Understanding CTCF boundaries controlling Hox gene expression Summary Spatial and temporal control of gene expression is crucial for the development of multicellular organisms. Although changes in looping interactions between enhancers and transcription start sites is an acknowledged mode of gene regulation, the contribution of larger 3D genomic reorganizations to gene expression and normal development is largely obscure. We propose experiments to clarify how the CTCF transcription factor controls chromatin structure at the Hox clusters to ensure proper Hox gene expression and thus, body patterning. During embryonic development, precise expression of Hox genes instructs cells to recognize their relative position in body axes. Hox genes are organized in four clusters with individual genes in these clusters expressed in patterns that are spatially and temporally collinear with their physical chromosomal organization. Collinear Hox gene expression along the spinal cord controls motor neuron (MN) subtypes and thus their connectivity. During MN differentiation, the Hox clusters undergo a chromatin and 3-D reorganization from a single repressed state to two domains harboring either transcribed or repressed genes. The two chromatin states are insulated by CTCF binding at the boundary, maintaining stable Hox chromatin states inherited though development to ensure proper MN connectivity. Of relevance, we recently demonstrated that the CTCF boundary is essential to normal body patterning during embryonic development in vivo. To understand how CTCF maintains insulated chromatin and 3-D boundaries at Hox clusters we propose: 1) To understand how disrupting the CTCF-mediated chromatin boundary affects subtype identity of spinal MNs; 2) To determine the molecular basis of establishing a CTCF-dependent boundary; 3) An advanced proteomics study to identify factors required by chromatin associated CTCF for its insulator activity, emphasizing those whose interaction is RNA-dependent.