The Role Of Drosophila PTIP In PcG/trxG Transcriptional Regulation

PAX transcription activation domain interacting protein (PTIP), as known as PAXIP1 in human genome, has six BRCT domains and a polyQ region. It is known that PTIP interacts with a variety of proteins to form distinct complexes that are involved in different biological functions. For example, the N-terminal two BRCTs may recruit PA1 to mediate the class switch recombination in the expression of immunoglobulin heavy chain. The C-terminal BRCTs interact with 53BP1 and are involved in DNA damage response and repair. They also interact with histone modification proteins, such as ALR (MLL2), MLL3/MLL4 and UTX, all of which possess histone methyltransferase (HMT) or histone demethylase enzymatic activities, suggesting that PTIP may play a role in transcriptional regulation.We have previously identified a single ptip orthologous gene in Drosophila genome demonstrated by the fully rescue of the lethal phenotype of a fly tip mutant with mouse Ptip expression. We also showed that ptip plays profound roles in fly development including transcriptional regulation of many developmental genes, such as even skipped, fushi tarazu and Engrailed, consistent with the recent finding that PITP belongs to a unique TRR/COMPASS complex.In this study, to further clarify the role of ptip along the epigenetic control of gene expression by PcG/trxG genes, we used fly genetics to explore the genetic interaction between ptip and Polycomb (Pc) in wing development. We also used cell model to explore the ptip role in transcriptional regulation. The results are as follows.1. ptip enhanced the wing phenotypes of Pc.As we already know that loss of PcG genes causes extra sex combs in the second and/or the third legs, a phenotype referred as homeotic transformation. Loss just one copy of ptip does not show any homeotic phenotypes. However, previously in our lab we have found that ptip enhances the extra sex combs caused by loss of PcG genes. Based on this finding we utilized a PcG related wing phenotype to further examination the role of ptip in PcG regulation. We found that both a ptip mutant allele,ptipPbac, and a ptip deficiency allele could enhance the Pc3 wing phenotype. In addition, importantly we explored the wing defects at Ubx gene expression level, we found that ptip heterozygosity greatly enhanced the ectopic Ubx expression presumed by derepression of Pc. We thus further confirmed the synergistic function of ptip in PcG repression and provide an explanation of this genetic interaction at a molecular/gene level.2. ptip affects House-keeping genes expression in Drosophila Kc cells.We wanted to study the mechanism on how ptip affects PcG/trxG transcriptional regulation from mRNA level in Kc cells by RNAi. As we already know that ptip knockdown (KD) influences the expression of many PcG target genes. Here, we showed that ptip is also required for expression of many house-keeping genes, such as tubulin, actin, gapdh, rp49 and tbp. These results indicate ptip may be generally required for gene activation. In the case of the derepression of Ubx caused by KD of PcG genes, we found co-KD of ptip dramatically enhanced the derepression when Ubx level was low. This is in contrast with our previous conclusion that ptip is required for derepression but consistent with the genetic studies in the wing in this study. We reasoned that these results may indicate the complexity of ptip function and might suggest that ptip could be important for the balance-play in PcG/trxG transcriptional regulation.Together, these in vivo and cells experiments suggest that PTIP is essential for the cell growth and/or differentiation. The role of PTIP can affect House-keeping genes expression that could explain the early lethal phenotype of ptip gene in both flies and mammals. At present, we are greatly interested in PTIP target genes and its specific functions which are our future directions for studying PTIP, therefore our results are helpful in further understanding the PTIP function.