Prof. Ju-Min Zhou, Principle Investigator. We are interested in the epigenetic mechanisms of higher order chromatin organization, long distance enhancer-promoter interactions, viral host interactions and DNA double strand break repair. Our work has been published in Cell, Molecular Cell, Genes Development, PNAS, Development, MCB, J Virology and Developmental Biology.
We are interested in the epigenetic basis cell fate determination, the role of chromatin in DNA damage response, viral latency and viral-host interactions. Recent research progress:
CTCF participates in DNA double strand break (DSB) repair. CTCF is an essential epigenetic regulator with multiple nuclear functions including chromatin insulation, long-range regulatory interactions, higher order chromosome conformation, imprinting, X-inactivation, V(D)J recombination, RNA pol II pausing, alternative splicing and enhancer function. Mutations in human and knock out in mice point to a defect in cell proliferation, phenotypes that could not be easily explained by the reported activities of CTCF. We studied the role of CTCF in genotoxic stress, and found that CTCF knock down increased the number of chemotherapy drug induced 53BP1 and gH2AX foci. Comet assay indicated that CTCF knock down increased DNA damage after UV irradiation. Immunofluorescent staining revealed colocalization of CTCF and 53BP1 in DSB-inducing drugs treated cells. Laser lesion of cell nucleus resulted in CTCF recruitment to the damaged areas marked by gH2AX or 53BP1. In these experiments, CTCF recruitment depends on the ATM pathway and the zinc finger region of CTCF. We next examined CTCF recruitment to the DSB ends in SceI-inducible DSB systems by ChIP, and found that, upon SceI induction, CTCF was recruited to DNA sequences located near DSB ends. Adding ATM inhibitor, Ku55933, CTCF binding was abolished, echoing the result from immunofluorescence study. To directly demonstrate the function of CTCF in DNA damage repair, we monitored the DSB repair efficiencies after CTCF knock down, and found that both HR and NHEJ were suppressed in knock down U2OS cells. Consistent with these results, CTCF knock down increased apoptosis and caused cell cycle arrest at G1 phase. These data together strongly argue for a role of CTCF in the DNA double strand break repair, and provide a direct link between higher chromatin organization to DNA damage response and maintenance of genome stability.
CTCF organizes HSV-1 replication center. The Herpes family of DNA viruses infects many cell types and establishes life long infection by latently infecting appropriate cell types. The neurotropic Herpes Simplex virus type I (HSV-1) enters productive infection in non-neuronal cells. In an attempt to determine the global chromatin structure change in HSV-1 infected cells, we found that 90% of host genomic CTCF binding signal disappeared in 6 hours after infection in BJ cells. Next we did immunofluorescent staining to determine the location of CTCF after HSV-1 infection, and found that CTCF is recruited to HSV-1 replication foci. To determine the role of CTCF in HSV-1 replication, we did CTCF knock down, and found that the knock down greatly reduced the number and intensity of HSV-1 replication centers. gH2A.X, a histone variant specific to DSB foci is found to surround HSV replication compartment. In CTCF KD cells, rH2AX is found to infiltrate HSV foci suggesting that CTCT is recruited to protect the viral genome from the silencing activity of gH2A.X. Consistent with this possibility, we found that viral transcription, viral genome copy number and viral yield are impaired by CTCF knock down. We next mapped the domains within CTCF that is necessary for the recruitment and found that the zinc finger region is necessary and sufficient for the recruitment, suggesting that DNA binding to the viral genome is directly responsible for CTCF recruitment to HSV-1 replication centers. ChIP-seq of CTCF revealed that CTCF interacts with over 20 distinct sites in the HSV-genome. Finally, we found that CTCF knock down reduced the binding of RNA pol II to the viral genome. These findings strongly support an important role of CTCF in HSV-1 transcription and viral replication center organization.
HSV-1 latency in tree shrews. The tree shrew (Tupaia belangeri chinensis) is a small mammal indigenous to southwest Asia. At behavioral, anatomical, genomic and evolutional levels, tree shrews are much closer to primates than rodents. In an attempt to develop the tree shrew into a model to study herpes virus infection, we were able to establish latent infections with HSV-1 17+ following ocular innoculation. In situ hybridization and qRT-PCR demonstrated that HSV-1 latently infects neurons of the trigeminal ganglion. Explant co-cultivation experiments, of trigeminal ganglia were performed. Virus was recovered after 5 days co-cultivation with high efficiency. Swabbing of the cornea of the infected tree shrews revealed that the tree shrews occasionally shed virus spontaneously. Interestingly the cornea surface, where viruses were inoculated display consistent keratitis similar to acute human viral keratitis. These observations suggest that the tree shrew is a viable model to study herpes virus latency and acute infection in the eye. It has the advantage of spontaneous shedding from the eye compared to the mouse and low cost compared to the rabbit.