Mutations in the A-type lamin (LMNA) gene are associated with age-associated degenerative disorders of mesenchymal tissues, such as dilated cardiomyopathy, Emery-Dreifuss muscular dystrophy, and limb-girdle muscular dystrophy. The molecular mechanisms that connect mutations in LMNA with different human diseases are poorly understood. Here, we report the identification of a Muscle-enriched A-type Lamin-interacting Protein, MLIP (C6orf142 and 2310046A06rik), a unique single copy gene that is an innovation of amniotes (reptiles, birds, and mammals). MLIP encodes alternatively spliced variants (23–57 kDa) and possesses several novel structural motifs not found in other proteins. MLIP is expressed ubiquitously and most abundantly in heart, skeletal, and smooth muscle. MLIP interacts directly and co-localizes with lamin A and C in the nuclear envelope. MLIP also co-localizes with promyelocytic leukemia (PML) bodies within the nucleus. PML, like MLIP, is only found in amniotes, suggesting that a functional link between the nuclear envelope and PML bodies may exist through MLIP. Down-regulation of lamin A/C expression by shRNA results in the up-regulation and mislocalization of MLIP. Given that MLIP is expressed most highly in striated and smooth muscle, it is likely to contribute to the mesenchymal phenotypes of laminopathies.

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Muscle-enriched A-type lamin-interacting protein (Mlip) is a recently discovered Amniota gene that encodes proteins of unknown biological function. Here we report Mlip’s direct interaction with chromatin, and it may function as a transcriptional co-factor. Chromatin immunoprecipitations with microarray analysis demonstrated a propensity for Mlip to associate with genomic regions in close proximity to genes that control tissue-specific differentiation. Gel mobility shift assays confirmed that Mlip protein complexes with genomic DNA. Blocking Mlip expression in C2C12 myoblasts down-regulates myogenic regulatory factors (MyoD and MyoG) and subsequently significantly inhibits myogenic differentiation and the formation of myotubes. Collectively our data demonstrate that Mlip is required for C2C12 myoblast differentiation into myotubes. Mlip may exert this role as a transcriptional regulator of a myogenic program that is unique to amniotes.

Specific missense mutations LMNA have been identified to be associated with muscular dystrophy suggesting that LMNA interacts with a muscle specific factor(s). A yeast two‐hybrid screen with LMNA as bait was employed to identify muscle specific proteins. A previously uncharacterized cDNA clone was identified that interacts with LMNA in muscle, MLIP. Preliminary results show MLIP protein is primarily expressed in brain, heart and skeletal muscle. No structural or functional domains have been identified within MLIP.Objective: To define the function of MLIP in muscle.Results: MLIP is expressed endogenously in a mouse myoblast cell line (C2C12) and is co‐localized to the nuclear envelope and PML bodies. Initiation of C2C12 differentiation led to a 3‐fold increase in MLIP expression peaking at 24hrs and MLIP continued to be expressed in differentiated myotubes. Specific knockdown of MLIP in C2C12 cells by shRNAi led to significant (p<0.01) reduction in Pax7 expression with a concurrent reduction in Myf5, MyoD and myogenin with Pax3 expression being unaffected. To determine whether MLIP is necessary and/or sufficient for myogenic differentiation, stable C2C12 cell lines are being generated that either knockdown MLIP or over‐express MLIP.Conclusion: MLIP is a novel LMNA interacting protein that may regulate Pax7 a myogenic determinant during regenerative myogenesis. Funding: CIHR operating grant to PGB.

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