Functional Characterization of the Liver-Enriched Transcription Factor Creb-H

This dissertation, "Functional Characterization of the Liver-enriched Transcription Factor CREB-H" by King-tung, Tony, Chin, 錢景彤, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Functional characterization of the liver-enriched transcription factor CREB-H submitted by King-Tung Chin, Tony for the Degree of Doctor of Philosophy at the University of Hong Kong April 2005 The establishment and maintenance of a differentiated phenotype in the liver require a specific gene expression program coordinated by a group of liver-specific transcription factors. Dysregulation of liver-specific transcription is a hallmark of hepatocellular carcinoma. We have previously characterized transcription factor LZIP to be a candidate tumor suppressor targeted by an oncoprotein encoded by hepatitis C virus. Similarity of LZIP to Drosophila fat body-specific transcription factor called box B-binding factor 2 suggests the existence of additional LZIP-like liver-enriched proteins in mammals. In search of proteins closely related to LZIP, we have identified a novel liver-enriched transcription factor CREB-H. This thesis reports on the functional characterization of CREB-H. First I performed bioinformatic analysis of CREB-H-related sequences. LZIP and CREB-H represent a new subfamily of bZIP proteins distinct to other well- characterized members of the family such as CREB, C/EBPα and ATF6. One notable feature of this new subfamily of bZIP factors is the existence of a unique putative transmembrane domain in the middle of protein right next to the highly conserved bZIP region. viiiNext I characterized the properties of CREB-H protein in cultured hepatoma cells using a combination of biochemical and cell biological methods. Reporter transcriptional assays indicated that CREB-H has one activation domain and two repression domains. Electrophoretic mobility shift assays and analysis of transcriptional activities demonstrated that CREB-H activates transcription by binding to cAMP responsive element, box B, and ATF6-site/UPRE-binding element. Confocal microscopy revealed that CREB-H localizes ambiently to the endoplasmic reticulum. In addition, I provided the first evidence for proteolytic processing of CREB-H in cultured mammalian cells. Importantly, I observed that proteolytic cleavage that removed the transmembrane domain led to nuclear translocation and activation of CREB-H. To investigate the roles of CREB-H in liver-specific transcription, I asked whether CREB-H regulates the promoter of hepatic gluconeogenic enzyme phosphoenolpyruvate carboxykinase. Analysis of transcriptional activities using reporter assays and chromatin immunoprecipitation assays verified the activation of phosphoenolpyruvate carboxykinase promoter by CREB-H. Interestingly, I found that this activation by CREB-H can be further stimulated by cAMP and protein kinase A. Another salient finding in this thesis is the underexpression of CREB-H in liver cancer. Northern blotting, Western blotting and semi-quantitative reverse transcription-polymerase chain reaction analyses indicated that CREB-H is exclusively abundant in adult liver but aberrantly underexpressed in hepatoma tissues and cells. Consistent with this expression pattern, the enforced expression of CREB-H suppressed the proliferation of cultured hepatoma cells. ixIn summary, my findings suggest that the liver-enriched bZIP transcription factor CREB-H is a growth suppressor that plays an important role in hepatic physiology and pathology.