LAB-1 targets PP1 and restricts Aurora B kinase upon entrance into meiosis to promote sister chromatid cohesion

Abstract

Successful execution of the meiotic program depends on the timely establishment and removal of sister chromatid cohesion. LAB-1 has been proposed to act in the latter by preventing the premature removal of the meiosis-specific cohesin REC-8 at metaphase I in C. elegans, yet the mechanism and scope of LAB-1 function remained unknown. Here we identify an unexpected earlier role for LAB-1 in promoting the establishment of sister chromatid cohesion in prophase I. LAB-1 and REC-8 are both required for the chromosomal association of the cohesin complex subunit SMC-3. Depletion of lab-1 results in partial loss of sister chromatid cohesion in rec-8 and coh-4 coh-3 mutants and further enhanced chromatid dissociation in worms where all three kleisins are mutated. Moreover, lab-1 depletion results in increased Aurora B kinase (AIR-2) signals in early prophase I nuclei, coupled with a parallel decrease in signals for the PP1 homolog, GSP-2. Finally, LAB-1 directly interacts with GSP-1 and GSP-2. We propose that LAB-1 targets the PP1 homologs to the chromatin at the onset of meiosis I, thereby antagonizing AIR-2 and cooperating with the cohesin complex to promote sister chromatid association and normal progression of the meiotic program.

Figure 1. LAB-1 is required for homologous pairing.

(A) High-magnification images of DAPI stained mid-pachytene nuclei (blue) hybridized with FISH probes targeting the pairing center region on chromosome I (red). Nuclei with either one, two, or three signals (foci) are depicted. Bars, 3 µM. (B) Diagram of a C. elegans germline indicating the position of the zones scored in the analysis of the progression of homologous pairing. (C, D) Graphs depicting the percentage of nuclei showing one, two, and three to four foci within each zone in control and lab-1(RNAi) gonads hybridized with FISH probes recognizing the chromosome I pairing center (C) and the X chromosome pairing center (D).

Figure 2. LAB-1 is required for double-strand break repair progression, crossover control, and complete synapsis.

(A–B) Histograms depict the quantification of RAD-51 foci in (A) control and (B) lab-1(RNAi) germlines. The number of RAD-51 foci per nucleus is categorized according to the color code shown on the right. The percent of nuclei observed for each category (y-axis) is depicted for each zone along the germline axis (x-axis). Insets represent examples of late pachytene nuclei co-stained with DAPI (blue) and RAD-51 (red). Bars, 3 µM. (C) Germline diagram indicates the eight zones throughout which RAD-51 foci were scored for all nuclei. (D) Quantification of germline apoptosis. Error bars represent standard deviation of the mean. Asterisk indicates statistically significant increase in the number of apoptotic nuclei (p<0.0001, by the two-tailed Mann-Whitney test, 95% C.I.). n, number of gonad arms scored. (E–F) Histograms depict the quantification of (E) ZHP-3::GFP and (F) MSH-5 foci in late pachytene nuclei of control and lab-1(RNAi) germlines. Between 63 and 75 nuclei were scored from 5 to 7 gonads for each genotype. Insets depict representative nuclei (DAPI, blue; ZHP-3::GFP, green; and MSH-5, red). Bars, 3 µM. (G) Mid-pachytene nuclei in control and lab-1(RNAi) gonads co-stained with SYP-1 (red), HTP-3 (green), and DAPI (blue). Arrowheads indicate HTP-3-stained tracks that lack SYP-1 signal. Bars, 4 µM.

Figure 3. LAB-1 localization is dependent on the cohesin complex.

LAB-1 (red) and DAPI (blue) staining of early pachytene, late pachytene, and −1 oocytes at diakinesis in the germlines of the indicated genotypes. Bars, 4 µM.

Figure 4. Both LAB-1 and REC-8 are required for SMC-3 and SYP-1 localization onto chromosomes.

Transition zone, mid-pachytene, and late pachytene nuclei in the germlines of the indicated genotypes co-stained with SMC-3 (red), SYP-1 (green), and DAPI (blue). Bars, 4 µM.

Figure 5. lab-1 depletion weakens sister chromatid cohesion.

Number of DAPI-stained bodies in the −1 oocyte at diakinesis in the indicated genotypes. The p values were calculated by (a) the Wilcoxon signed-rank test and by (b) the two-tailed Mann-Whitney test, 95% C.I. Bars, 4 µM.

Figure 6. LAB-1 binds GSP-1 and GSP-2 and is required for correct AIR-2 and GSP-2 localization on early meiotic nuclei.

(A) Far-western analysis of in vitro binding of purified recombinant LAB-1, LAB-1 PP1 putative motif mutants, GSP-1, and GSP-2. Purified bacterially expressed proteins were transferred to membranes, incubated with the indicated binding protein, and probed with appropriate antibodies. BSA was used as control. (B) The yeast two-hybrid system was used to test the protein interactions between LAB-1, LAB-1 PP1 putative motif mutants, GSP-1, and GSP-2. Proteins were fused to either the DNA binding domain (DB) or the activation domain (AD) of GAL4. Interactions were scored by growth on SC-Leu-Trp-Ade plates. Growth on SC-Leu-Trp was used as a control. Negative (No. 1) and positive (No. 2–6) controls are shown. Positive interactions are shaded in gray. (C) Transition zone nuclei in control and lab-1(RNAi) gonads co-stained with AIR-2 (red) and DAPI (blue). (D) Transition zone nuclei in control and lab-1(RNAi) gonads co-stained with H3S10ph (red) and DAPI (blue). A quantification plot is presented below where each dot represents the ratio between the level of fluorescence detected for an individual nucleus and that of the adjacent background. Error bars represent standard deviation of the mean. Asterisk indicates statistically significant increase in fluorescence (p<0.0001, by the two-tailed Mann-Whitney test, 95% C.I.). (E) Transition zone nuclei in control and lab-1(RNAi) gonads co-stained with GSP-2 (red) and DAPI (blue). Bars, 4 µM.

Figure 7. LAB-1 utilizes a similar mechanism to protect SCC during two different meiotic stages.

In wild type, LAB-1 starts associating with the chromosomes at the entry into meiosis and transiently targets GSP-2 to the chromosomes. This targeting antagonizes AIR-2 and maintains SCC. During diakinesis, LAB-1 localizes to the long arms, where it specifically antagonizes AIR-2 and protects REC-8 from premature removal. When lab-1 is depleted, AIR-2 associates with the chromosomes as early as transition zone, and SCC is perturbed. The weakened SCC prevents successful binding of homologs and many reach diakinesis as either univalents or detached chromatids.