Nuclear myotubularin and its role in muscle development and the pathogenesis of X-linked myotubular myopathy - X-linked myotubular myopathy (XLMTM) is a devastating childhood muscle disease characterized clinically by severe weakness and early death and pathologically by small myofibers that contain disorganized organelles and myonuclei with aberrant appearance and localization. XLMTM is caused by mutations in the MTM1 gene. How MTM1 mutations cause these phenotypes is not well known, and this lack of knowledge presents a key barrier for disease understanding and therapy development. Myotubularin (MTM1) encodes a 3-position phosphoinositide phosphatase that in vitro localizes to the endosome and has role(s) in regulating endosomal vesicular sorting. In exciting new data, we have discovered for the first time subpopulation(s) of MTM1 at the nuclear envelope and within the nucleus. We also reveal that Mtm1 knockout mice have structurally abnormal myonuclei, display an arrest in muscle development and abnormalities in the myogenic transcriptome, and have altered genome organization and a shift in chromatin accessibility. Further, we show that expression of MTM1 exclusively in the nucleus can rescue aspects of the knockout phenotype. Based on these data, and our previous work, we hypothesize that MTM1 regulates nuclear architecture and regional chromatin modifications, and thereby participates as a key modulator of myogenesis. We additionally hypothesize that a critical consequence of MTM1 mutation is to alter genome topography and interrupt the normal progression of the myogenic expression program, resulting in failure of muscle development and disruption of myofiber growth. We will test these novel, conceptually innovative hypotheses using our mouse model of XLMTM in three specific aims. Aim 1 will characterize the localization and interactome of nuclear MTM1, and determine how these change with Mtm1 knockout. Aim 2 will define the alterations in nuclear structure, genome organization, and myogenic transcription associated with MTM1 mutation. Aim 3 will test the impact of nuclear vs non-nuclear MTM1 during muscle development using a series of AAV driven rescue constructs. These Aims utilize state- of-the field experimental approaches, including 3D block face electron microscopy, in vivo miniTurboID, spatial transcriptomics and single nucleus RNAseq, and a novel HiC approach (scHiCAR) for defining genome architecture. Findings from this project will be highly significant as they will advance understanding of the role of MTM1 in normal nuclear function and in the regulation of an understudied area of muscle development (postnatal sequential progression of the myogenic transcriptional program). In addition, this study will improve our knowledge of XLMTM pathogenesis by addressing a key fundamental unknown (i.e. the contribution of the pathognomonic nuclear abnormalities to the disease process) and by providing new knowledge that will lay the groundwork for therapy development. To ensure success of this project and enable its highest impact, we have assembled a collaborative investigator team expert in XLMTM and the key experimental approaches.
