Functional Study On The Role Of PTIP Along PcG And TrxG Pathways

Gene expression and its regulation play central roles in cellular activities.During developmental process,the activated or silenced pattern of target genes were established at early stage by transcriptional factors,and the chromatin structure modifiers Pc G/trx G proteins play crucial roles in the maintenance.However,it remains to be elucidated on how do these two categories of transcriptional regulators cooperate with each other to define the precise gene expression profiles for certain cell-lineages.In this study,we focused on Pax2 Transactivation domain Interacting Protein(PTIP,also known as PAXIP1 in mammals)and found that PTIP potentially participates in Pc G targets regulation.PTIP exerts a broad range of biological functions and participate in many biological processes,such as DNA damage response,DNA recombination,and epigenetic modification.Previous research revealed that PTIP is a member of COMPASS complex and required for embryonic development of Drsophila.Based on previous research,we sought to further study PTIP and obtained the following results.To gain deep insight of molecular mechanism of PTIP participates in Pc G/trx G target genes regulation,we first generated PTIP tagged knock in flies and performed ChIP-seq by using early-stage embryos.Then we performed analysis of ChIP-seq data and compared with existed data,we found that PTIP colocalized with most chromatin modification factors,especially closely associated with Pc G and Trx G complexes.At the genome scale,we sorted given PTIP binding peaks into two groups: 2722 PTIP/TRR-cobound and 1080PTIP/PC-cobound peaks.In particular,we found that PTIP mediates the molecular switch between H3K4me3/H3K27 ac and H3K27me3 histone modifications at TRR or PC occupied regions.Further Gene Ontology analysis showed that PTIP,TRR and PC common targets are mainly involved in transcriptional regulation and development,most of which are homeotic genes(Hox genes).In cell model,we found that PTIP is required for the transrepression of many Pc G-repressed transcription factors.Taken together,PTIP is required for regulation of the Pc G targets related to embryonic development.At the genetic level,by observing the sex comb and wing phenotype of heterozygotic Pc G,we found that PTIP genetically enhance Pc G function in Drosophila.To explore the molecular mechanism of the antagonism between PTIP and PC,immunostaining was carried out in the third instar leg/wing imaginal discs,and we found that PTIP was required for the derepression of Scr/Ubx.Thus,PTIP might directly enhance Pc G repression.Moreover,heterozygotic mutants of the trx G conponents such as trx bring about an abdominal tergite defect phenotype,the proportion of which was evidently increased by heterozygotic PTIP mutant or deficiency.Thus,PTIP could antagonize trx G gene function,which also indirectly supports the enhance function between PTIP and Pc G.In summary,based on the alignment of ChIP seq data,we found that PTIP colocalized with many chromatin regulators.To gain further insight into the mechanism underlying the transcriptional activation role of PTIP,we carried out ChIP-seq analyses and we found that PTIP might mediate the molecular switch between H3K27me3 and H3K4me3.In Kc cells,by RNAi based RNA-seq and bioinformatics analysis,we have shown that PTIP might also be required for the repression or activation of Pc G target genes,such as homeotic genes.Finally,we unexpectedly found that ptip genetically enhance Pc G while antagonize trx G function through phenotype analyses,which was further confirmed by immunostaining of the targets in imaginal discs.All the results support a model that PTIP is widely required for transcriptional activation and repression.Taken together,we have revealed a new aspect of PTIP in regulation of PcG/TrxG target genes.PTIP mediated a wide range of developmental processes and were enriched in Hox clusters,which were related by Pc G.PTIP mediates the molecular switch from H3K27me3 to H3K4me3 and H3K27 ac histone modifications at PTIP/PC-cobound regions.Furthermore,analysis of PTIP target genes indicates that the majority of Hox genes are PTIP,PC,and TRR common targets,which reveals that PTIP functions to coordinate with Pc G and Trx G during the developmental process.Thus,analyzing the mechanism of PTIP in Pc G target gene transcriptional regulation will help provide the necessary theoretical basis for developmental gene expression as well as understanding Pc G/trx G pathways.