Defects in lamin B1 expression or processing affect interphase chromosome position and gene expression

Abstract

Radial organization of nuclei with peripheral gene-poor chromosomes and central gene-rich chromosomes is common and could depend on the nuclear boundary as a scaffold or position marker. To test this, we studied the role of the ubiquitous nuclear envelope (NE) component lamin B1 in NE stability, chromosome territory position, and gene expression. The stability of the lamin B1 lamina is dependent on lamin endoproteolysis (by Rce1) but not carboxymethylation (by Icmt), whereas lamin C lamina stability is not affected by the loss of full-length lamin B1 or its processing. Comparison of wild-type murine fibroblasts with fibroblasts lacking full-length lamin B1, or defective in CAAX processing, identified genes that depend on a stable processed lamin B1 lamina for normal expression. We also demonstrate that the position of mouse chromosome 18 but not 19 is dependent on such a stable nuclear lamina. The results implicate processed lamin B1 in the control of gene expression as well as chromosome position.

Figure 1.

The stability of lamin B1 interactions is dependent on posttranslational modification by Rce1. (a) FLIP in WT mouse embryonic fibroblasts, Rce^−/−, and Icmt^−/− cells expressing GFP–lamin B1. A specific ROI was photobleached at full laser power for 250 s. The postbleach image shows the extent of fluorescence loss in the nonbleached area, which is indicative of the stability of GFP–lamin B1 at the nuclear periphery. Bar, 10 μm. (b) Quantitative FLIP. An ROI outside the photobleached area shown in panel a was used to measure fluorescence loss after photobleaching (mean values ± SD; n = 5). The background intensity was subtracted and the values normalized by dividing by the intensities of an ROI in an adjacent control cell to account for photobleaching of the 5% laser power used for scanning the nonbleached areas. GFP–lamin B1 interactions at the nuclear periphery are more stable in WT and Icmt^−/− (c) than in Rce^−/− (b), indicating that carboxymethylation has little or no effect on the stability of the nuclear lamina. (d) FLIP of GFP–lamin B1 in the Lmnb1^−/− cells used in the current study does not show any significant difference from its behavior in WT cells, indicating that the difference observed in the Rce1^−/− cells is due to a lack of processing and not another cell-specific factor.

Figure 2.

A summary of the gene expression changes in cells lacking full-length lamin B1, Rce1, or Icmt. (a) A tree view diagram showing the fold change of genes that are differentially expressed in Lmnb1^−/−, Rce1^−/−, and Icmt^−/− cells. (b) A detailed view of the 16 genes with coordinately altered expression. The top box shows genes up-regulated in both Lmnb1^−/− and Rce1^−/−, and the bottom box shows genes down-regulated in both these knockout (KO) cells. Fold changes are shown as KO/WT. (c) A Venn diagram showing the proportion of genes that are regulated by 1.5-fold because of abnormalities in lamin B1 processing. The percentages represent the proportion of up-regulated genes in relation to all dysregulated genes within a specific category. (d) Summary of changes in gene expression in Lmnb^−/− and Rce1^−/− cells, showing changes unique to one knockout cell type.

Figure 3.

Genes that are dysregulated in the absence of full-length lamin B1 or Rce1 do not show any functional clustering. A tree view of genes that are changed by 1.5-fold or more in Rce^−/− and Lmnb1^−/− cells (red indicates up-regulated genes, and green indicates down-regulated genes), illustrating that there is no functional clustering based on the following GO entries: Calcium ion binding (0005509), transcription (0003700), cell adhesion (0007155), zinc ion binding (0008270), heparin binding (0008201), development (0007275), growth factor activity (0008083), and transferase activity (0016740). Note that some genes are represented more than once because they have multiple entries in the GO database.

Figure 4.

The distribution of genes that are dysregulated in the absence of full-length lamin B1 or Rce1 shows some positional clustering. Genes that are differentially expressed by at least 1.5-fold (red indicates up-regulated genes, and blue indicates down-regulated genes) in both Lmnb1^−/− and Rce1^−/− cells were mapped to their specific chromosomal positions using Ensembl KaryoView. Note that the cluster on chromosome 18 contains three up-regulated genes not resolved at this scale.

Figure 5.

Endoproteolyzed but not carboxymethylated lamin B1 is required for the peripheral localization of mouse chromosome 18. (a) Adherent cells were fixed in methanol acetic acid and hybridized with probes to mouse chromosomes 18 (red) and 19 (green). Nuclei were then counterstained with DAPI (blue). (b) 50 nuclei of normal morphology from each cell type were used to quantitate the distribution of chromosomes 18 and 19 in five concentric shells eroded from the nuclear periphery (left) to the center (right). The y axis shows the proportion of FISH signal in each of five zones; error bars show SEM. (c) Con A labeling (blue) alongside chromosome 18 FISH (red) shows that chromosome 18 is in close contact with the nuclear periphery.

Figure 6.

Farnesylated lamin B1 is required for the peripheral localization of mouse chromosome 18. (a) FISH analysis of chromosome 18 position after FTI treatment, which disrupts the first step of lamin B1 processing, shows that loss of farnesylated lamin B1 has an effect that is similar to that of loss of endoproteolysis. Bar, 10 μm. (b) Quantitative analysis of chromosome 18 position as described above after FTI treatment shows loss of peripheral localization. Error bars indicate SEM.