NON PHOSPHORYLATED CREB REPRESSES GENE TRANSCRIPTION BY RECRUITING HDAC1

SIRKIN, PABLO; CERUTI, JULIETA; SCASSA, MARÍA; CANEPA, EDUARDO TOMAS

Iguazú, Misiones, Argentina

Congreso; XL Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2004

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Transcription factor CREB, phosphorylated in S133, induce the activity of a great variety of CRE-containing gene promoters. On the contrary, unphosphorylated CREB cause an inhibitory effect on transcription in several genes like 5-aminolevulinate synthase (ALAS). The molecular basis of this inhibitory effect remains unclear. Transfection experiments in HepG2 cells show that CREB overexpression inhibits basal activity of ALAS/CAT fusion gene and that this effect is blocked by cotransfection with CBP. As we show using a DHAT-CBP mutant, HAT activity is crucial for CBP action. Overexpression of HDAC1 counteracts the CBP rescue effect. Similar results were observed on endogenous ALAS mRNA in HepG2 cells as assessed by Northern blot. Coimmunoprecipitation reveals a CREB-HDAC interaction in HepG2 cells that is strongly impaired after cAMP stimulation. We utilize a chromatin reconstitution approach on ALAS promoter using Drosophila extracts and recombinant proteins to further explore the CREB effects. Promoter activity were analysed by in vitro transcription and primer extension. We show that P-CREB and CBP increase ALAS expression. Excess of recombinant CREB diminishes the transcriptional activity and HDAC co-addition enhanced this inhibitory effect. We propose that CREB affects gene expression in a phosphorylation status-dependent manner. Non phosphorylated CREB recruits HDAC in order to maintain a repressive condition. Phosphorylation at S133 disrupts HDAC association and allows CBP engagement to render a productive transcriptional initiation complex.