Chromatin Immunoprecipitation (ChIP) Protocols for the Cancer and Developmental Biology Laboratory.

Chromatin immunoprecipitation assays permit the isolation and subsequent identification of genomic DNA (gDNA) fragments bound directly or indirectly to proteins of interest, including transcription factors, co-factors, or chromatin remodeling proteins. These isolated DNA fragments may include gene regulatory regions from enhancers, super-enhancers, promoters, and/or insulators. Cells of interest can be obtained from embryonic tissues at various developmental time points or cancer cells from patients or derived from model systems, including patient-derived xenotransplants and primary cancer stem cells and cell lines. ChIP variants include ChIP-reChIP to identify targets bound to different transcription factors or members of protein complexes or, alternatively, to characterize the histone modifications accompanying occupation of specific regulatory regions by the protein of interest, such as a transcription factor. Subsequent analysis of ChIP experiments includes standard PCR, quantitative PCR (qPCR), and ChIPseq.

The role of homeobox genes in retinal development and disease.

Homeobox genes are an evolutionarily conserved class of transcription factors that are critical for development of many organ systems, including the brain and eye. During retinogenesis, homeodomain-containing transcription factors, which are encoded by homeobox genes, play essential roles in the regionalization and patterning of the optic neuroepithelium, specification of retinal progenitors and differentiation of all seven of the retinal cell classes that derive from a common progenitor. Homeodomain transcription factors control retinal cell fate by regulating the expression of target genes required for retinal progenitor cell fate decisions and for terminal differentiation of specific retinal cell types. The essential role of homeobox genes during retinal development is demonstrated by the number of human eye diseases, including colobomas and anophthalmia, which are attributed to homeobox gene mutations. In the following review, we highlight the role of homeodomain transcription factors during retinogenesis and regulation of their gene targets. Understanding the complexities of vertebrate retina development will enhance our ability to drive differentiation of specific retinal cell types towards novel cell-based replacement therapies for retinal degenerative diseases.