The focus of experiments presented in this dissertation is to determine how signals created by exposure to environmental stimuli are integrated at the level of transcription, resulting in the generation of specific patterns of gene expression. The model system used was expression of the neurotensinl neuromedin N (NT/N) neuropeptide gene in the neuroendocrine PC12 cell line. This gene is synergistically activated in PC12 cells in response to nerve growth factor, lithium, glucocorticoids, and activators of adenylate cyclase. Several cis-regulatory elements were identified within a 200 bp regulatory region, including AP-1, CRE, and GRE-like elements. Mutational analysis confirmed the importance of these elements for responses to inducer combinations. The primary objective was to identify proteins that interact with NT/N promoter sequences and determine if they are important in mediating responses to inducer combinations. The first set of experiments was designed to investigate changes in AP-1 binding activity. Previous analysis had shown that mutation of the AP-1 site severely curtails responses to all inducer combinations indicating that AP-1 plays a pivotal role in NT/N gene activation. DNA binding studies using in vitro synthesized AP-1 proteins revealed that all heterodimeric combinations could bind both the AP-1 and JARE sites; however, these complexes displayed a higher affinity for the AP-1 site. c-Jun homodimers were also found to bind both these sites albeit with a lower affinity and with a preference for the JARE site. These studies revealed that specificity is probably not at the level of DNA binding. Therefore, it was possible that only a subset of AP-1 proteins were activated upon stimulation. DNase I footprint analysis using nuclear extracts from PC12 cells showed changes in protection at the consensus AP-1 site upon treatment with inducers suggesting changes in AP-1 binding activity. It was found that AP-1 binding activity was increased upon stimulation, with the major component being Jun B. However, substantial levels of c-Fos and c-Jun were also detected at some time points. These results coupled with transfection data demonstrating that forced expression of c-Jun and c-Fos result in potent synergistic activation of the NT/N promoter support the hypothesis that c-Jun and c-Fos are also involved in NT/N gene activation. DNase I footprinting studies using PC12 nuclear extracts also revealed substantial areas of protection surrounding the CRE element. This result, along with the high degree of conservation of these sequences between human and rat, suggested they play a role in the regulation of the NT/N gene in PC12 cells. Mutational analysis of this region showed that sequences upstream of the CRE were important for full activation of the NT/N promoter. Specific mutation of the CRE resulted in a 75% decrease in activity upon induction, a level similar to that observed previously with less precise linker scanner mutations. This site had also been shown to be critical for c-Jun mediated NT/N activation, even though c-Jun homodimers do not bind this site in vitro. Therefore, nuclear extracts from PC12 cells were tested for the presence of proteins which could bind this site. Complexes composed of both c-Jun and ATF-2 were found in extracts from both uninduced and induced PC12 cells. ATF-2 could mediate both the recruitment of c-Jun to this site as well as mediate the effect of activators of adenylate cyclase, since ATF-2 has been shown to be a target for protein kinase A in vitro. Expression of ATF-2 in PC12 cells resulted in a modest increase in NT/N promoter activation. The significant levels of endogenous ATF-2 protein in PC12 cells most likely accounts for the relatively small magnitude of this effect. Experiments with the closely related protein, ATF-a2, revealed that it potently antagonizes c-Jun activation while forced expression of ATF-2 did not affect c-Jun activation under the conditions analyzed. Therefore, ATF proteins could be involved in both activation and repression of the NT/N gene. Both c-Jun and ATF-2 have been shown to be activated by c-Jun N-terminal kinase (JNK) in response to environmental stress or cytokine activation. Therefore, the ability of inducers to activate the previously described N-terminal ATF-2 activation domain was investigated using a GAL4-ATF-2 (1-109) chimer construct. This construct was not significantly activated by inducer combinations that result in high level NT/N gene expression, indicating that activation of ATF-2 through this pathway is not involved in NT/N gene activation. Also activation of JNK, a MAPK which activates both c-Jun and ATF-2, only partially substituted for NGF indicating that NGF activates an additional pathway. The data presented here support a model involving synergistic transcriptional activation of the NT/N promoter by c-Jun/c-Fos, ATF-2, ATF-2/c-Jun and the GR. ATF-2 was found to enhance NT/N promoter activation while a splice variant (ATF-2 195) lacking a central portion of ATF-2 that is rich in Ser/Thr residues had no effect suggesting that this region could be important for ATF-2 activation in PC12 cells. The identification of the signaling pathways that mediate the effects of inducer combinations on NT/N gene activation will be an important future goal and should provide insights into the control of neuronal gene expression.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 135-149). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 133-147). Also available on the Internet.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves : 144-159). Also available on the Internet.

The antibody class switch is achieved by DNA recombination between the sequences called switch (S) regions located 5' to immunoglobulin (Ig) heavy chain constant (CH) region genes. This process can be induced in cultured B cells by polyclonal stimulation and switching can be directed to specific antibody classes by certain lymphokines. These stimuli may regulate the accessibility of CH genes and their S regions to a recombinase as indicated by hypomethylation and transcriptional activity. For example, RNAs transcribed from specific unrearranged (germ-line) CH genes are induced prior to switching under conditions that promote subsequent switching to these same CH genes. The function of transcription of these germ-line CH genes is unknown. How stimuli regulate the accessibility of CHgenes is also unclear. I report in this dissertation the structure of the RNA transcribed from the unrearranged Cγ1 gene in mouse spleen cells treated with LPS plus a HeLa cell supernatant containing recombinant interleukin 4 (rIL-4). I will also show that an 150-bp region upstream of the first initiation site of germ-line γ1 RNA contains promoter and enhancer elements responsible for basal level expression and inducibility by phorbol 12-myristate 13-acetate (PMA) and synergy with IL-4 in an IgM+ B cell line, L10A6.2, and an IgG2a+B cell line, A20.3. The germ-line γ1 RNA is initiated at multiple start sites 5' to the tandem repeats of the γ1 switch (Sγ1) region. As is true for analogous RNAs transcribed from other unrearranged genes, the germ-line γ1 RNA has an I exon transcribed from the region 5' to the Sγ1 region.. The Iγ1 exon is spliced at a unique site to the Cγ1 gene. The germ-line γ1 RNA has an open-reading frame (ORF) that potentially encodes a small protein 48 amino acids in length. Elements located within the 150 bp region 5' to the first initiation site of germ-line γ1 RNA are necessary and sufficient to confer inducibility by PMA and synergy with IL-4 to a minimal thymidine kinase (TK) promoter in L10A6.2 cells but are not sufficient to confer this inducibility in A20.3 cells. Linker-scanning mutations demonstrated that these multiple elements function in a mutually dependent manner as indicated by the fact that mutation of any single element will decrease constitutive expression and inducibility by PMA and PMA plus IL-4. This 150-bp region contains several consensus sequences that bind to known or putative transcription factors, including a C/EBP binding site/IL-4 response element (in the promoter for Ia Aαkgene), four CACCC boxes, a PU box, a TGFβ inhibitory element (TIE), an interferon-αβ response element (αβIRE), and an AP-3 site. My results begin to provide a description of the mechanism of regulation of the accessibility of unrearranged germ-line Sγ1-Cγ1 gene. By activating the germ-line γ1 promoter, IL-4 induces transcription of germ-line γ1 RNA, thereby inducing accessibility of the Sγ1-Cγ1 gene. By inhibiting expression of the germ-line γ1 promoter, IFNγ and TGFβ down-regulate transcription of germ-line γ1 RNA, thus reducing the accessibility of the Sγ1-Cγ1 gene. My results also suggest that signaling via the antigen receptor on B cells may be involved in induction of switch to IgG1. Furthermore, this is the first case reported in which multiple functionally interdependent elements are needed to respond to PMA.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.