Insulators: A “Safety Guard” for Genome Stability in <i>Drosophila melanogaster</i>

Chromatin insulators are DNA-protein complexes that assist in the formation of chromatin loop structures by mediating long-range contacts between distant sites, which regulate proper organization of the chromatin fiber within the tridimensional space of the nucleus. It is considered that this function of insulators is required for the regulation of gene expression during development and in differentiated cells. This thesis focuses specifically in the Suppressor of Hairy wing [Su(Hw)] insulator of Drosophila and its associated proteins, and explores the possibility that chromatin insulators are not only significant for regulation of gene expression, but are also essential for DNA replication and for the maintenance of genome stability.

Su(Hw) is one of the best characterized insulators in Drosophila and requires the insulator proteins Su(Hw), Modifier of mdg4 [Mod(mdg4)] and Centrosomal protein 190 (CP190) to accomplish its function.

Traditionally, there are two well-known properties that are shared by chromatin insulators: they have the ability to block the communication between enhancers and promoters when located between them and they can function as heterochromatin barriers. However, previous studies have revealed that not all insulator sites in the genome share the enhancer-promoter blocking property, leaving open the possibility that, in addition to the establishment of chromatin loops and gene transcription regulation, insulators might play yet unexplored roles in the genome.

Chapter one introduces a new role for the phosphorylation of H2Av (γH2Av), traditionally a marker for DNA damage, in insulator function. This work shows that γH2Av is required for the Su(Hw) enhancer-blocking function, and suggests that the ATM and ATR kinases modulate insulator function through phosphorylation of H2Av at insulator sites. Chapter two explores the possibility that Su(Hw) and HIPP1 (HP1-Insulator Protein Partner 1) may play a role in the regulation of DNA replication in the genome. Findings in this work suggest that insulators regulate activation of origins of replication by cooperation with other proteins such as HP1 and H4K20me1. Altogether these findings provide new insights into insulator function and suggest that Su(Hw) and likely other insulators are critical for DNA replication and for the maintenance of genome stability.