Abstract
Abstract: :
Purpose: Crx is a transcription factor required for expression of many photoreceptor genes. The mechanism by which Crx activates transcription is unclear. Transcriptional activation begins with a de–repression process in which highly condensed chromatin is converted into a less condensed configuration that is competent for transcription. De–repression is often associated with the covalent modifications of histones at the regulatory region of specific genes. The goal of this study was to test whether Crx promotes the de–repression of target genes by recruiting histone acetyl–transferases (HATs). Methods: FLAG–Crx pull–down and immunoprecipitation assays were performed to test if Crx recruits protein complexes containing HATs, such as GCN5 and CBP. Chromatin immunoprecipitation (ChIP) and real–time PCR were used to detect the binding of Crx, specific HATs and acetylated histone H3 (Ac–H3) to the promoter/enhancer regions of selected photoreceptor genes in the retinas of Crx–/–, Crx+/– and Crx+/+ mice at postnatal day 14. Results: (i) GCN5 or CBP co–activator complexes co–immunoprecipitated with Crx from cell extracts. (ii) The regulatory regions of the Crx target genes, rod and cone opsins and IRBP, were present in immunoprecipitated chromatin from wild–type mouse retinas using antibodies to Crx, GCN5, CBP and Ac–H3. The association of the four factors with their target gene chromatin is retina– and regulatory region–specific, as it is absent in the brain and in the 3’ region of the same genes in the retina. (iii) In the Crx–/– retina, the association of Ac–H3, GCN5 and CBP with the opsin genes was markedly reduced. These genes are either not transcribed or transcribed at a low level in the absence of Crx. In contrast, transcription of the IRBP gene is unchanged in Crx–/– retina and there is no change in the association of HATs with the IRBP promoter. Conclusions: Retinal–specific transcription of Crx–regulated photoreceptor genes correlates with hyperacetylation of histones on their promoters/enhancer regions. Hyperacetylation is important for transcriptional activation and requires the function of Crx and its interaction with HAT–containing co–activator complexes.
Keywords: transcription factors • transcription • photoreceptors