Functional dissection of MEL-28, a chromatin-binding protein with essential roles in nuclear envelope function and chromosome segregation

Resumen del póster presentado al 19th International C. elegans Meeting, celebrado en Los Angeles, California (US) del 26 al 30 de junio de 2013.-- et al.MEL-28/ELYS is a large AT-Hook protein required for the proper structure and function of the nuclear envelope during interphase in nematodes and vertebrates. In addition, MEL-28 has critical roles in chromosome congression and segregation during mitosis. MEL-28 localizes to nuclear pores and chromatin during interphase and shuttles to the kinetochore during cell division. Other than the AT-Hook domains, which suggest that MEL-28 binds DNA directly, primary structure analysis has not revealed functional domains. Biochemical studies have demonstrated that MEL-28 interacts with the NUP107 subcomplex at nuclear pores, but its targeting mechanism to kinetochores is unknown. We are interested in understanding 1) the function and mode of MEL-28 chromatin binding and 2) which regions of the MEL-28 protein are required for its different functions. To this end we have used DamID to define the chromatin regions with which MEL-28 associates. Interestingly, MEL-28 is enriched in active chromatin, suggesting that it may be involved in regulation of gene expression. In addition we are using a structure/function approach to determine which domains of the MEL-28 protein are required for MEL-28 localization and function. We have generated truncated versions of MEL-28 that lack different domains and fused these to GFP to track their localization in live embryos. Biological function of these fusions is evaluated by crossing them into mel-28 mutants and scoring for rescue of embryonic lethality. In doing this we have defined separate domains that are implicated in localization of MEL-28 to the nuclear envelope, chromatin and kinetochores. Surprisingly, we have found that removal of the AT Hooks does not affect MEL-28 localization although it does disrupt MEL-28 function. These studies illuminate the functioning of an essential and conserved protein.Peer Reviewe