Functional Analysis of Vertebrate Msl2
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hMSL2 (Male Specific Lethal 2, human) is a RING finger protein with E3 ubiquitin ligase activity. Although it has been shown to target histone H2B at lysine 34 and p53 at lysine 351, suggesting roles in transcription regulation and apoptosis, its molecular contribution to these and other processes remains poorly defined. To better understand the function of this protein in vertebrates we decided to take a knockout approach using the DT40 model system. We first confirmed the expression of Msl2 in chicken DT40 cells, designed a strategy to target this gene and then confirmed its successful disruption. Msl2-/- cells are viable, with minor growth retardation and mitotic defects. Biochemical analysis of the chromatin in these cells revealed aberrations in the levels of several histone modifications involved in DNA damage response pathways. To ensure that any phenotypes observed in the Msl2-/- cells were due to loss of Msl2 and more specifically, due to its E3 ligase activity, Msl2-rescue, Msl2-[delta]R and Msl2-C44R cell lines were made to re-express full length wild-type Msl2, 1-107 amino acids truncated Msl2 or Msl2 with a point mutation C44R respectively. We found that the above defects could be rescued in the Msl2-rescue cell line. Interestingly, when treated with either of the damaging agents ionizing radiation (IR) or methyl-methanesulfonate (MMS), the Msl2-rescue cell line showed increased Msl2 protein stability. In summary, these data identify a novel role for Msl2 in the cellular response to DNA damage. Moreover, Msl2 plays a role in maintaining normal histone modification profiles, which may also contribute to the DNA damage response.