Part 1: Role of the histone kinase MSK1 on MLL gene regulation & Part 2: the molecular basis of inhibitory siganlling during neuronal regeneration.

[摘要] Part 1: The mixed-lineage leukaemia (MLL) protein is a histone methyl-transferase, that deposits the trimethylation mark on Lysine 4 of Histone 3 (H3K4me3) and that is often mutated in certain forms of leukaemia. MLL is normally associated with a cohort of other proteins and cofactors, but the mechanisms regulating MLL activity remain unclear. The H3K4me3 mark, as deposited by MLL‘s SET domain, which is found in many proteins and mediate lysinedirected histone methylation, is asociated with actively transcribed genes. Here we examine the role of Msk1, a downstream kinase of the MAP-kinase pathway, in regulating MLL1 activity. Msk1 is known to deposit phosphorylation marks on H3 and these were found on MLL1 target genes and the same target site like MLL1. It could be demonstrated that in Msk1 knock-down cells the MLL1 target genes are down-regulated. Furthermore during the cell cycle MLL1 and Msk1 show the same varying distribution. These findings suggest that MLL1 is regulated by extracellular signals via the MAP-kinase pathway and Msk1. Part 2: Limited neuronal regeneration following CNS injury is due to interactions between the myelin derived inhibitors and the Nogo-receptor (NgR) ternary complex, consisting of NgR, p75 and Lingo1. The inhibitory role of Lingo-1 presents a novel target for nerve repair and remyelination therapies. Lingo1 is one member of a large group of CNS enriched membrane proteins, with a LRR and an Ig domain in their ectodomain. Recently, other members of the LRR-Ig protein family, namely Amigo-proteins, have been reported in the context of neuronal development, survival and regeneration. This study has combined biochemical, binding and structural approaches to further characterise the function of Amigo proteins, with a particular emphasis on whether Amigo proteins can substitute for Lingo1 in forming a ternary NgR complex. The ectodomains of Amigo1 and Amigo3 were expressed and successfully purified by Ni-NTA affinity and gel filtration chromatography. Based on BIAcore binding analyses, whereas Amigo 1 and Amigo 3 demonstrated no binding with p75, weak interactions were found with NgR. In addition, initial cross-linking experiments with BS\(^3\) indicated, that Amigo1, but not Amigo 3, can self-associate forming dimers in solution. Finally, crystallisation trials for Amigo1 ectodomain identified a condition that yielded suitable sized crystals for preliminary X-ray diffraction experiments.

[发布机构] University:University of Birmingham;Department:Medical School

[关键词] Q Science;QH Natural history;QH426 Genetics