undertaken to determine which of the five Rel proteins, p50/p65/p52/c-Rel/RelB, play such a role. Mutation of the nonconsensus κB site did not abolish binding of C/EBPβ to its binding site, indicating that this site was not necessary for the formation of C/EBPβ-complexes. Depletion of Rel proteins from Hep3B nuclei led to decreased formation of C/EBPβ-complexes on a CRP promoter-derived oligonucleotide that contained only the intact C/EBP binding site but not the nonconsensus κB site. This finding indicates that Rel proteins are involved in the binding of C/EBPβ to its binding site by a κB site-independent mechanism. Electrophoretic mobility shift assays (EMSAs) revealed that it was c-Rel that facilitated formation of C/EBPβ-complexes and that c-Rel bound directly to C/EBPβ-complexes formed on the C/EBP site. Cotransfection of c-Rel enhanced the induction of CRP promoter-driven luciferase activity and enhanced endogenous CRP expression in cells transfected with C/EBPβ. We conclude that c-Rel regulates CRP expression without the requirement of binding to a κB site, and binds directly to C/EBPβ to facilitate the binding of C/EBPβ to the CRP promoter.

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<p>A microarray analysis of RNA from contact inhibited CEF indicated a hypoxic signature in the contact inhibition program of gene expression (Ghosh <em>et al</em>., 2009). The purpose of this thesis was to investigate whether GAS genes known to be induced during contact inhibition are inducible by hypoxia. The gene p20K was selected as the model for this investigation because it is a growth arrest specific (GAS) gene with a well-characterized promoter (Mao <em>et al</em>., 1993). p20K expression was shown to be positively regulated in hypoxia. It was then determined by transient expression assay that this induction occurred at the promoter level. Interestingly by dissecting the promoter it was found that the quiescent responsive unit (QRU) was required for promoter induction during hypoxia. It has previously been shown that the QRU was required for contact inhibition induction of p20K in a C/EBPβ dependent manner (Mao <em>et al</em>., 1993; Kim <em>et al</em>., 1999).</p> <p>The mechanism behind hypoxic induction of the QRU was then investigated. The kinetics of HIF1α and p20K induction during hypoxia demonstrated that HIF1α was transiently expressed between 2-8 hrs of hypoxia while p20K was induced after 8 hrs of hypoxia. Co-Immuniprecipitation assay was also used to determine if a HIF1α-C/EBPβ interaction occurred, however, this molecular interaction could not be shown. These experiments suggests that HIF1α is not involved with the induction of the QRU. Over-expression of the dominant negative C/EBPβΔ184 repressed p20K induction, thus implicating C/EBPβ in activation in both contact inhibition and hypoxia. We also observed by western blot analysis that the C/EBP family member CHOP was repressed during hypoxia, causing a decrease in the amount of CHOP-C/EBPβ complexes in the cell. It was also found that over-expression of CHOP antagonized the induction of p20K by hypoxia. In conclusion hypoxia represses CHOP levels resulting in an increase of potent C/EBPβ homodimers at the expense of the inactive CHOP-C/EBPβ heterodimers.</p> / Master of Science (MSc)

<p>The study of growth arrest specific (GAS) genes is critical for our understanding of quiescence cell states. C/EBP-β is a transcriptional activator which is central to the expression of GAS genes in growth arrested cells. C/EBP-β is involved in the activation of numerous pathways, including mitogenesis, cytokine signaling, stress response, etc. Thus, it requires signaling cues which confer specificity in terms of gene expression.</p> <p>Here we used the p20K gene in chicken embryonic fibroblasts as a model system to study the control mechanisms of GAS genes. p20K is expressed in conditions such as contact inhibition mediated growth arrest and mild hypoxia. Here we explored the control mechanism mediated by ERK2 at the p20K promoter (QRU), as a mode of regulation which confers C/EBP-β binding specificity.</p> <p>In this study we demonstrate that ERK2 is recruited to the QRU in proliferative cells, i.e. where p20K is repressed. Using ChIP analysis we show that ERK2 binds directly to the QRU in proliferative cell states, but not in growth arrested cell conditions. Using a similar approach we demonstrate that ERK2 binding to the QRU is lost in states of hypoxia, where p20K is strongly induced. Furthermore, we show that this interaction is specific to ERK2 and is not observed with the related ERK1 kinase. Lastly, we employed transient expression assays to illustrate that ERK2 acts as a transcriptional repressor of the QRU. Through these experiments we have illustrated that ERK2 mediated transcriptional repression is a novel control mechanism at the QRU which skews C/EBP-β mediated signaling networks in proliferating cells.</p> / Master of Science (MSc)

We reported previously that prostaglandin E2 (PGE2) upregulates IL-23 in vitro in bone marrow-derived dendritic cells (DC), and in vivo in models of collagen-induced arthritis and inflammatory bowel disease, leading to preferential Th17 development and activity. There is very little information on the molecular mechanisms involved in the PGE2-induced upregulation of Il23a gene expression. In the present study we investigated the signaling pathways and transcription factors involved in the stimulatory effect of PGE2. Although PGE2 does not induce IL-23p19 expression by itself, it synergizes with both extra- and intracellular TLR ligands and with inflammatory cytokines such as TNFα. We established that the effect of PGE2 in conjunction with either LPS or TNFα is mediated through the EP4 receptor and the cAMP-dependent activation of both PKA and EPAC. Using the EP4 agonist PGE1OH in conjunction with TNFα, we found that PKA-induced PCREB and EPAC-induced PC/EBPβ mediate the stimulatory effect of PGE2 on IL-23p19 expression. This is the first report of CREB and C/EBPβ involvement in Il23a promoter activation. Mutation within the putative CREB and C/EBP sites combined with in vivo DNA binding (ChIP) assays identified the distal CREB site (-1125) and the two proximal C/EBP sites (-274 and -232) as essential for PKA-activated CREB and EPAC-activated C/EBPβ induced IL-23p19 expression. / Microbiology and Immunology