Mechanisms of Eukaryotic Transcriptional Regulation Abstract In eukaryotes, gene regulation is largely controlled at the transcriptional level through multiple, distinct mechanisms. Transcription initiation by RNA polymerase II involves the assembly of general transcription factors on the core promoter to form a preinitiation complex (PIC). A variety of studies indicate that promoter-specific activator proteins (activators) work, at least in part, by increasing PIC assembly, which results from a direct interaction between the activator and one or more components of the transcription machinery. It has become increasingly evident that transcription is also regulated by alterations in the general transcription machinery. Our laboratory was the first to identify a vertebrate-specific TATA-box-binding protein (TBP)-related factor, TRF3. During the past funding period we have found that Trf3-depleted zebrafish embryos exhibit multiple developmental defects and, in particular, fail to undergo hematopoiesis. We have identified a single TRF3 target gene, mespa, which is required for early embryonic development and commitment of mesoderm to the hematopoietic lineage. We will continue to study the role of TRF3 and TRF3-containing complexes in transcription regulation, early development and hematopoiesis. We seek to understand how TRF3 is selectively recruited to specific target genes, identify the components of the TRF3 complex, and determine how TRF3 and TBP are incorporated into distinct multisubunit complexes. Recently, we have found that alterations in the general transcription machinery may play an important role in human embryonic stem cells (hESCs). Specifically, we have obtained several lines of evidence that hESCs lack the canonical TFIID complex and instead use an alternative TFIID complex for expression of protein- coding genes. Experiments are proposed to characterize and study this alternative TFIID complex and its role in hESC biology. We will determine the composition of the alternative TFIID complex, and the role of its individual components in hESC self- renewal and differentiation. In mammalian cells, the transcriptional activity of many genes is controlled by epigenetic mechanisms that involve histone modifications and DNA methylation. The detailed mechanisms and pathways by which epigenetic repression is established and maintained on specific genes remain to be elucidated. During the past funding period, we have performed a genome-wide shRNA screen to identify 28 factors required for epigenetic silencing of Fas, and a select group of other genes, in ras-transformed NIH 3T3 cells. This model system, in conjunction with the experimental reagents we have developed, provides a unique opportunity to study the detailed mechanisms by which an epigenetically repressed state is established and maintained on a select group of genes. Experiments are proposed to understand how multiple factors act cooperatively to establish and maintain an epigenetically repressed state and the basis by which only a small subset of genes are silenced. PUBLIC HEALTH RELEVANCE: In eukaryotes, gene regulation is largely controlled at the transcriptional level through multiple, distinct mechanisms. Transcriptional regulation is highly relevant to numerous human diseases, including inborn errors of metabolism, birth defects, viral infections and cancer. The experiments proposed in this application will increase our understanding of eukaryotic transcriptional regulatory mechanisms. A specific objective of the application is to study the mechanism by which tumor suppressor genes become transcriptionally silenced during cancer development.