Deciphering the molecular mechanisms of the transcriptional regulation mediated by orphan nuclear receptor COUP-TFI and C2H2 zinc finger protein Ctip2

Graduation date: 2010COUP-TFI, an orphan nuclear receptor of the steroid/thyroid hormone receptor superfamily, plays important roles in homeostasis and the CNS development, including differentiation, patterning, axonal projection, cell migration, cortical arealization and the temporal specification of neural stem cells. A number of COUP-interacting proteins have been described previously, and the majority of these proteins were identified and/or characterized by yeast two-hybrid systems. None of these COUP-interacting proteins, to our knowledge, have been demonstrated to be recruited to the promoter of target genes in a COUP-dependent manner, and a systematic study of COUP-TFI complexes in mammalian cells has not been conducted.\ud In our study, we have identified a number of COUP-TFI-interacting proteins in HeLa S3 cells by a tandem affinity purification procedure. We found that COUP-TFI associated with transcriptional regulatory proteins, including the nuclear receptor corepressor (NCoR) and TIF1β, and a proapoptotic protein DBC1. In vitro experiments revealed that COUP-TFI interacted directly with NCoR but in a manner different from that of other nuclear receptors. DBC1 stabilized the interaction between COUP-TFI and NCoR by interacting directly with both proteins. The gene encoding the anti-apoptotic protein TNFAIP8 (TNFα-induced protein 8) was identified as being repressed by COUP-TFI in a manner that required several of the component proteins of the COUP-TFI complex. Our studies also highlight a central role for COUP-TFI in the induction of the TNFAIP8 promoter by TNFα.\ud Ctip2, a C2H2 zinc finger transcription factor, plays crucial roles in the development of the CNS, and in the immune and cutaneous systems, and is essential for post-natal life. Germline deletion of Ctip2 results in arrest of early T cell development with the complete absence of αβ T cells. In contrast, deletion of Ctip2 at the CD4+ CD8+ double-positive (DP) stage allows development of DP cells but not transition to the CD4+ or CD8+ single positive (SP) stage. Thus, Ctip2 plays an important role in at least two stages of T cell development: a very early stage prior to the development of the DP cell and also in the DP → CD4+ or CD8+ single positive (SP) transition.\ud In the effort to understand the influence of cell signaling pathways on the transcriptional regulatory activity of Ctip2 in thymocytes, we adapted an ex vivo system in which the combination of phorbol ester PMA and calcium ionophore A23187 (P/A) triggered differentiation of primary mouse thymocytes. We have discovered a dynamically regulated pathway in stimulated mouse DP cells involving sequential and linked post-translational modifications (PTMs) of Ctip2, as follows: Phosphorylation → Dephosphorylation → SUMOylation. SUMOylation of Ctip2 is dynamic and is tightly regulated by the phosphorylation status of Ctip2. Our data also suggest that SUMOylation of Ctip2 may serve as a molecular switch that converts Ctip2 from a repressor to an activator of the promoter of Id2 gene, a newly identified Ctip2-targeted gene. We have also demonstrated the molecular basis for the output of this switch: P/A treatment promotes interaction of Ctip2 with the transcriptional coactivator p300 on the Id2 promoter.\ud These results described herein provide a framework for understanding the mechanisms underlying the transcription regulatory activities of COUP-TFI and Ctip2, and how these two transcription factors are regulated by the TNFα signaling pathway and the T cell signaling pathway, respectively. These studies may contribute to a better understanding of the molecular and cellular basis for COUP-TFI and Ctip2 function in vivo