Anchorage of H3K9-methylated heterochromatin to the nuclear periphery helps mediate P-cell nuclear migration though constricted spaces in Caenorhabditis elegans

Abstract

Nuclei adjust their deformability while migrating through constrictions to enable structural changes and maintain nuclear integrity. The effect of heterochromatin anchored at the nucleoplasmic face of the inner nuclear membrane on nuclear morphology and deformability during in vivo nuclear migration through constricted spaces remains unclear. Here, we show that abolishing peripheral heterochromatin anchorage by eliminating CEC-4, a chromodomain protein that tethers H3K9-methylated chromatin to the nuclear periphery, disrupts constrained P-cell nuclear migration in Caenorhabditis elegans larvae in the absence of the established LINC complex-dependent pathway. CEC-4 acts in parallel to an actin and CDC-42-based pathway. We also demonstrate the necessity for the chromatin methyltransferases MET-2 and JMJD-1.2 during P-cell nuclear migration in the absence of functional LINC complexes. We conclude that H3K9-nethylated chromatin needs to be anchored to the nucleoplasmic face of the inner nuclear membrane to help facilitate nuclear migration through constricted spaces in vivo.

Figure 1:. The cec-4 null mutation enhances the P-cell nuclear migration defect observed in unc-84 and unc-84; cgef-1 null mutants.

A) Images of L4 animals expressing GFP from the oxIs12[punc-47::gfp] marker in P-cell derived GABAergic neurons. Dorsal is up, ventral is down. The numbers of GABAergic neurons present in the ventral cord were scored and are numbered from anterior (left) to posterior. Scale bar: 100 µm. B) Plot of the number of missing GABAergic neurons from the 19 observed in wild type (UD87 RFP+ animals) and null mutant lines unc-84 (UD87 RFP-), cec-4 (UD462 RFP+), cgef-1 (UD285 RFP+), cec-4; unc-84 (UD462 RFP-), cgef-1, unc-84 (UD285 RFP-), cec-4; cgef-1 (UD795 RFP+), cec-4; cgef-1, unc-84 (UD795 RFP-). Each dot represents a single worm. Three different temperatures are shown: 25° C (salmon), 20° C (violet), and 15° C (blue). n=40. Means and 95% confidence intervals (CI) are shown. Tests for statistical significance are shown to compare cec-4 to wild type, cec-4; unc-84 double mutants to unc-84 single mutants, and cec-4; cgef-1, unc-84 triple mutants to cgef-1, unc-84 double mutants. Results were corrected using a Tukey HSD test. ***p≤0.001

Figure 2:. Inhibiting H3K9 methylation enhances the P-cell nuclear migration defects observed in unc-84 mutants.

Plot of the number of missing GABAergic neurons observed in wild type (UD87 RFP+ animals) and null mutant lines unc-84 (UD87 RFP-), met-2; unc-84 (UD906 RFP-), cgef-1, unc-84 (UD285 RFP-), met-2; cgef-1 (UD957 RFP+), met-2; cgef-1, unc-84 (UD957 RFP-). Each dot represents a single worm. Three different temperatures are shown: 25° C (salmon), 20° C (violet), and 15° C (blue). n=40. Means and 95% CI are shown. Significance indicated is compared to unc-84(n369) at each temperature. Results were corrected using a Tukey HSD test. ***p≤0.001.

Figure 3:. The presence of one copy of the jmjd-1.2 mutation enhances the P-cell nuclear migration observed in unc-84 mutant animals.

A plot of the number of missing GABAergic neurons of wild type (UD87 RFP+ animals) and the following null mutant lines: unc-84 (UD87 RFP-), cgef-1 (UD285 RFP+), +/jmjd-1.2 (UD996 RFP+) cgef-1, unc-84 (UD285 RFP-), +/jmjd-1.2; unc-84 (UD996 RFP-), +/jmjd-1.2; cgef-1 (UD997 RFP+), +/jmjd-1.2; cgef-1, unc-84 (UD997 RFP-). Each dot represents a single worm. Three different temperatures are shown: 25° C (salmon), 20° C (violet), and 15° C (blue). n=40. Means and 95% CI are shown. Significance indicated is compared to unc-84(n369) at each temperature. Results were corrected using a Tukey SD test. ***p≤0.001.