UC Riverside

ABSTRACT OF THE DISSERTATION

Mechanisms of Core Promoter Sequence-dependent RNA Polymerase II Transcription

by

Muyu Xu

Doctor of Philosophy, Graduate Program in Biochemistry and Molecular Biology

University of California, Riverside, December 2012

Dr. Ernest Martinez, Chairperson

The TATA-box, Initiator (INR), Downstream Promoter Element (DPE), Motif Ten Element (MTE), TFIIB Recognition Element (BRE) and the other core promoter elements contribute to the diverse architecture of core promoters and are paramount for transcriptional activation. Diverse core promoters can communicate with enhancer-bound activators to contribute in the second level gene expression regulation. However, the mechanisms of transcription initiation catalyzed by different core promoters remain unknown and/or controversial. Because TFIID and TFIIB bind most of the core promoter elements, many scientists believe that different core promoters are regulated by the same set of general transcription factors. In contrast, other scientists including us insist that additional core promoter sequence-specific transcription factors besides the general transcription machinery are required to regulate transcription from different core promoters. Several lines of evidence support this hypothesis: a TAFs and Initiator dependent Cofactor 1 (TIC1) fraction requires for the TATA/INR synergy; a TIC2 fraction supports TATA-less core promoter directed transcription; CK2, PC4 and Mediator facilitate the Sp1-activated INR/DPE transcription in mammalian system and NC2 mediates the INR/DPE transcription in Drosophila system. Here, we further purify the TIC1 fraction and identified HMGA1 and Mediator as the effective components that support TATA/INR synergy in vitro. In addition, we also verify the TATA/INR specific role of HMGA1 in mammalian cells. Furthermore, we demonstrate HMGA1 interacts with TFIID and Mediator, and the acidic COOH-tail of HMGA1 is required but not sufficient for HMGA1 to interact with TFIID and Mediator. Accordingly, HMGA1 COOH-tail is also required to support the maximal transcriptional synergy between the TATA-box and the INR. Finally, analysis of activated transcription by Gal4-fusion activators and the beta-Actin gene (ACTB) promoter upstream activating sequences further demonstrates that preferential communication between activators and core promoters contributes to the gene expression regulation. Amazingly, a strict TATA-specificificity by the ACTB upstream activating sequences is revealed for the first time.