In the course of our work, we discovered that CTCF is not solely a marker for terminal differentiation since it is nucleolar in many non-differentiated cell types. We found that CTCF binds to at least one specific site within the transposable elements of the repeated rDNA cistron, which contributes to a model for regional regulation of rDNA expression. We used RNAi-mediated reduction of gene activity, mutation of the gene in whole animals, and disruption of the poly- ADP-ribosylation pathway that modifies CTCF to directly determine if endogenous CTCF is necessary for normal rDNA silencing, and showed that all treatments resulted in both increased rDNA expression, expression of rDNA-associated transposable elements and transgenic marker gene expression, and increased nucleolar instability. To determine if CTCF plays a role in rDNA regulation in Drosophila, we first had to ascertain whether it could be detected in nucleoli of numerous different cell types, and moreover to determine if it is TH-302 generally used to regulate rDNA. We observed strong immunofluorescence signal for CTCF in the nucleoli of differentiated salivary glands of third instar Drosophila larvae, showing that it is cytologically associated with the rDNA, and supporting our belief that the biology of Drosophila CTCF may be similar to that of mammals. Even in occasional nuclei with multiple nucleoli, CTCF was found to overlap with all focal localization of fibrillarin, a marker for the fibrillary component of the nucleolus. Nucleolar localization of CTCF was in addition to a focal nucleoplasmic staining. At higher magnification, we observed that CTCF did not conform to any obvious landmarks of DNA within the nucleolus, although it was largely excluded from the visible DNA threads and foci. Unlike mammalian Fingolimod tissue culture and nervous tissue, nucleolar CTCF did not require terminal differentiation and cessation of division in Drosophila since we observed CTCF in the nucleoli of undifferentiated cycling interphase S2 tissue culture and larval neuroblast cells. The amount of nucleolar CTCF differed in those cell types, in the former it was neither enriched nor excluded but appeared similar to levels in the non-nucleolar chromatin, while in the latter it was moderately enriched over the amount found in the chromatin. Many non-nucleolar nuclear proteins are seen to be excluded from the nucleolus, and so the lack of CTCF exclusion is indicative of some localization even if it is not enriched in this compartment; this is especially true given the thousands of euchromatic binding sites to which it is being compared.