Both sequence-specific transcriptional factors/cofactors and the chromatin structure modifiers PcG/trxG proteins play crucial roles in the developmental gene regulation. However, it remains to be elusive on how do these two categories of transcriptional regulators cooperate with each other to define the precise gene expression profiles for certain cell-lineages. The evolutionarily conserved C-terminal binding protein (CtBP) is a well-characterized context-dependent transcriptional cofactor in developmental processes. The prevailing model of CtBP actions is gene repression, and CtBP is reported to interact with PcG members. However, the mechanism of CtBP involved in PcG targets regulation is still unclear. In this study, we unexpectedly found that CtBP genetically antagonized PcG, suggesting activation function might be more pronounced for the role of CtBP in an epigenetic context. Thus, we furthered our activation research of CtBP and obtained the following results.
At the genetic level, by observation of the sex comb and wing phenotype of heterozygotic PcG, we found that CtBP genetically antagonize PcG function in Drosophila. To explore the molecular mechanism of the antagonism between CtBP and Pc, immunostaining was carried out in the third instar leg/wing imaginal disc, which found that CtBP was required for the activation of Scr/Ubx. Thus, CtBP might directly antagonize PcG repression. And heterozygotic mutants of the trxG conponents such as trx, Ash1 and brm bring about an abdominal tergite defect phenotype, the propotion of which was evidently increased by heterozygotic CtBP mutant or deficiency. Above all, CtBP could enhance trxG gene function, which also indirectly support the antagonism between CtBP and PcG.
We furtherly carried out more experiments in cells. In Drosophila Kc cells, Knock down of PcG members resulted in a great depression of the targets, a good model to study the activation regulation of target genes. Thus, the PcG conponents ph-p and E(z) were simutanously knocked down and sustained for transcriptome sequencing. Through the subsequent bioinformatic analysis, 381 genes were greatly depressed, among which, 153 genes were not able to derepress with the co-knock down of CtBP, suggesting that CtBP is required for activation of these genes. Further Gene Ontology analysis showed that these targets are mainly involved in transcriptional regulation and development, most of which are HOX genes. Taken together, CtBP is required for activation of the PcG targets relative to embryonic development.
To explore the function of CtBP in typical PcG targets regulation, 73 of the 381 genes were conducted to be PcG targets through Venn diagram analysis. Over 90 percent (66 genes) of the targets were activated by CtBP. GO analysis of the 66 targets showed the same enrichment pattern with the former 153 CtBP co-activated genes, most of which were HOX genes. To verify the results of the RNA-seq and GO analysis, four PcG targets (Ubx, Antp, abd-A and Abd-B) were selected for further qPCR verifications. All four targets were derepressed for at least 40 folds in ph-p and E(z) knocked down cells, which were remarkably receded by CtBP co-knock down. The same results were also observed in Pc and CtBP RNAi-ed cells. Thus, CtBP could promote the activation of PcG targets in Kc cells, especially HOX genes.
To further explore the molecular mechanism of targets activation by CtBP, ChIP analysis was carried out to detect the DNA recruitments of the PcG and trxG components as well as the histone modificaitons in different PcG and CtBP RNAi-ed Kc cells. The binding of Pc was greatly reduced after PcG RNAi, which was recovered in CtBP and PcG simutaneously RNAi-ed cells, indicating CtBP antagonize Pc DNA binding. On the other hand, the DNA bindings of trxG components UTX and CBP were sharply increased by PcG RNAi, which was removed by the addition of CtBP RNAi. These results suggesting that CtBP help recruit the trxG to DNA to activate the target genes. Besides, we also detected the DNA bindings of histone modifications and different forms of RNA Pol II. In PcG RNAi-ed Kc cells, the H3K27me3 levels were greatly decreased in both of the Promoter and PRE regions, where the H3K27ac levels were generally increased, which was disappeared in PcG and CtBP double RNAi-ed cells, suggesting that CtBP is required for the swith of histone modifications in PcG reduced background. Besides, knock down of PcG caused an increased promoter binding of RNA Pol II, all of which was removed by simutanously knock down of CtBP and PcG, demonstrating that CtBP is required for transcriptional activation and elongation. ChIP-seq data alignment between CtBP and many acitive hisone marks from ModENCODE showed the genomewide coocupation of CtBP with many acitve marks. We also carried out co-IP experiments, which showed that CtBP indeed physically interacts with the acitve histone marks.
Above all, we unexpectedly found that CtBP genetically antagonize PcG while enhance trxG function through phenotype analysis, which was furtherly confirmed by immunostaining of the targets in imaginal discs. In Kc cells, by RNAi based RNA-seq and bioinformatics analysis, we have shown that CtBP might also be required for the derepression of PcG target genes, such as homeotic genes. To get further insight into the mechanism underlying the transcriptional activation role of CtBP, we carried out ChIP analyses and found that CtBP might help recruiting proteins with activation roles, including UTX, CBP and RNA Pol II. Besides, CtBP may be an essential player in the histone modification dynamics. As such, CtBP is required for the switch of the methylated H3K27 into acetylated one. In addition, based on the alignment of publically available ChIP seq data, we found that CtBP might colocalized with many chromatin regulators. Also, our Co-IP assays indicated that CtBP might indeed physically interact with these proteins. All the results support a model that CtBP is widely required for transcriptional activation.
Taken together, we have revealed a new aspect of CtBP in contributing epigenetic regulation of gene transcription. In addition, a series of researches indirectly support the relevence between CtBP or PcG and cancers such as breast, colon and stomach cancers. Thus, analyzing the mechanism of CtBP in PcG target gene transcriptional regulation will help provide the necessary theoretical basis for developmental gene expression as well as the relevance between CtBP and cancers.