DNA damage and lamins

Abstract

The spatial and temporal organization of the genome has emerged as an additional level of regulation of nuclear functions. Structural proteins associated with the nuclear envelope play important roles in the organization of the genome. The nuclear lamina, a polymeric meshwork formed by lamins (A- and B-type) and lamin-associated proteins, is viewed as a scaffold for tethering chromatin and protein complexes regulating a variety of nuclear functions. Alterations in lamins function impact DNA transactions such as transcription, replication, and repair, as well as epigenetic modifications that change chromatin structure. These data, and the association of defective lamins with a whole variety of degenerative disorders, premature aging syndromes, and cancer, provide evidence for these proteins operating as caretakers of the genome. In this chapter, we summarize current knowledge about the function of lamins in the maintenance of genome integrity, with special emphasis on the role of A-type lamins in the maintenance of telomere homeostasis and mechanisms of DNA damage repair. These findings have begun to shed some light onto molecular mechanisms by which alterations in A-type lamins induce genomic instability and contribute to the pathophysiology of aging and aging-related diseases, especially cancer.

Fig. 1

A-type lamins function as a scaffold that ensures nuclear function. A-type lamins are a special class of intermediate filaments that form the nuclear lamina, a protein meshwork underlying the inner nuclear membrane, which can also extend throughout the nucleoplasm. They serve as a scaffold for tethering chromatin and proteins to specific nuclear subcompartments. A whole variety of functions have been ascribed to A-type lamins including processes such as DNA replication, transcription, and DNA repair. For the most part, the molecular mechanisms behind these functions remain poorly understood

Fig. 2

Schematic representation of lamin A structure and posttranslational processing. Lamins consist of a central rod domain, flanked by a globular head and a globular tail domain. Lamin A is synthesized as a prelamin A precursor which undergoes processing of its C-terminus. The C-terminal -CAAX motif (blue) undergoes farnesylation (green), cleavage of the last three amino acids (red), followed by carboxymethylation (purple) of the new terminal cysteine. A second cleavage by the metalloprotease Zmpste24 (red) removes another 15 amino acids, including the farnesyl group, rendering mature lamin A. Sites of interaction with gene transcriptional regulators such as Rb family members, lamin-associated proteins such as LAP2α, inner nuclear membrane proteins such as emerin, or chromatin have been described that involve primarily the C-terminal part of the protein and the central rod domain

Fig. 3

Processes essential for maintenance of genome stability. Maintenance of telomere homeostasis and mechanisms of DNA DSB repair is critical for genome stability. DSBs resulting from telomere dysfunction or exogenous or endogenous insults are repaired primarily by two mechanisms: nonhomologous end-joining (NHEJ) and homologous recombination (HR)

Fig. 4

Important factors in the DNA damage response (DDR). The presence of a DNA DSB is sensed by the MRN complex, which recruits and activates ATM, a master regulator of DDR. ATM phosphorylates H2AX, which targets MDC1 and other mediator factors to the break. These factors attract RNF8 and RNF168 which ubiquitylate histones around the break. These histone modifications facilitate recruitment of factors that participate in DNA DSB repair such as 53BP1 and BRCA1. 53BP1 promotes recruitment of the NHEJ machinery and leads to end-joining of the break. In contrast, BRCA1 promotes end-resection of the break, which initiates the complex process of recombination. The presence of DSBs can be easily visualized by performing immunofluorescence, as shown for γ-H2AX ionizing radiation-induced foci (IRIF)

Fig. 5

A-type lamins are essential for NHEJ of dysfunctional telomeres. (a) Telomere dysfunction due to overexpression of a dominant-negative version of the telomere-binding protein TRF2 causes chromosome end-to-end fusions, a process that requires a functional NHEJ mechanism of repair. (b) Graph shows that loss of A-type lamins reduces significantly the frequency of chromosome end-to-end fusions by NHEJ. (c) Table shows the total number of fusions observed as well as the percentage of metaphases with fusions in Lmna^+/+ and Lmna^−/− MEFs. These data demonstrated an unprecedented role for A-type lamins in NHEJ of dysfunctional telomeres

Fig. 6

Loss of A-type lamins impacts on 53BP1 stability and IRIF formation. Immunofluorescence showing a marked decrease in global levels of 53BP1 protein in Lmna^−/− MEFs and reduced intensity of 53BP1 labeling of DNA repair foci at 1 h postirradiation. Thus, loss of A-type lamins decreases the accumulation of 53BP1 protein at DNA repair foci, which explains reduced NHEJ in lamin-deficient cells

Fig. 7

Lamin-deficient cells exhibit defects in the repair of DNA DSBs induced by IR. (a) Images of neutral comet assays. Lmna^+/+ and Lmna^−/− MEFs were irradiated and subjected to single cell electrophoresis at different times postirradiation. Note how the comet tail decreases over time due to repair of DSBs. (b) Olive moment measures unrepaired DNA damage. A bimodal form of DNA DSB repair is clearly observed in Lmna^+/+ fibroblasts, with the fast phase corresponding to NHEJ occurring within the first hour after DNA damage. Lmna^−/− fibroblasts exhibited a greatly reduced rate of the fast component of DSB repair, indicating that these proteins play a role in the NHEJ repair mechanism

Fig. 8

Model of roles of A-type lamins in DNA repair. Loss of A-type lamins upregulates CTSL expression, resulting in elevated protein levels both in the nucleus and in the lysosomes. CTSL participates in the degradation of 53BP1, and the Rb family members pRb and p107, favoring the formation of p130/E2F4 repressor complexes, which in turn inhibit BRCA1 and RAD51 gene expression. The decrease in 53BP1 hinders NHEJ and the decrease in BRCA1 and RAD51 impairs HR repair. Other factors are likely to contribute to genomic instability in different laminopathies such as the accumulation of XPA at DSBs, the deficiency in DNA-PK, the increase in generation of ROS, epigenetic alterations, and loss of telomere homeostasis (not shown in the model). Importantly, vitamin D treatment inhibits CTSL-mediated degradation of 53BP1 and upregulates transcription of BRCA1 and RAD51, thus providing a potential therapeutic strategy