Coordination Of Mediator Complex And Histone Acetyltransferase CBP/p300 In Transcriptional Regulation

Transcriptional regulation,which is precise and complex,is the fundamental process of multiple biological functions.In the cells,the genetic information carrier-DNA forms the nucleosome by wrapping around the histone octamer;the nucleosome is highly compacted to form chromatin with a complex superstructure.In general,the transcriptional regulation contains two levels.Firstly,the chromatin structure is regulated and the target genes become accessible.Subsequently,the transcriptional machine and the RNA polymerase Pol II assemble in the transcription site to initiate the transcription.CBP/p300 is one group of histone acetyltransferases,which catalyze the acetylation of histone lysine in order to loosen the chromatin structure and promote transcription.The multiple-subunits complex MEDIATOR is important for the transcription initiation through participating in the assembly of transcriptional pre-initiation complex(PIC).Through the affinity purification coupled with mass spectrometry,we identified the composition of the complex involved with CBP/p300.It was found that the HAC1 and HAC5 could be co-purified with the MEDIATOR complex in the Arabidopsis thaliana.By using yeast-two-hybrid system,we found that MED8 and MED25 subunits physically interact with HAC1 and HAC5.The double mutant hac1hac5 and med8med25 show similar phenotypes,including serious late flowering and reduced fertility.RNA-seq data indicated that MEDIATOR and CBP/p300 co-regulate a large number of genes.Furthermore,H3Ac ChIP-seq data showed that the H3 acetylation level is downregulated in the med8med25 mutant as well as in hac1hac5 mutant,indicating that MEDIATOR complex plays an important role in regulating the histone H3 acetylation.Besides,we found that MEDIATOR and CBP/p300 were enriched in the API,ALG8,APL,LAA29,and GAI genes.In hac1hac5 and med8med25 mutants,the Pol ? enrichment on those genes was significantly reduced,but the H3Ac level did not change obviously.These results suggested that CBP/p300 and MEDIATOR regulate transcription through affecting Pol ?enrichment in transcription initiation directly,independent of H3 acetylation activity.MED8 protein form the liquid puncta in vitro and in vivo,exhibiting the canonical prosperity of liquid-liquid phase separation(LLPS).MED8 can co-localize with HAC1 protein in vitro and in partial cells in vivo.The N-terminal prion-like domain of MED8 is rich in glutamine,which is essential for the phase separation.The MED8 protein lacking PrD domain or without glutamine could not phase separate.These results indicates that MEDIATOR and CBP/p300 participate in the LLPS organization to regulate the transcription.In summary,our study identified that MEDIATOR interacts with HAC1 and HAC5,and participate in the regulation of transcription and histone acetylation coordinately,thereby regulating the development of Arabidopsis.Besides,MEDIATOR,HAC1,and HAC5 may be organized together by the LLPS to regulate transcription.