Release of CHK-2 from PPM-1.D anchorage schedules meiotic entry

Abstract

Transition from the stem/progenitor cell fate to meiosis is mediated by several redundant posttranscriptional regulatory pathways in Caenorhabditis elegans. Interfering with all three branches causes tumorous germ lines. SCF^PROM-1 comprises one branch and mediates a scheduled degradation step at entry into meiosis. prom-1 mutants show defects in the timely initiation of meiotic prophase I events, resulting in high rates of embryonic lethality. Here, we identify the phosphatase PPM-1.D/Wip1 as crucial substrate for PROM-1. We report that PPM-1.D antagonizes CHK-2 kinase, a key regulator for meiotic prophase initiation, including DNA double-strand breaks, chromosome pairing, and synaptonemal complex formation. We propose that PPM-1.D controls the amount of active CHK-2 via both catalytic and noncatalytic activities; notably, noncatalytic regulation seems to be crucial at meiotic entry. PPM-1.D sequesters CHK-2 at the nuclear periphery, and programmed SCF^PROM-1-mediated degradation of PPM-1.D liberates the kinase and promotes meiotic entry.

Fig. 1.. Loss of SCF^PROM-1 activity at meiotic entry is rescued by mutating ppm-1.D.

(A) Schematic diagram of the SCF^PROM-1 complex. (B) Left: Immunodetection of WAPL-1 (cyan) and PROM-1::HA (magenta) in the progenitor zone, at the distal end of the C. elegans germ line. Arrows mark the entry into meiosis, which occurs at the leptonema-zygonema. Scale bar, 5 μm. Right: Normalized levels of PROM-1::HA (magenta) throughout the progenitor cell zone, measured as cell diameters from the distal tip; the end of the progenitor zone (cyan) is marked. Error bars, SD. DAPI, 4′,6-diamidino-2-phenylindole; a.u., arbitrary units. (C) Gonads displaying prophase I for the indicated genotypes. Scale bar, 10 μm. Boxed insets show representative diakinesis chromosomes. (D) Insets showing staining for HTP-3 (magenta), SYP-1 (cyan), and REC-8 (yellow) for the depicted zones. Scale bar, 10 μm.

Fig. 2.. PPM-1.D is a conserved PP2C phosphatase and its expression is controlled by SCF^PROM-1.

(A) Phylogenetic tree of PPM-1.D. (B) Gene structure of ppm-1.D, with domains, exons/introns, and alleles depicted (top) and alignment of PPM-1.D protein sequences (bottom; amino acid range, 498 to 530) for selected organisms to highlight conservation of the PP2C domain. Asterisk marks the conserved aspartic acid necessary for phosphatase activity. (C) Immunodetection of PPM-1.D::HA (yellow) and SUN-1 (magenta) in the progenitor zone (top) and at diplonema (middle) and diakinesis (bottom). Scale bars, 5 μm. (D) Dissected gonads stained for DAPI (top) and PPM-1.D::HA (bottom) in the wild type (left) and prom-1 mutant (right). Scale bar, 10 μm. (E) Left: Western blot of trichloroacetic acid (TCA)–precipitated proteins from yeast expressing PPM-1.D::LexA, PROM-1::HA in the absence or presence of the proteasome inhibitor, MG132. For full photographs of the blots, see fig. S5A. Right: Quantification of PPM-1.D::LexA in Western blots (n = 2) normalized to the level of PPM-1.D::LexA when both PROM-1 and PPM-1.D are expressed.

Fig. 3.. PPM-1.D and CHK-2 colocalize in the progenitor zone and interact physically.

(A) Western blot of cellular fractions (cytosolic, soluble nuclear, and insoluble nuclear) with the specified antibodies for the indicated genotypes. For full photographs of the blots, see fig. S5B. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Stimulated emission depletion (STED)–visualized immunostaining of CHK-2::3×FLAG (magenta) and PPM-1.D::HA (yellow; top); straightened profiles of the signals (bottom). tM, automatic threshold Manders colocalization coefficient. Scale bar, 5 μm. (C) Left: Raw electron microscopy image of one nucleopore with gold particles indicating CHK-2. Right: With annotated nuclear membranes (cyan) and CHK-2 (magenta). Scale bar, 10 nm. (D) Left: Raw electron microscopy image of one mitotic nucleus with gold particles indicating CHK-2. Right: With annotated nuclear membranes (cyan) and CHK-2 (magenta). Scale bar, 100 nm. (E) Scheme used to divide the three nuclear zones of equal area (zones 1 to 3) and the outer vicinity of the nucleus (zone 0). (F) Distribution of CHK-2 gold particles in the four different zones. (G) Top: Western blot analysis after amylose purification of the indicated proteins expressed in E. coli. For full photographs of the blots, see fig. S5C. (H) Quantification of the FLAG signal (CHK-2) normalized to the HIS signal for the indicated colysed samples (n = 2 Western blots).

Fig. 4.. Regulation of CHK-2 localization and activity by PPM-1.D.

(A) Gene structure of ppm-1.D, showing the domain and exon/intron structure and alleles (top left), and genotypes suppressing the prom-1 phenotype. DAPI staining (white) and HA immunostaining (yellow) in the progenitor zone for the indicated genotypes. The jf182[D274A] allele encodes catalytically inactive PPM-1.D. Scale bar, 5 μm. (B) Left: DAPI staining and HA immunodetection (cyan) in the progenitor zone for the indicated genotypes. Scale bar, 5 μm. Right: Average line profile analysis of HA signal intensity centered on the nucleus for the indicated genotypes (n = 25 nuclei from the progenitor zone). (C) Left: RNA quantification for ppm-1.D for the indicated genotypes. Data for the wild type are the same as in fig. S3A. Center: Western blot analysis of HA and histone H3 in whole-worm extracts for the indicated genotypes. For full photographs of the blots, see fig. S5D. Right: Quantification of the ratio of the HA signal intensity to histone H3 signal intensity for the indicated genotypes. n.s., not significant. (D) Left: DAPI staining and immunostaining of PPM-1.D::HA (cyan) and SUN-1(S8Pi) (magenta) in the distal tip for the indicated genotypes, showing the average line profile analysis of HA signal intensity centered on the nucleus for the indicated genotypes (n = 25 nuclei from the mitotic zone). Right: Number of cell rows before entry into meiotic prophase for the indicated genotypes. Scale bar, 10 μm. (E) DAPI staining and immunodetection of SUN-1(S8Pi) (magenta), FLAG (yellow), and HA (cyan) at transition from the progenitor zone to entry into leptonema-zygonema (at around 20 cell rows from the distal tip cell) for the indicated genotypes. Scale bar, 5 μm. Right: Average line profile analysis of HA signal intensity centered on the nucleus for the indicated genotypes (n = 25 nuclei from the mitotic zone).

Fig. 5.. Premature meiotic entry in ppm-1.D mutants.

(A) Top: Immunostaining of CYE-1 (magenta) and SUN-1(S8Pi) (green) in the progenitor zone for the indicated genotypes. Scale bar, 10 μm. Bottom: Distribution of the overlap between CYE-1 and SUN-1(S8Pi) staining in cell diameters, for the genotypes shown. **P < 0.01 and ****P < 0.0001 for the Mann-Whitney test. (B) Left: EdU incorporation into replicating DNA (yellow) and SUN-1(S8Pi) staining (magenta) for the indicated genotypes. Blue arrows in the inset highlight nuclei with both significant EdU incorporation (indicating ongoing meiotic S phase) and SUN-1(S8Pi) staining (indicating CHK-2 activity and meiotic entry). Scale bar, 10 μm. Right: Quantification of double-positive nuclei for the indicated genotypes. ****P < 0.0001 for the Mann-Whitney test. (C) Left: DAPI staining and immunostaining of HIM-3 (yellow) and SUN-1(S8Pi) (magenta). Scale bar, 5 μm. Right: The cell row number at which HIM-3 and SUN-1(S8Pi) appear in the germ line, for the indicated genotypes. Cell rows were counted as positive when more than half of the cells showed positive staining. **P < 0.01. (D) Top: Immunostaining of HA in gld-1(q485) gld-2(q497); ppm-1.D::ha mutant worms. Scale bar, 50 μm. Insets show a higher magnification for nuclei stained with DAPI (white) and for HA (yellow), HIM-3 (cyan), and pHIM-8/ZIMs (magenta) in the boxed zone in the top picture. Scale bar, 5 μm. (E) Dissected gonads were divided into six zones of equal length. The percentage of nuclei with a paired FISH signal (5S probes on chromosome V) in each zone for the indicated genotypes. *P < 0.05 and ****P < 0.0001 for the Fisher’s exact test. (F) Percentage of nuclei with the specified numbers of RAD-51 foci in each zone for the indicated genotypes. P values for the Fisher’s exact test are shown in table S5.

Fig. 6.. PPM-1.D functions in the DNA damage response.

Quantification of apoptotic corpses (scatter and means ± SD) for the indicated genotypes. ***P < 0.001 and ****P < 0.0001 for the Mann-Whitney test. Diagonal matrix with P values for Mann-Whitney test for all genotypes is in table S6.

Fig. 7.. Model of control of meiotic entry by PPM-1.D.

Entry of PPM-1.D into the nucleus is mediated by nucleopores in the progenitor zone. Inside the nucleus, the C terminus of PPM-1.D interacts directly with CHK-2 and inhibits CHK-2 by both sequestering it at the nuclear periphery and dephosphorylation. At meiotic entry, SCF^PROM-1 degrades PPM-1.D. After the scheduled degradation of PPM-1.D, CHK-2 is released from the nuclear periphery and gains access to its substrates, thereby launching the initial events of meiotic prophase. C-term, C terminus; ONM, Outer nuclear membrane; INM, Inner nuclear membrane; NPC, Nucleo Pore Complex; SUN, Sad-1 and UNC-84; KASH, Klarsicht; ANC-1, SYNE homology.