Biochemical and functional analysis of the human transcription factor CA150

The human transcription factor CA150 is a promoter-specific repressor of RNA polymerase II(RNAPII) transcription. We found that CA150 associates with a large RNAPII complex that contains positive transcription elongation factors including several factors necessary for activation of transcription by the HIV-1 Tat protein (P-TEFb, Tat-SF1, and TFIIF). Over-expression of CA150 was previously shown to inhibit the elongation of transcripts produced from the HIV-1 promoter. We purified a CA150 complex from HeLa cells and tested its activity in an in vitro transcription elongation assay. This CA150 fraction inhibited elongation of transcripts from the HIV-1 promoter, further demonstrating that CA150 is a negative transcription elongation factor. To understand the pathway of CA150-mediated repression, we identified several proteins that bind to CA150. The recently identified FF repeats in the carboxyl-terminal half of CA150 bind directly to the phosphorylated Carboxyl-Terminal Domain of the largest subunit of RNAPII. We determined that this interaction was required but not sufficient for CA150-mediated repression of the human alpha-4 integrin promoter. These findings provide the first known function of FF repeats. Additional functional determinants present in the amino-terminal half of CA150 were required for repression. A protein that interacted directly with the WW1 and WW2 domains of CA150 was identified as the pre-mRNA splicing factor SF1, an RNA-binding protein. Previous studies have demonstrated a role for SF1 in transcription repression. We found that binding of SF1 to CA150 WW1 and WW2 domains correlated with the requirement of these domains for CA150-mediated repression. These findings suggest that the interaction of CA150 with SF1 is important for repression by CA150. We characterized the ligand-binding specificity of the CA150 WW domains, which was unique compared to previously identified classes of WW domains. Furthermore, we mapped the CA150 binding site within the carboxyl-terminal half of SF1 (amino acids 420–500), which contains a novel type of proline-rich motif. These results support a model for CA150-mediated repression in which a complex of CA150 and SF1 targets RNAPII elongation complexes by binding to the phosphorylated CTD and the nascent transcript. An additional implication is the possible role of the CA150-SF1 interaction in co-transcriptional splicing.