A Drosophila Insulator Interacting Protein Suppresses Enhancer-Blocking Function and Modulates Replication Timing

Insulators play important roles in genome structure and function in Drosophila and mammals. More than six different insulator proteins are required in Drosophila for normal genome function, whereas CTCF is the only identified protein contributing to insulator function in mammals. Interactions between a DNA binding insulator protein and its interacting partner proteins define the properties of each insulator site. The different roles of insulator protein partners in the Drosophila genome and how they confer functional specificity remain poorly understood. Functional analysis of insulator partner proteins in Drosophila is necessary to understand how genomes are compartmentalized and the roles that different insulators play in genome function. In Drosophila, the Suppressor of Hairy wing [Su(Hw)] insulator is targeted to the nuclear lamina, preferentially localizes at euchromatin/heterochromatin boundaries, and is associated with the Gypsy retrotransposon. The properties that the insulator confers to these sites rely on the ability of the Su(Hw) protein to bind the DNA at specific sites and interact with Mod(mdg4)-67.2 and CP190 partner proteins. HP1 and insulator partner protein 1 (HIPP1) is a recently identified partner of Su(Hw), but how HIPP1 contributes to the function of Su(Hw) insulators has not yet been elucidated. Here, we find that mutations in the HIPP1 crotonase-like domain have no impact on the function of Su(Hw) enhancer-blocking activity but do exhibit an impaired ability to repair double-strand breaks. Additionally, we find that the overexpression of each HIPP1 and Su(Hw) causes defects in cell proliferation by limiting the progression of DNA replication. We also find that HIPP1 overexpression suppresses the Su(Hw) insulator enhancer-blocking function.

Mutations in the Insulator Protein Suppressor of Hairy Wing Induce Genome Instability

Chromatin insulator proteins mediate the formation of contacts between distant insulator sites along chromatin fibers. Long-range contacts facilitate communication between regulatory sequences and gene promoters throughout the genome, allowing accurate gene transcription regulation during embryo development and cell differentiation. Lack of insulator function has detrimental effects often resulting in lethality. The Drosophila insulator protein Suppressor of Hairy wing [Su(Hw)] is not essential for viability, but plays a crucial role in female oogenesis. The mechanism(s) by which Su(Hw) promotes proper oogenesis remains unclear. To gain insight into the functional properties of chromatin insulators, we further characterize the oogenesis phenotypes of su(Hw) mutant females. We find that mutant egg chambers frequently display an irregular number of nurse cells, have poorly formed microtubule organization centers (MTOC) in the germarium, and show mislocalized Gurken (Grk) in later stages of oogenesis. Furthermore, eggshells produced by partially rescued su(Hw) mutant females exhibit dorsoventral patterning defects that are identical to defects found in spindle mutants or in piRNA pathway mutants. Further analysis reveals an excess of DNA damage in egg chambers, which is independent of activation of transposable elements, and that Gurken localization defects and oogenesis progression are partially rescued by mutations in mei-41 and chk1 genes. In addition, we show that Su(Hw) is required for chromosome integrity in dividing neuroblasts from larval brains. Together, these findings suggest that Su(Hw) plays a critical role in maintaining genome integrity during germline development in Drosophila females as well as in dividing somatic cells.

Zinc Finger Protein CG9890 - New Component of ENY2-Containing Complexes of Drosophila

In previous studies, we showed that the insulator protein Su(Hw) containing zinc finger domains interacts with the ENY2 protein and recruits the ENY2-containing complexes on Su(Hw)-dependent insulators, participating in the regulation of transcription and in the positioning of replication origins. Here, we found interaction between ENY2 and CG9890 protein, which also contains zinc finger domains. The interaction between ENY2 and CG9890 was confirmed. It was established that CG9890 protein is localized in the nucleus and interacts with the SAGA, ORC, dSWI/SNF, TFIID, and THO protein complexes.

Depletion of the Insulator Protein CTCF Results in Herpes Simplex Virus 1 ReactivationIn Vivo

ABSTRACTHerpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in host peripheral neurons, including the neurons of the trigeminal ganglia (TG). HSV-1 can reactivate from neurons to cause recurrent infection. During latency, the insulator protein CTCF occupies DNA binding sites on the HSV-1 genome, and these sites have been previously characterized as functional enhancer-blocking insulators. Previously, CTCF was found to be dissociated from wild-type virus postreactivation but not in mutants that do not reactivate, indicating that CTCF eviction may also be an important component of reactivation. To further elucidate the role of CTCF in reactivation of HSV-1, we used recombinant adeno-associated virus (rAAV) vectors to deliver a small interfering RNA targeting CTCF to peripheral neurons latent with HSV-1 in rabbit TG. Our data show that CTCF depletion resulted in long-term and persistent shedding of infectious virus in the cornea and increased ICP0 expression in the ganglia, indicating that CTCF depletion facilitates HSV-1 reactivation.IMPORTANCEIncreasing evidence has shown that the insulator protein CTCF regulates gene expression of DNA viruses, including the gammaherpesviruses. While CTCF occupation and insulator function control gene expression in DNA viruses, CTCF eviction has been correlated to increased lytic gene expression and the dissolution of transcriptional domains. Our previous data have shown that in the alphaherpesvirus HSV-1, CTCF was found to be dissociated from the HSV-1 genome postreactivation, further indicating a global role for CTCF eviction in the transition from latency to reactivation in HSV-1 genomes. Using an rAAV8, we targeted HSV-1-infected peripheral neurons for CTCF depletion to show that CTCF depletion precedes the shedding of infectious virus and increased lytic gene expressionin vivo, providing the first evidence that CTCF depletion facilitates HSV-1 reactivation.