4 research outputs found
Investigation of the Role of LAP1B in Transcriptional Regulation of Muscle Cells
The loss-of-function of
the inner nuclear membrane protein LAP1B (lamina-associated polypeptide 1, isoform
B) causes muscular dystrophy and cardiomyopathy in humans. The function of LAP1B
in muscle is still unknown. The goal of this thesis is to contribute to the understanding
of the molecular pathogenesis underlying muscular dystrophy caused by LAP1B, by
determining transcriptional changes occurring throughout differentiation of muscle
cells lacking LAP1B. For this purpose, primary fibroblasts isolated from healthy and
one patient affected by LAP1B-related muscular dystrophy were immortalized and
myoconverted with inducible MyoD gene transfer. Whereas control cells formed
mature myotubes within eight days, mutant cells demonstrated very low fusion
potential and failed to fully differentiate. Mutations causing knockdown of LAP1A/B
expression were created in C2C12 mouse myoblasts and similar results were observed.
By RNA sequencing, genes differentially expressed in control and mutant cells within
the transcriptome and enriched pathways were identified. Muscle contraction, cell
cycle, mitotic chromatid segregation and extracellular matrix organization were among
the most significantly enriched pathways. It was shown by cell cycle assay that despite
downregulation of p21 expression, mutant cells withdrew from the cell cycle. Finally,
upregulation of p53 expression and increase in the number of micronuclei in mutant
cells were related to cellular stress and DNA damage. These findings demonstrated
that LAP1B is not involved in cell cycle exit but might suggest a role in DNA damage
repair necessary for the induction of myogenin expression. Identification of previously
unknown pathways for LAP1 will contribute to the discovery of novel targets for
therapy.Çekirdek iç membran proteini LAP1B’nin (lamina-ilişkili polipeptit 1, izoform B)
işlev kaybı insanda kas distrofisi ve kardiyomiyopatiye neden olmaktadır. LAP1B’nin
kas dokusundaki işlevi henüz bilinmemektedir. Tezin amacı, LAP1B’yi ifade etmeyen
kas hücrelerinin farklılaşma sürecinde transkriptom düzeyindeki değişikliklerin
belirlenmesi ile, insanda neden olduğu kas distrofisinin moleküler patogenezinin
anlaşılmasına katkı sağlamaktır. Bu amaçla in vitro model olarak kullanılmak üzere
kontrol ve bir LAP1B-ilişkili kas distrofisi hastasına ait primer fibroblastlar
ölümsüzleştirilmiş ve uyarılabilir MyoD gen transferi gerçekleştirilmiştir. Kontrol
hücrelerin sekiz günde olgun miyotüpler oluşturduğu gözlenirken, mutant hücrelerin
füzyon kapasitelerinin çok düşük olduğu ve farklılaşmayı tamamlayamadıkları
bulunmuştur. Ayrıca C2C12 fare miyoblastlarında LAP1A/B ifadesinin
baskılanmasına neden olan mutasyonlar oluşturulmuş ve aynı bulgular gözlenmiştir.
RNA dizileme yöntemi ile kontrol ve mutant hücrelerde tüm transkriptom içerisinde
ifadesi değişen genler ve görev aldıkları yolaklar belirlenmiştir. Bu yolaklar arasından
kas kasılması, hücre döngüsü, mitozda kromatit segregasyonu ve hücre dışı matriks
organizasyonu öne çıkmıştır. Mutant hücrelerin p21 ifadesinin baskılanmış olmasına
rağmen hücre döngüsünden çıktıkları hücre döngüsü analizi ile belirlenmiştir. Son
olarak mutant hücrelerde p53 ifadesinde ve mikroçekirdek sayısında gözlenen artışın,
hücresel stres ve DNA hasarı ile ilişkili olduğu görülmektedir. Bu çalışmadan elde
edilen bulgular, LAP1B’nin kas farklılaşmasının erken evresinde hücre döngüsü
çıkışında rolünün olmadığı ancak miyogenin ifadesinin uyarılması için DNA hasarının
tamirinde görevli olabileceğine işaret etmektedir. LAP1’in yeni yolaklardaki olası
rolünün tanımlanması, tedavide hedeflenebilir yolakların keşfedilmesine de olanak
sağlayacaktır
Otozomal Resesif Limb-Girdle Kas Distrofisi Tanısı Alan Ailelerde Yeni Gen Araştırılması
Limb-girdle muscular dystrophies (LGMD) are a clinically and genetically heterogeneous group of hereditary muscle disorders characterized by symmetric, proximal and progressive muscular weakness. In this thesis, four consanguineous families diagnosed with autosomal recessive LGMD (LGMD2) which are not linked to any of the known LGMD2 loci were studied. The aim of this thesis was to identify the chromosomal localization of a novel mutant gene responsible for LGMD2 by performing genome-wide homozygosity mapping using 250K NspI SNP array data. In one family, the disease was mapped to a homozygous haplotype in chromosomal region 1q25. DNA sequencing of TOR1AIP1 encoding LAP1B (lamina-associated polypeptide 1B) in this critical chromosomal region showed a homozygous c.186delG mutation (p.E62fsTer25) in the three affected individuals. Relative quantitation of TOR1AIP1 mRNA by quantitative real-time PCR showed that the level of expression in the skeletal muscle of the patient was 5,88-fold lower than in the control sample. Western blot and immunofluorescent staining demonstrated that LAP1B was absent in the patient's skeletal muscle fibres. Ultrastructural examination showed alterations of the nuclear envelope including nuclear fragmentation and altered chromatin condensation. LAP1B is an integral protein of the inner nuclear membrane that binds to both A-type and B-type lamins, and is involved in the regulation of torsin A ATPase. This thesis expands the spectrum of genes associated with nuclear envelopathies and underlies a critical role for LAP1B in striated muscle.Limb-girdle kas distrofileri (LGMD) proksimal kaslarda simetrik olarak ilerleyen kas güçsüzlüğünün görüldüğü, klinik ve genetik heterojenite ile karakterize olan kalıtsal kas hastalığı grubudur. Bu tezde, otozomal resesif LGMD (LGMD2) klinik tanısı alan ve bilinen LGMD2 lokuslarına bağlantı göstermeyen, ebeveynlerin akraba olduğu dört farklı aile çalışılmıştır. Tez çalışmasında, 250K NspI çipleri ile yapılan genom boyu SNP (tek nükleotid polimorfizmi) genotipleme verileri kullanılarak homozigotluk haritalaması yaklaşımı ile LGMD2'den sorumlu yeni bir mutant genin kromozomdaki lokalizasyonunun saptanması amaçlanmıştır. Üç hasta bireyin bulunduğu bir ailede, 1q25 kromozom bölgesinde kısa bir homozigot haplotip tespit edilmiştir. DNA dizi analizi sonucunda, LAP1B (lamina-associated polypeptide 1B) proteinini kodlayan TOR1AIP1 (torsin A-interacting protein 1) geninde hastalarda homozigot c.186delG mutasyonu (p.E62fsTer25) saptanmıştır. Kantitatif gerçek zamanlı PZR ile hasta çizgili kas dokusunda TOR1AIP1 mRNA ifadesinin 5,88 kat azaldığı tespit edilmiştir. Western blot ve immünfloresan boyama çalışmaları ile hastada 66,3kDa büyüklüğündeki LAP1B izoformunun kas hücrelerinde ifade olmadığı saptanmıştır. Kas liflerinin çekirdeklerinde, çekirdek membranı parçalanması ve anormal kromatin yoğunlaşması gibi yapısal bozukluklar gözlenmiştir. LAP1B çekirdek iç membranında yerleşim gösteren, A ve B laminlere bağlanan ve torsin A ATPazının regülasyonunda görevli bir integral membran proteinidir. Bu tez çalışması, çekirdek zarfı hastalıklarına neden olan mutant gen spektrumunu genişletmiş ve LAP1B'nin çizgili kasta kritik bir role sahip olduğunu göstermiştir
Loss of the Nuclear Envelope Protein LAP1B Disrupts the Myogenic Differentiation of Patient-Derived Fibroblasts
Lamina-associated polypeptide 1 (LAP1) is a ubiquitously expressed inner nuclear membrane protein encoded by TOR1AIP1, and presents as two isoforms in humans, LAP1B and LAP1C. While loss of both isoforms results in a multisystemic progeroid-like syndrome, specific loss of LAP1B causes muscular dystrophy and cardiomyopathy, suggesting that LAP1B has a critical role in striated muscle. To gain more insight into the molecular pathophysiology underlying muscular dystrophy caused by LAP1B, we established a patient-derived fibroblast line that was transdifferentiated into myogenic cells using inducible MyoD expression. Compared to the controls, we observed strongly reduced myogenic differentiation and fusion potentials. Similar defects were observed in the C2C12 murine myoblasts carrying loss-of-function LAP1A/B mutations. Using RNA sequencing, we found that, despite MyoD overexpression and efficient cell cycle exit, transcriptional reprogramming of the LAP1B-deficient cells into the myogenic lineage is impaired with delayed activation of MYOG and muscle-specific genes. Gene set enrichment analyses suggested dysregulations of protein metabolism, extracellular matrix, and chromosome organization. Finally, we found that the LAP1B-deficient cells exhibit nuclear deformations, such as an increased number of micronuclei and altered morphometric parameters. This study uncovers the phenotypic and transcriptomic changes occurring during myoconversion of patient-derived LAP1B-deficient fibroblasts and provides a useful resource to gain insights into the mechanisms implicated in LAP1B-associated nuclear envelopathies
Knockout of zebrafish desmin genes does not cause skeletal muscle degeneration but alters calcium flux
Desmin is a muscle-specific intermediate filament protein that has fundamental role in muscle structure and force transmission. Whereas human desmin protein is encoded by a single gene, two desmin paralogs (desma and desmb) exist in zebrafish. Desma and desmb show differential spatiotemporal expression during zebrafish embryonic and larval development, being similarly expressed in skeletal muscle until hatching, after which expression of desmb shifts to gut smooth muscle. We generated knockout (KO) mutant lines carrying loss-of-function mutations for each gene by using CRISPR/Cas9. Mutants are viable and fertile, and lack obvious skeletal muscle, heart or intestinal defects. In contrast to morphants, knockout of each gene did not cause any overt muscular phenotype, but did alter calcium flux in myofibres. These results point to a possible compensation mechanism in these mutant lines generated by targeting nonsense mutations to the first coding exon