Expression of disease-causing lamin A mutants impairs the formation of DNA repair foci

A-type lamins are components of the nuclear lamina. Mutations in the gene encoding lamin A are associated with a range of highly degenerative diseases termed laminopathies. To evaluate sensitivity to DNA damage, GFP-tagged lamin A cDNAs with disease-causing mutations were expressed in HeLa cells. The inner nuclear membrane protein emerin was mislocalised upon expression of the muscular dystrophy mutants G232E, Q294P or R386K, which aberrantly assembled into nuclear aggregates, or upon expression of mutants causing progeria syndromes in vivo (lamin A del50, R471C, R527C and L530P). The ability of cells expressing these mutants to form DNA repair foci comprising phosphorylated H2AX in response to mild doses of cisplatin or UV irradiation was markedly diminished, unlike the nearly normal response of cells expressing wild-type GFP-lamin A or disease-causing H222P and R482L mutants. Interestingly, mutants that impaired the formation of DNA repair foci mislocalised ATR (for 'ataxia telangiectasia-mutated and Rad3-related') kinase, which is a key sensor in the response to DNA damage. Our results suggest that a subset of lamin A mutants might hinder the response of components of the DNA repair machinery to DNA damage by altering interactions with chromatin

Lamin A/C speckles mediate spatial organization of splicing factor compartments and RNA polymerase II transcription

The A-type lamins have been observed to colocalize with RNA splicing factors in speckles within the nucleus, in addition to their typical distribution at the nuclear periphery. To understand the functions of lamin speckles, the effects of transcriptional inhibitors known to modify RNA splicing factor compartments (SFCs) were examined. Treatment of HeLa cells with α-amanitin or 5,6-dichlorobenzimidazole riboside (DRB) inhibited RNA polymerase II (pol II) transcription and led to the enlargement of lamin speckles as well as SFCs. Removal of the reversible inhibitor DRB resulted in the reactivation of transcription and a rapid, synchronous redistribution of lamins and splicing factors to normal-sized speckles, indicating a close association between lamin speckles and SFCs. Conversely, the expression of NH2-terminally modified lamin A or C in HeLa cells brought about a loss of lamin speckles, depletion of SFCs, and down-regulation of pol II transcription without affecting the peripheral lamina. Our results suggest a unique role for lamin speckles in the spatial organization of RNA splicing factors and pol II transcription in the nucleus

Lamin misexpression upregulates three distinct ubiquitin ligase systems that degrade ATR kinase in HeLa cells

Lamins are the major structural components of the nucleus and mutations in the human lamin A gene cause a number of genetic diseases collectively termed laminopathies. At the cellular level, lamin A mutations cause aberrant nuclear morphology and defects in nuclear functions such as the response to DNA damage. We have investigated the mechanism of depletion of a key damage sensor, ATR (Ataxia-telangiectasia-mutated-and-Rad3-related) kinase, in HeLa cells expressing lamin A mutants or lamin A shRNA. The degradation of ATR kinase in these cells was through the proteasomal pathway as it was reversed by the proteasomal inhibitor MG132. Expression of lamin A mutants or shRNA led to transcriptional activation of three ubiquitin ligase components, namely, RNF123 (ring finger protein 123), HECW2 (HECT domain ligase W2) and the F-box protein FBXW10. Ectopic expression of RNF123, HECW2 or FBXW10 directly resulted in proteasomal degradation of ATR kinase and the ring domain of RNF123 was required for this degradation. However, these ligases did not alter the stability of DNA-dependent protein kinase, which is not depleted upon lamin misexpression. Although degradation of ATR kinase was reversed by MG132, it was not affected by the nuclear export inhibitor, leptomycin B, suggesting that ATR kinase is degraded within the nucleus. Our findings indicate that lamin misexpression can lead to deleterious effects on the stability of the key DNA damage sensor, ATR kinase by upregulation of specific components of the ubiquitination pathway

A rare mutation in lamin A gene is associated with dilated cardiomyopathy in Indian patients

Mutations in lamin A gene (LMNA) are associated with a number of genetic diseases that are collectively termed laminopathies. Most LMNA mutations cause muscular dystrophies and cardiomyopathies. The incidence of LMNA mutations in familial dilated cardiomyopathy (DCM) patients is 5-8 % in Caucasian populations. However, there is no large scale study of LMNA mutations in Indian DCM patients. Hence, we have carried out sequence analysis of LMNA in 239 Indian DCM patients. We have identified a rare non-synonymous mutation c.1873A>T in one patient, which predicted a change in the amino acid serine to cysteine at residue 625. In addition we also identified 20 synonymous single nucleotide polymorphisms in 28 patients. The c.1873A>T mutation was absent in 156 healthy and ethnically matched controls. The serine at position 625 has been earlier identified as a mitotic phosphorylation site in lamin A. Expression of mutant lamin S625C in cultured cells led to a decrease in levels of cyclin dependent kinase inhibitor p21, suggesting compromised cell cycle regulation in these cells. Our study provides important information on the extent of variations in LMNA in Indian DCM patients and identifies a rare mutation in LMNA that is likely to cause deleterious effects on cellular functions