Role of lamin b1 in chromatin instability

Abstract

Nuclear lamins play important roles in the organization and structure of the nucleus; however, the specific mechanisms linking lamin structure to nuclear functions are poorly defined. We demonstrate that reducing nuclear lamin B1 expression by short hairpin RNA-mediated silencing in cancer cell lines to approximately 50% of normal levels causes a delay in the cell cycle and accumulation of cells in early S phase. The S phase delay appears to be due to the stalling and collapse of replication forks. The double-strand DNA breaks resulting from replication fork collapse were inefficiently repaired, causing persistent DNA damage signaling and the assembly of extensive repair foci on chromatin. The expression of multiple factors involved in DNA replication and repair by both nonhomologous end joining and homologous repair is misregulated when lamin B1 levels are reduced. We further demonstrate that lamin B1 interacts directly with the promoters of some genes associated with DNA damage response and repair, including BRCA1 and RAD51. Taken together, the results suggest that the maintenance of lamin B1 levels is required for DNA replication and repair through regulation of the expression of key factors involved in these essential nuclear functions.

FIG 1

Reduction of LB1 levels slows proliferation and DNA replication in U-2-OS and HCT116 cell lines. (A) LB1, LB2, LA, and LC protein levels were measured by immunoblotting at PD3 following silencing and selection. (B) The proliferation rates of shLB1 and Sc cells were compared for 5 days following silencing and selection. Proliferation rates were determined as described in Materials and Methods. U-2-OS: shLB1-1 (n = 9), P = 2.21 × 10⁻⁵; shLB1-2 (n = 3), P = 1.46 × 10⁻⁴. HCT116 (n = 3), P = 1.87 × 10⁻⁵. Error bars represent standard deviations. (C) FACS analysis of the complete cell cycle. U-2-OS: shLB1-1 (n = 5), P = 1.6 × 10⁻³; shLB1-2 (n = 3), P = 2.2 × 10⁻⁴. HCT116 (n = 3), P = 2.2 × 10⁻³. (D) FACS analysis of G₁ and S phases in Sc and shLB1 cells. U-2-OS shLB-1 (n = 3): early S, P = 4.8 × 10⁻³, late S, P = 2.3 × 10⁻⁴. shLB-2 (n = 3): early S, P = 2.39 × 10⁻³; late S, P = 4.1 × 10⁻⁴. HCT116 shLB-1 (n = 4): early S, P = 3.72 × 10⁻³; late S, P = 2.75 × 10⁻³. (E) Detection of LB1 (red) and BrdU (green) incorporation into replicating DNA following 40 min of labeling in the nuclei of Sc and shLB1 cells (DNA stained with Hoechst, blue). More than 600 cells of each type were counted, and images of single representative nuclei are shown. U-2-OS: shLB-1 (n = 4), P = 2.3 × 10⁻²; shLB-2 (n = 3), P = 4.7 × 10⁻³. HCT116 shLB-1 (n = 3), P = 2.12 × 10⁻³. (F) Detection of BrdU by ELISA. Sc and shLB1 cells where fractioned according to cell cycle phase by FACS and labeled with BrdU for 40 min. BrdU was detected in isolated genomic DNA by ELISA as described in Materials and Methods. The experiment was repeated five times, and each DNA sample was subjected to ELISA in quadruplicate. U-2-OS shLB-1 (n = 5): all, P = 0.0024; early S, P = 0.00012; late S, P = 0.00048. shLB-2 (n = 3): all, P = 0.0039; early S, P = 0.0023; late S, P = 0.0011. HCT116 shLB1 (n = 3): all, P = 0.0054; early S, P = 0.009; late S, P = 0.0032. Error bars represent standard deviations. The asterisks in panels C to F indicate statistically significant differences.

FIG 2

Association of lamins with actively replicating chromatin. (A) Analysis of DNA replication at nine origins of replication in Sc and shLB1 cells. After 40 min of labeling with BrdU, DNA was precipitated from a soluble chromatin fraction with specific anti-BrdU antibodies. Input and precipitated DNA samples were analyzed by qPCR. This experiment was repeated three times with four technical repeats of the qPCR analysis. The decrease in the precipitation of eight origins of replication was significant in shLB1 (n = 3, P < 0.003). (B) BrdU ChIP analysis of proteins associated with replicating chromatin. Nonsilenced U-2-OS cells were labeled with BrdU for 40 min. Soluble and insoluble chromatin fractions were prepared from nuclei as described in Materials and Methods. Proteins were analyzed by immunoblotting with specific antibodies. The experiment was repeated five times. (C) Direct interaction of LB1 and LA/C with actively replicating chromatin in Sc and shLB1 cells. Soluble chromatin fractions were immunoprecipitated with specific anti-LB1 or LA/C antibodies. Input and precipitated DNA samples were analyzed with qPCR. This experiment was repeated four times with five technical repeats of qPCR analysis. Error bars represent standard deviations. Asterisks indicate statistically significant differences.

FIG 3

Analysis of the elongation phase and replication fork stability. (A, B) Analysis of replication in Sc and shLB1 cells by single-label DNA fiber analysis. Incorporated BrdU was detected in extracted DNA fibers with specific anti-BrdU antibodies following 40 min or 2 h of labeling. Fiber length was measured with 250 fibers analyzed for each cell type (n = 7; 40 min, P = 0.0004; 2 h, P = 0.002). Representative images are shown. (C) Analysis of replication fork stability. Sc and shLB1 cells were incubated with CldU for 40 min; this was followed by the addition of HU and 4 h of incubation to deplete the nucleotide pool and cause replication fork stalling. HU was then removed, and the cells were labeled with CldU for 40 min. DNA fiber extraction and double staining were done as described in Materials and Methods. CldU (red) incorporation represents DNA replication before replication fork stalling was induced. IdU (green) represents recovery of stalled replication forks after HU removal. In each experiment, 300 fibers were analyzed for each cell type and the length of the separated track was measured. U-2-OS shLB1-1 (n = 5), P = 2.74 × 10⁻³; shLB-2 (n = 3), P = 2.53 × 10⁻³; HCT116 shLB-1 (n = 3), P = 2.15 × 10⁻³. Representative images are shown. Asterisks indicate statistically significant differences. (D) Immunoblot analyses of proteins associated with replication in total Sc and shLB1 cell lysates. GAPDH served as a loading control. (E) Analysis of proteins associated with replicating chromatin in U-2-OS cells. Sc and shLB1 cells were incubated with BrdU for 40 min or 2 h. Soluble chromatin fractions were prepared from nuclei as described in Materials and Methods for BrdU ChIP. Proteins were analyzed by immunoblotting following BrdU ChIP.

FIG 4

Reduction of LB1 levels leads to accumulation of DNA damage and activation of a persistent DNA damage response. (A) Sc and shLB1 cells were fixed at PD3 and stained with antibodies specific to LB1 (red) and γH2AX (green) or 53BP1 (red) and LB1 (green). Images of single representative nuclei are shown. (B) DNA damage assayed by localization of 53BP1 or γH2AX in the nuclei of Sc and shLB1 cells at PD3. More than 700 nuclei were examined for each protein. (n = 4; 53BP1, P = 0.0018; γH2AX, P = 0.0075). (C) Average sizes of γH2AX and 53BP1 foci in nuclei of Sc and shLB1 cells at PD3. The diameters of the foci were measured with ImageJ software. More than 400 nuclei were examined for each protein, and at least 20 foci/nucleus were measured (n = 4; γH2AX, P = 0.0102; γH2AX, P = 0.0013). Error bars represent standard deviations. Asterisks indicate statistically significant differences.

FIG 5

(A) Color-coded map showing genes differentially regulated in Sc and shLB1 cells. The fold change is absolute with P values of <0.05. (B) Gene ontology analysis of differentially regulated genes in Sc and shLB1 cells showing functional clusters of genes. The functional clusters were obtained with DAVID. Significant clusters (P < 0.05; FDR, <0.05) were plotted with KRONA (78). The number of genes in each cluster is shown. (C) Significant pathways (P < 0.05; FDR, <0.05) were plotted as a pie chart for differentially regulated genes by DAVID and KEGG (79) pathway analysis. The number of genes in each pathway is shown.

FIG 6

Immunoblotting of DNA damage response- and repair-associated proteins. (A) Sc and shLB1 cells were harvested at PD3 after selection, and total cell lysates were analyzed. GAPDH served as a loading control. (B) Analysis of EGFP expression facilitated by activation of HR or NHEJ as described in Materials and Methods. U-2-OS (n = 6): HR, P = 0.0025; NHEJ, P = 0.0097. HCT116 (n = 3): HR, P = 0.0045; NHEJ, P = 0.01. (C) Detection of apoptosis in Sc and shLB1 cells after treatment with 0.25 μM ST, 5 μg/ml BLM, or 20 μM CMPT. Sc and shLB1 cells were harvested at 24 h after treatment, stained for annexin V/PI, and examined by FACS. U-2-OS shLB1-1 (n = 5): BLM, P = 0.036; CMPT, P = 0.0023. shLB1-2 (n = 3): BLM, P = 0.013; CMPT, P = 0.0076. HCT116 (n = 3): BLM, P = 0.018; CMPT, P = 0.0045. Error bars represent standard deviations. Asterisks indicate statistically significant differences. (D) Clonogenicity assay of Sc and shLB1 cells treated with 2.5 μg/ml BLM or 10 μM CMPT for 48 h. Colonies were counted on day 10. U-2-OS shLB1-1 (n = 6): BLM, P = 0.004; CMPT, P = 0.0013. shLB1-2 (n = 3): BLM, P = 0.008; CMPT, P = 0.005. HCT116 (n = 3): BLM, P = 0.0024; CMPT, P = 0.0089. (E) Formation of DNA repair foci in Sc and shLB1 cells. Cells were treated with 20 μM CMPT for 4 h, fixed, and stained with antibodies to LB1 (green) and MRE11 (red) or pRPA32 (red). Images of single representative nuclei are shown. ct in panel C and Ct in panel D, control.

FIG 7

Reduced LB1 levels cause increased chromosome instability. (A) Aneuploidy in Sc and shLB1 cells was analyzed by FACS of PI-stained cells. U-2-OS (n = 5), P < 0.021; HCT116 (n = 3), P = 0.017. The formation of micronuclei was determined by direct counting of cells stained with antilamin or anti-γH2AX antibodies and Hoechst staining of DNA. U-2-OS (n = 4; number of nuclei examined, >600), P < 0.0101; HCT116 (n = 3; number of nuclei examined, >500), P < 0.022. The appearance of lagging chromosomes was analyzed with the same antibody and DNA stain combination. U-2-OS (n = 5); number of nuclei examined, >800), P < 0.003; HCT116 (n = 3; number of nuclei examined, >600), P < 0.0042. Error bars represent standard deviations. Asterisks mark statistically significant differences. (B) Representative images of nuclei showing micronuclei and lagging chromosomes.

FIG 8

Analyses of LB1 interactions with specific gene promoters by lamin ChIP-qPCR. LB1-associated chromatin from a soluble nuclear chromatin fraction isolated from Sc and shLB1 cells was immunoprecipitated with specific antibodies to LB1. Input and precipitated DNA samples were analyzed by qPCR. The experiment was repeated five times with five technical repeats of qPCR analysis. BRCA1, P = 0.006; RAD51, P = 0.0011; DDB1, P = 0.0003. Error bars represent standard deviations. Asterisks indicate statistically significant differences.