Modeling Human Laminopathies In C.Elegans And Molecular Mechanism Study

In higher eukaryotic cells,beneath the inner nuclear membrane(INM)lies a filamentous protein meshwork known as the nuclear lamina(NL),which is assembled by lamin proteins.Lamin is not only the sole intermediate filament(IF)member located in nuclei,but the ancestral type of the IF superfamily.Lamin proteins are widely present in metazoans and highly conserved during evolution.There are three lamin genes in human genome,LMNA,LMNB1 and LMNB2.Through alternative splicing,LMNA generates two major A-type products,LMNA and LMNC.Its expression is developmentally regulated,whereas the other two B-type genes essential for cell viability are constitutively expressed.Except LMNC,these products are subjected to complex post-translational modifications at the C-termini.Phosphorylation is important in regulating the activities of lamin proteins,which realizes the reversible assembly of the NL during cell cycle.Lamins can interact,directly or indirectly,with various INM or nucleoplasmic proteins.Together with these partners,besides the structural role,lamins are essential in multiple cellular functions,such as DNA replication,DNA repair,chromatin organization,gene regulation,and signal transduction.Mutations or aberrant expression of human lamin genes,particularly the LMNA gene,can result in a diverse set of genetic disorders,collectively referred to as laminopathies.Examples comprise autosomal Emery-Dreifuss muscular dystrophy(EDMD)and limb girdle muscular dystrophy type 1B(LGMD1B)both characterized by progressive muscle wasting,as well as Hutchinson-Gilford progeria syndrome(HGPS)that can lead a severe form of premature ageing to children.Despite that LMNA/C is expressed in most adult tissues,laminopathies actually exhibit a relatively tissue-specific pattern.According to the tissues affected,laminopathies can be classified into 5 categories,including striated muscle disease,lipodystrophy,neuropathy,progeria,and overlapping type.Loss of the histone chaperone RBBP4/7 is a notable event in both HGPS and normal ageing cells.Abnormal expression or localization of RBBP4/7 has also been observed in primary myoblasts isolated from two patients diagnosed as EDMD and LGMD1B,respectively.These suggest that RBBP4/7 might be a common pathogenic factor in many laminopathies.In model organism Caenorhabditis elegans,there are two homologs of RBBP4/7,LIN-53 and RBA-1.Interestingly,depletion of lin-53 by RNAi or mutation leads to muscle dysfunction and lifespan shortening,which mimic laminopathic phenotypes.Thus,we were wondering whether C.elegans could be used as a disease model for human laminopathies to dissect the link between lin-53/rba-1 and laminopathies.We also aimed to take advantage of C.elegans to gain more insights into the molecular mechanisms underlying laminopathies,as well as to provide more direction and possibility for the treatment.Notably,C.elegans body wall muscle is analogous to vertebrate skeletal muscle,rendering the feasibility for modeling human striated muscle diseases in C.elegans.Therefore,we introduced EDMD-and LGMD1B-causing mutations into the single lamin gene lmn-1 of C.elegans and generated via micro injection muscle-specific extrachromosomal lines,which were subsequently integrated after y irradiation.The severe muscle phenotypes observed in patients were recapitulated in our lamin mutants as revealed by swimming assay,antibody staining and the Pmyo-3::GFP reporter,demonstrating that we obtained worm disease models for EDMD and LGMD1B.Besides,lifespan assay indicated that the viability of the mutants was less affected.We also employed MosSCI to achieve single copy insertion of the transgene in a well-defined genomic locus.The MosSCI mutants exhibited mild muscle phenotypes after crossed into the lmn-1-/-background.Moreover,we also generated transgenic lines carrying mutations driven by a ubiquitous promoter,in which misshapen intestinal nuclei were observed.According to the expression patterns of LIN-53 and RBA-1 revealed by immunostaining,the former is ubiquitously present,whereas the latter is restricted to germline in adult N2 hermaphrodites,implying that LIN-53 is solely functioning in adult muscle cells.Interestingly,exogenous expression of the functional GFP::LIN-53::2xFLAG fusion protein was beneficial for muscle homeostasis.Actually,in EDMD and LGMD1B mutants as well as lmn-1-/-homozygotes showing premature ageing,loss of LIN-53 was observed,suggesting that LIN-53 might also be a common pathogenic factor in many laminopathies,which seems to be an evolutionarily conserved mechanism.Furthermore,we carried out RNAi screen based on MH chromatin sub-library composed of～700 worm genes and identified three factors that were able to ameliorate the severe muscle phenotype of our lamin mutants upon depletion,as validated by swimming assay.To sum up,we established C.elegans disease models for EDMD and LGMD1B,discovered the histone chaperone LIN-53 to be a possible common pathogenic factor,and identified another three genes implicated in laminopathies.These provide basis for further study to dissect the molecular mechanisms of laminopathies and develop therapies in the future.