Drosophila HP1 and Insulator Partner Protein 1 (HIPP1) is the homologue of the human co-repressor Chromodomain Y family of proteins that repress neuronal gene expression in mammals. HIPP1 was identified by its extensive co-localization with Heterochromatin Protein 1a (HP1a) in heterochromatic regions of the genome and insulator binding proteins in euchromatic regions. The majority of HIPP1 binding to euchromatin is at binding sites for Drosophila Suppressor of Hairy-wing [Su(Hw)]. Su(Hw) is a zinc finger DNA binding protein that functions as an insulator, activator, and repressor. Transcriptional regulation by Su(Hw) is essential in the ovary and testis, where Su(Hw) functions primarily as a repressor of neuronal genes. However, the mechanism of Su(Hw) dependent repression is not clear. The focus of my thesis work has been defining the role of HIPP1 in development and its contribution to Su(Hw) function and heterochromatin formation. As part of this work, CRISPR was used to generate multiple Hipp1 null alleles and a tagged derivative of the endogenous gene (Hipp1GFP). Hipp1 null flies were found to be viable. Study of HIPP1 expression revealed it is present in most tissues and restricted to the nucleus. HIPP1 showed limited colocalization with HP1a, and tests of repression of transgenes in heterochromatin suggested that HIPP1 is not required for heterochromatin formation. Investigations of HIPP1 binding revealed that Su(Hw) is responsible for the majority of HIPP1 recruitment to euchromatin. Despite this, HIPP1 was found to be dispensable for the transcriptional and insulator functions of Su(Hw) as well as for female and male fertility. These data indicate that HIPP1 is not a critical Su(Hw) cofactor. Further studies are needed to clarify the role of HIPP1 in Drosophila development.

Chromatin insulators play a role in gene transcription regulation by defining chromatinboundaries. Genome-wide studies in Drosophila have shown that a large proportion of insulator sites are found in intergenic DNA sequences, supporting a role for these elements as boundaries. However, approximately 40% of insulator sites are also found in intragenic sequences, where they can potentially perform as yet unidentified functions. Here we show that multiple Su(Hw) insulator sites map within the 110 kb sequence of the muscleblind gene (mbl), which also forms a highly condensed chromatin structure in polytene chromosomes. Chromosome Conformation Capture assays indicate that Su(Hw) insulators mediate the organization of higher-order chromatin structures at the mbl locus, resulting in a barrier for the progression of RNA polymeraseII (PolII ), and producing a repressive effect on basal and active transcription. The interference of intragenic insulators in PolII progression suggests a role for insulators in the elongation process. Supporting this interpretation, we found that mutations in su(Hw) and mod(mdg4) also result in changes in the relative abundance of the mblD isoform, by promoting early transcription termination. These results provide experimental evidence for a new role ofintragenic Su(Hw) insulators in higher-order chromatin organization, repression of transcription, and RNA processing.