Dr. Amira Fitieh

BMI-1 is an essential regulator of transcriptional silencing during development. Recently, the role of BMI-1 in the DNA damage response has gained much attention, but the exact mechanism of how BMI-1 participates in the process is unclear. Here, we establish a role for BMI-1 in the repair of DNA double-strand breaks by homologous recombination (HR), where it promotes DNA end resection. Mechanistically, BMI-1 mediates DNA end resection by facilitating the recruitment of CtIP, thus allowing RPA and RAD51 accumulation at DNA damage sites. Interestingly, treatment with transcription inhibitors rescues the DNA end resection defects of BMI-1-depleted cells, suggesting BMI-1-dependent transcriptional silencing mediates DNA end resection. Moreover, we find that H2A ubiquitylation at K119 (H2AK119ub) promotes end resection. Taken together, our results identify BMI-1-mediated transcriptional silencing and promotion of H2AK119ub deposition as essential regulators of DNA end resection and thus the progression of HR.

The polycomb group (PcG) proteins are a class of transcriptional repressors that mediate gene silencing through histone post-translational modifications. They are involved in the maintenance of stem cell self-renewal and proliferation, processes that are often dysregulated in cancer. Apart from their canonical functions in epigenetic gene silencing, several studies have uncovered a function for PcG proteins in DNA damage signaling and repair. In particular, members of the poly-comb group complexes (PRC) 1 and 2 have been shown to recruit to sites of DNA damage and mediate DNA double-strand break repair. Here, we review current understanding of the PRCs and their roles in cancer development. We then focus on the PRC1 member BMI1, discussing the current state of knowledge of its role in DNA repair and genome integrity, and outline how it can be targeted pharmacologically.

Double-strand breaks and stalled replication forks are a significant threat to genomic stability that can lead to chromosomal rearrangements or cell death. The protein CtIP promotes DNA end resection, an early step in homologous recombination repair, and has been found to protect perturbed forks from excessive nucleolytic degradation. However, it remains unknown how CtIP's function in fork protection is regulated. Here, we show that CtIP recruitment to sites of DNA damage and replication stress is impaired upon global inhibition of SUMOylation. We demonstrate that CtIP is a target for modification by SUMO-2 and that this occurs constitutively during S phase. The modification is dependent on the activities of cyclin-dependent kinases and the PI-3-kinase-related kinase ATR on CtIP's carboxyl-terminal region, an interaction with the replication factor PCNA, and the E3 SUMO ligase PIAS4. We also identify residue K578 as a key residue that contributes to CtIP SUMOylation. Functionally, a CtIP mutant where K578 is substituted with a non-SUMOylatable arginine residue is defective in promoting DNA end resection, homologous recombination, and in protecting stalled replication forks from excessive nucleolytic degradation. Our results shed further light on the tightly coordinated regulation of CtIP by SUMOylation in the maintenance of genome stability.

In a previous attempt to generate a protective vaccine against Candida albicans, a β-mannan tetanus toxoid conjugate showed poor immunogenicity in mice. To improve the specific activation toward the fungal pathogen, we aimed to target Dectin-1, a pattern-recognition receptor expressed on monocytes, macrophages, and dendritic cells. Laminarin, a β-glucan ligand of Dectin-1, was incorporated into the original β-mannan tetanus toxoid conjugate providing a tricomponent conjugate vaccine. A macrophage cell line expressing Dectin-1 was employed to show binding and activation of Dectin-1 signal transduction pathway by the β-glucan-containing vaccine. Ligand binding to Dectin-1 resulted in the following: 1) activation of Src family kinases and Syk revealed by their recruitment and phosphorylation in the vicinity of bound conjugate and 2) translocation of NF-κB to the nucleus. Treatment of immature bone marrow-derived dendritic cells (BMDCs) with tricomponent or control vaccine confirmed that the β-glucan-containing vaccine exerted its enhanced activity by virtue of dendritic cell targeting and uptake. Immature primary cells stimulated by the tricomponent vaccine, but not the β-mannan tetanus toxoid vaccine, showed activation of BMDCs. Moreover, treated BMDCs secreted increased levels of several cytokines, including TGF-β and IL-6, which are known activators of Th17 cells. Immunization of mice with the novel type of vaccine resulted in improved immune response manifested by high titers of Ab recognizing C. albicans β-mannan Ag. Vaccine containing laminarin also affected distribution of IgG subclasses, showing that vaccine targeting to Dectin-1 receptor can benefit from augmentation and immunomodulation of the immune response.