Altered Chromosomal Positioning, Compaction, and Gene Expression with a Lamin A/C Gene Mutation

Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression.To investigate the hypothesis that mutant lamin A/C changes the lamina's ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction.These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered

Altered Chromosomal Positioning, Compaction, and Gene Expression with a Lamin A/C Gene Mutation

Background: Lamins A and C, encoded by the LMNA gene, are filamentous proteins that form the core scaffold of the nuclear lamina. Dominant LMNA gene mutations cause multiple human diseases including cardiac and skeletal myopathies. The nuclear lamina is thought to regulate gene expression by its direct interaction with chromatin. LMNA gene mutations may mediate disease by disrupting normal gene expression.Methods/Findings: To investigate the hypothesis that mutant lamin A/C changes the lamina's ability to interact with chromatin, we studied gene misexpression resulting from the cardiomyopathic LMNA E161K mutation and correlated this with changes in chromosome positioning. We identified clusters of misexpressed genes and examined the nuclear positioning of two such genomic clusters, each harboring genes relevant to striated muscle disease including LMO7 and MBNL2. Both gene clusters were found to be more centrally positioned in LMNA-mutant nuclei. Additionally, these loci were less compacted. In LMNA mutant heart and fibroblasts, we found that chromosome 13 had a disproportionately high fraction of misexpressed genes. Using three-dimensional fluorescence in situ hybridization we found that the entire territory of chromosome 13 was displaced towards the center of the nucleus in LMNA mutant fibroblasts. Additional cardiomyopathic LMNA gene mutations were also shown to have abnormal positioning of chromosome 13, although in the opposite direction.Conclusions: These data support a model in which LMNA mutations perturb the intranuclear positioning and compaction of chromosomal domains and provide a mechanism by which gene expression may be altered.</p

Model of chromatin positioning and gene expression.

<p>In the case of the <i>LMNA</i> E161K mutation, two distinct loci on chromosome 13 were displaced to a more intranuclear position (right). We hypothesize that loss of interaction with the lamina (blue) prevents interaction with active chromatin complexes (black) and reduces gene expression.</p

Genomic clusters of misexpressed genes.

<p>Shown are genes that are misexpressed in the LMNA mutant heart that are colocalized in the same genomic interval. The chromosome position is shown on the left. The two genes within each interval are indicated in the subsequent columns.</p

The lamina is intact in <i>LMNA</i> E161K heart and fibroblasts.

<p>(A) Electron microscopy illustrates the electron dense lamina in both the <i>LMNA</i> E161K and <i>LMNA</i> normal hearts, and shows no appreciable difference. N = nucleus, red arrows indicate nuclear membrane. Scale bar  = 2 µm. (B) The LINC complex proteins localize normally in <i>LMNA</i> E161K mutant heart. Sections from <i>LMNA</i> E161K mutant and <i>LMNA</i> normal heart were analyzed by immunofluorescence microscopy using antibodies for lamin A/C (green), nesprin-1, emerin and SUN1 (red). DAPI is shown in blue. Scale bar  = 10 µm. (C) Lamin A/C (green) localization was determined using immunofluorescence microscopy in <i>LMNA</i> E161K mutant and <i>LMNA</i> normal fibroblasts. DAPI shown in blue. Scale bar  = 10 µm.</p

Genes misexpressed in both LMNA mutant heart and fibroblasts.

<p>Genes misexpressed in both LMNA mutant heart and fibroblasts.</p

Increased distance between clusters 13A and 13B in <i>LMNA</i> E161K mutant nuclei.

<p>(A) The distance between Clusters 13A and 13B was measured in <i>LMNA</i> normal and <i>LMNA</i> E161K mutant nuclei (n = 56 and n = 94 respectively), 13A = red, 13B = green, anti-lamin B1 = blue. (B) The clusters are significantly further apart in the <i>LMNA</i> mutant nuclei than in the control nuclei consistent with a reduced compaction of the chromosome in this region, (*p = 0.0024). (C) The chromosome territory volume of chromosome 13 (green) and chromosome 7 (red) was reduced in <i>LMNA</i> E161K compared to <i>LMNA</i> normal fibroblasts. DAPI is blue. (D) Both chromosome 13 (left) and 7 (right) territories are significantly more compact in the <i>LMNA</i> E161K mutant fibroblasts. Scale bar = 10 µm.</p

The chromosome 13 territory is displaced in <i>LMNA</i> mutant nuclei.

<p>(A) Nuclear position of the chromosome 13 territory was determined by chromosomal painting, shown as green. The position of chromosome 13 is shown for <i>LMNA</i> normal and mutant nuclei. DAPI staining (blue). (B) Chromosome 13 territories are significantly displaced in the <i>LMNA</i> mutant nuclei compared to the control nuclei (n = 30 control nuclei and n = 15 <i>LMNA</i> E161K (p = 0.009), n = 12 Δ303, n = 10 D596N).</p

Chromosome 13 has a higher than expected percentage of misexpressed genes in both <i>LMNA</i> mutant heart and fibroblasts.

<p>Gene expression was compared between <i>LMNA</i> E161K mutant and <i>LMNA</i> normal hearts (A) or fibroblasts (B). Chromosomes are indicated along the x-axis. Percent misexpressed genes for entire genome is drawn as expected line. Percent misexpressed genes per chromosome is represented by gray bars. Arrows indicate chromosome 13.</p

Two gene clusters on chromosome 13 are displaced from the nuclear periphery in <i>LMNA</i> E161K cells.

<p>Gene expression profiling identified gene clusters that were misexpressed in <i>LMNA</i> E161K. Two clusters from chromosome 13, referred to as 13A and 13B were studied because they contain genes important for striated muscle function. (A) Cluster 13A contains <i>LMO7</i> which encodes a nuclear membrane associated emerin-interacting protein. Cluster 13B contains <i>MBNL2.</i> The intranuclear position of Cluster 13A (red, top) and Cluster 13B (green, bottom) is shown in <i>LMNA</i>-normal nuclei and <i>LMNA</i>-mutant nuclei. Anti-Lamin B-1 (αLMNB1) is shown in blue. (B) The nuclear position of Cluster A was displaced away from the nuclear periphery in <i>LMNA</i> mutant versus normal (n = 98 control nuclei and n = 64 E161K nuclei), (*p = 0.0001)(top). Similarly, the nuclear position of Cluster B was displaced towards the nuclear center in <i>LMNA</i> E161K mutant versus <i>LMNA</i> normal nuclei (n = 106 control nuclei and n = 66 for E161K nuclei) (*p = 0.02) (bottom). (C) The nuclear position of the <i>ACTB</i> gene encoding β-actin was examined as a control genomic locus and did not differ between mutant and normal. Anti-Lamin B-1 (αLMNB1) is shown in green and DAPI staining in blue. Scale bar = 10 µm. (D) There was no significant difference between the localization of the ACTB locus in E161K <i>LMNA</i> mutant versus <i>LMNA</i> normal nuclei.</p