Pax6 is a highly conserved developmental regulator with a complex temporal, spatial and quantitative expression pattern, that is crucial for correct development of the central nervous system, the eye, and pancreas. Accordingly, the Pax6 gene resides in a complex genomic locus containing a large array of long-range tissue-specific cis-regulatory elements primarily identified through multispecies sequence conservation and reporter studies. I have set out to understand how the chromatin architecture of the locus contributes to the mechanism and specificity of cis-regulatory interactions. As well as addressing whether the DNA looping model for regulatory interactions applies to the mouse Pax6 locus, I will identify which elements facilitate such interactions and if they vary between cell types. Utilising ChIP-array technology the distribution and variability of key regulatory histone modifications and factors were assessed in a set of Pax6 expressing and non-expressing mouse cell lines, acting as models for different regulatory states of the locus. Work in other loci suggests a key role for CTCF and cohesin (subunit Rad21) in chromatin organisation and long distance regulatory interactions. ChIP-chip for CTCF/Rad21 across the Pax6 locus identified numerous sites within the gene and at distal regulatory locations. The majority of these sites are cell type invariable. The active enhancer modification H3K27ac identified both known and several novel putative enhancer elements distributed through the locus that are highly cell type specific. A subset of CTCF/Rad21 sites also acquire the active enhancer modification H3K27ac in a cell type dependent manor, suggesting that CTCF/Rad21 may facilitate looping to the target gene from these sites. Using reporter based assays, putative regulatory elements marked by the looping factors and active histone modifications showed a diverse range of functional activities. Unexpectedly only 3 of the 7 CTCF sites tested showed classical insulator activity in an enhancer blocking reporter assay. Surprisingly the strongest insulator tested resided within intron 7 the Pax6 gene. Other CTCF/Rad21 sites were neutral or enhancers in the insulator assay. This reveals the disparity between predicting regulatory properties using ChIP binding profiles alone and the actual outcome of functional reporter experiments. A novel element, CTCF6 showed a ChIP signature of CTCF/Rad21/H3K27ac in all Pax6 expressing tissues, and functioned as a strong enhancer in transient transfection and stable LacZ reporter assays. CTCF6 recapitulated a broad range of Pax6 expression patterns, at multiple embryonic stages, including the brain, neural tube and pancreas. A second novel element, E-120 identified in the pancreatic derived cell line, drove stable embryonic reporter expression in the embryonic pancreas and sub set of brain regions. Together this has expanded the repertoire and size of Pax6’s regulatory landscape particular in the upstream region. Chromatin conformation capture (3C) was used to characterise the dynamic chromatin architecture of the locus and identify the interaction profiles from three CTCF/Rad21 binding regulatory locations within the Pax6 locus. This revealed a core set of regulatory interactions with the Pax6 gene, while individual elements showed a more variable set of cell type specific interactions. The CTCF6 enhancer showed highly cell type specific promoter interactions throughout the Pax6 gene, indicative of enhancer-promoter looping not detected in the non-expressing cells. While the downstream site CTCF5 at the edge of a cluster of regulatory elements known as the DRR (differentially regulated region), interacted with both the gene and an upstream element CTCF7 300 kb away only in the Pax6 expressing locus. Together these results reveal Pax6 has a chromatin hub structure with regulatory loops from upstream and downstream bringing distant yet variable active elements in to the vicinity of the Pax6 promoters where they can act. This work has revealed new roles for CTCF/cohesin sites in transcriptional regulation of Pax6 and how the cis-regulatory activity and structure of the locus varies across different cell types.