Abstract

The transcriptional co-repressor CtIP interacts with several tumor suppressor proteins including pRB family members, CtBP, and BRCA1 that participate in transcriptional regulation, cell cycle control and DNA damage response. Mouse embryos lacking both alleles of Ctip died extremely early during embryogenesis, with defects in cell proliferation. Here I show that CtIP may have a role in regulation of DNA replication through its direct interaction with PCNA. CtIP localizes at S-phase DNA replication foci through a novel motif containing 42-amino acid, which is able to bind to PCNA in vivo and in vitro. Down regulation of CtIP by its shRNA expression failed to block DNA replication and S-phase progression. However, overexpression of the PCNA-interacting domain from CtIP induced DNA double-strand breaks, activated replication checkpoint signaling and reduced UV triggered Chk1 activation, suggesting an intimate relationship of CtIP and PCNA for proper regulation of DNA replication in higher eukaryotic organisms.

The Myc/Max/Mad transcriptional regulation network functions in the regulation of various cellular behaviors, including cell proliferation, differentiation, transformation and apoptosis. A search for CtIP-interacting proteins using yeast two-hybrid system led to the isolation of Mad4, a basic helix-loop-helix/leucine zipper (bHLHZip) transcriptional repressor in the Myc/Max/Mad pathway that antagonizes the transcriptional activation activity of c-Myc. Overexpressed CtIP and Mad4 interacts in vivo. Subcellular localization analysis revealed that GFP-CtIP and Mad4-RFP, as well as RFP-CtIP and GFP-c-Myc, colocalize at discrete nuclear foci. In transcription reporter assays using a reporter regulated by canonical E-Box containing promoter, I show that expression of CtIP, or CtIP mutants that can not bind to CtBP, RB or BRCA1, represses the transcriptional activity, while the expression of CtIP shRNA that down regulates CtIP protein level de-represses/activates the E-Box promoter. Finally, by using the Adenovirus-mediated RNAi coupled in vitro mouse embryonic P19 cell neuronal differentiation system, we identified a requirement of CtIP in the Myc/Max/Mad regulated neuronal differentiation. Therefore, we propose that CtIP-Mad4 complex plays an important role in the initiation of cell differentiation during the neuronal differentiation program.