Interaction of Positive Coactivator 4 with Histone 3.3 Protein is Essential for Transcriptional Activation of the Luteinizing Hormone Receptor Gene

Abstract

The luteinizing hormone receptor (LHR) is a member of the G protein‐coupled receptor family which is essential for sexual development and reproduction in mammals. Previous studies from our laboratory established that Sp1 is important for basal LHR promoter activity and has central role in derepression of LHR gene transcription induced by the histone deacetylase inhibitor Trichostatin A (TSA) in MCF‐7 cancer cells. Moreover, our studies revealed that the co‐activator PC4 which associates directly with Sp1 at the LHR promoter is essential for TSA‐mediated LHR transcription. The present study explores interactions of PC4 with histone proteins which presumably triggers chromatin modifications during LHR transcriptional activation induced by TSA. TSA treatment of MCF‐7 cells transiently expressing PC4‐Flag protein induces acetylation of H3 and immunoprecipitation (IP) studies revealed its interaction with PC4‐Flag protein. Mass spectrometry analysis from the protein complex obtained after IP with PC4 flag in TSA treated MCF‐7 samples detected H3.3 acetylated at lysine residues (K9, K14, K18, K23, K27 and K36) as a PC4 interacting protein. This association of PC4 with H3.3 was corroborated by re‐ChIP. In subsequent studies by IP and reChip analysis using specific antibodies against acetylated H3 protein for the lysine residues, also confirmed PC4 interaction with acetylated H3 protein. Knockdown of PC4 in MCF‐7 cells significantly reduced H3 protein acetylation at these sites and LHR promoter activity in TSA treated cells despite an increase in H3 and H3.3, linking PC4 expression to H3.3 recruitment and its acetylation. Depletion of H3.3 A/B in MCF7 cells significantly impair recruitment of Pol II and TFIIB to the LHR promoter and its activation induced by TSA. In this study, we have shown interaction between PC4 and acetylated H3.3 in LHR induced derepression by TSA. Together these findings point to the critical role of PC4 and it is associated acetylated H3.3 protein in TSA‐induced LHR gene transcription.Support or Funding InformationIntramural Research Program, National Institute of Child Health and Human Development, NIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.