The development and maintenance of photoreceptor cell function require precisely regulated expression of photoreceptor-specific genes. The long-term goal of our research is to determine the molecular mechanisms regulating the transcription of photoreceptor genes and the role of transcription dysregulation in photoreceptor diseases. Regulation of transcription involves interactions among network transcription factors and their target genes. We are studying a photoreceptor-specific transcription factor, cone-rod homeobox (Crx) that is essential for the transcription of many photoreceptor genes and is associated with photoreceptor degenerative diseases. We have identified ten Crx interacting proteins (CIPs) that are expressed either ubiquitously in many cells or specifically (preferentially) in photoreceptors. We hypothesize that the interactions of Crx and these CIPs regulate transcription of target genes in specific types of photoreceptors. In this renewal application, we propose to test this hypothesis using both in vivo and in vitro approaches.
In Aim #1, we will focus on two specific CIPs, the nuclear receptor Nr2e3 and the zinc-finger transcription factor Sp4 and characterize their interactions with Crx in vivo using co-immunoprecipitation and coexpression studies in the mouse retina. We will also use cell transfections to determine if these interactions have functional significance on the transcription of rod- or cone-specific genes, such as opsins and the gene products identified in Aim #2.
In Aim #2, we will use chromatin immunoprecipitation assays (ChIP) to identify a variety of in vivo targets that are regulated by Crx and CIPs and that are important for the function and survival of photoreceptors. Candidate photoreceptor gene targets will be detected using PCR with primers corresponding to the regulatory regions of specific genes, while novel targets will be identified by screening genomic arrays (ChIP-Array) or by cloning (ChIP-Cloning).
Aim #3 is based on our recent findings that histones on the regulatory regions of several photoreceptor genes are hyper-acetylated (a chromatin modification that activates transcription) and that Crx interacts with ataxin-7 or CBP/p300, ubiquitous CIPs associated with co-activator complexes that catalyze histone acetylation. Thus, we hypothesize that hyper-acetylation of histones is important for activating transcription of the photoreceptor genes and is promoted by Crx interacting with ubiquitous CIPs. We will determine if the degree of histone acetylation affects photoreceptor gene expression by modifying the factors regulating this process. For example, histone deacetylase inhibitors should increase histone acetylation and therefore photoreceptor gene transcription in retinoblastoma cells, and Crx or ataxin-7 mutations should decrease histone acetylation and transcription of photoreceptor genes in the retina of Crx-/- or SCA7 mice. We will also determine if Crx recruits the co-activator complexes involved in histone acetylation in vivo. In each of the three aims, we will use mouse models of photoreceptor diseases to investigate how disease-causing mutations in Crx and its associated factors, alter the normal functions of these regulatory proteins in vivo. These studies will lead to a new level of understanding of the molecular mechanisms that regulate photoreceptor-specific gene expression in vivo and will provide new approaches for designing treatments for photoreceptor diseases. ? ?
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