Project Summary The formation of discrete chromatin-chromatin interactions in the genome during the differentiation of mammalian cells leads to the specific insulation of repressed chromatin domains from those actively engaged in transcription. This partitioning thwarts the inappropriate invasion of actively transcribing regions into those that must stay repressed⏤this process is highly regulated and diversified during development, giving rise to the specific gene expression profiles reflective of and inherent to markedly distinct cell types. The key proteins recognized as fostering chromatin-chromatin interacting structures are cohesin, CTCF, and the newly recognized MAZ. Cohesin is believed to extrude a loop of chromatin such that the endpoints of this loop merge with two chromatin-bound CTCF proteins, thereby anchoring cohesin. CTCF and MAZ interact independently with cohesin and exert their function through binding to specific pairs of CTCF and MAZ DNA elements, respectively. Notably, CTCF also interacts with RNA which leads to its multimerization and CTCF mutant in any of its RNA binding domains [Zinc Finger 1 (ZF1), ZF10, and RBRi] gives rise to disrupted chromatin loop formation. Of particular interest in this study is the CTCF mutant in its ZF1 as it retains all other CTCF functions. The approach here exploits the mammalian HoxA-D clusters as an ideal model for investigating the process of insulation as a function of differentiation as they exhibit linear and temporal regulation during development. This project proposes to expand upon previous successful investigations of Hox gene regulation, which led to the discovery of MAZ as a DNA-site-specific insulation factor, our recent findings of another possible “site-specific insulator”, PATZ1, the key role of CTCF/RNA interaction in fostering the integrity of chromatin boundaries, as well as the construction of synthetic HoxA clusters that when placed ectopically exhibit all the regulatory features inherent to the endogenous version. With this foundation, investigations of the features requisite to chromatin boundary formation as a function of differentiation will include: genetic manipulations in vivo in conjunction with molecular biological approaches to define the roles of MAZ and PATZ1 in insulating a portion of the HoxA cluster, the identification of other potential insulating proteins that function at the HoxA cluster posterior to that of MAZ and PATZ1, and an investigation as to whether RNAs interacting with CTCF are determinant to particular chromatin boundary formation. The results from these multi-pronged in vivo and in vitro approaches will reveal the regulatory and mechanistic basis by which chromatin boundaries are organized, thereby dictating appropriate gene expression.