Robust designation of meiotic crossover sites by CDK-2 through phosphorylation of the MutSγ complex

Crossover formation is essential for proper segregation of homologous chromosomes during meiosis. Here, we show that Caenorhabditis elegans cyclin-dependent kinase 2 (CDK-2) partners with cyclin-like protein COSA-1 to promote crossover formation by promoting conversion of meiotic double-strand breaks into crossover–specific recombination intermediates. Further, we identify MutSγ component MSH-5 as a CDK-2 phosphorylation target. MSH-5 has a disordered C-terminal tail that contains 13 potential CDK phosphosites and is required to concentrate crossover–promoting proteins at recombination sites. Phosphorylation of the MSH-5 tail appears dispensable in a wild-type background, but when MutSγ activity is partially compromised, crossover formation and retention of COSA-1 at recombination sites are exquisitely sensitive to phosphosite loss. Our data support a model in which robustness of crossover designation reflects a positive feedback mechanism involving CDK-2–mediated phosphorylation and scaffold-like properties of the MSH5 C-terminal tail, features that combine to promote full recruitment and activity of crossover–promoting complexes.

Sexually reproducing organisms rely on proper chromosome segregation during meiosis to produce gametes with a complete genome. During meiotic prophase I, chromosomes pair and undergo crossover recombination with their homologous partners. This process, together with sister chromatid cohesion, leads to the formation of physical linkages between the homologs and enables their separation during meiosis I. Defects in crossover formation are disastrous, leading to miscarriages and congenital disorders, such as Down syndrome (1).Meiotic recombination initiates with the generation of programmed DNA double-strand breaks (DSBs) by the topoisomerase-like enzyme Spo11 (2). DSBs are resected to yield two 3′-end single-stranded DNA (ssDNA) overhangs, which are rapidly coated by RecA recombinases Dmc1 and Rad51. This nucleoprotein filament then seeks out homology and invades a homologous template, forming a metastable single-end invasion intermediate (D-loop) (3). The invading strand primes DNA synthesis and extends the D-loop. If the extended D-loop is captured by ssDNA on the other side of DSBs in a process known as second-end capture, a double Holliday junction (dHJ) forms (4). While dHJs can be resolved biochemically as either crossovers or non–crossovers (5), during meiosis, the majority of dHJs are specifically resolved as crossovers through the activity of MutLγ (MLH1-MLH3) (6–8) or other structure-selective endonucleases. Although a multitude of DSBs are generated during meiotic prophase, strikingly few are ultimately selected to become crossovers. Early recombination intermediates pare down in pachytene until each homolog pair receives at least one crossover, while the majority of DSBs are repaired as non–crossovers via synthesis-dependent strand annealing (9). However, how meiotic DSBs are chosen to become crossovers remains poorly understood.Throughout eukaryotes, crossover recombination is primarily controlled by a group of proteins collectively termed “ZMM” (10). Notably, homologs of the yeast RING (Really interesting new gene) domain protein Zip3 [ZHP-1, ZHP-2, ZHP-3, and ZHP-4 in Caenorhabditis elegans (11–14), Drosophila Vilya and Narya/Nenya (15, 16), Hei10 in Arabidopsis (17), and Hei10 and RNF212 in mammals (18, 19)] initially localize as abundant foci or long stretches along the synaptonemal complex (SC) but eventually concentrate at crossover sites in late pachytene (20). These SUMO or ubiquitin ligases appear to promote crossover designation by stabilizing the ZMM proteins at crossover sites while removing them from other recombination intermediates (13, 14, 18, 19, 21, 22). Although many meiotic proteins are shown to be SUMO modified (23), key targets of the Zip3 family proteins remain largely unknown.The meiosis-specific MutS homologs MSH4 and MSH5 form a heterodimeric MutSγ complex and play essential roles in crossover formation in diverse eukaryotes (24–31). MutSγ localizes to recombination intermediates as numerous foci but ultimately accumulates at sites that are destined to become crossovers (32, 33). Biochemical analyses using recombinant MSH4 and MSH5 have shown that MutSγ recognizes single-end invasion intermediates and HJs in vitro (34, 35). HJs activate the ATP hydrolysis of MutSγ and promote the exchange of bound ADP for ATP, inducing the formation of a sliding clamp that dissociates from HJs (35, 36). By iterative loading and embracing DNA duplexes within a dHJ, MutSγ is thought to stabilize crossover–specific recombination intermediates (33, 35). In addition, MutSγ recruits and activates the resolvase activity of MutLγ, enabling biased processing of dHJs into crossovers during meiosis (37, 38).A genetic screen in C. elegans identified a cyclin-like protein COSA-1 as a component essential for processing meiotic DSBs into crossovers (39). The mammalian ortholog CNTD1 was subsequently identified (40), and both COSA-1 and CNTD1 have been shown to localize to crossover sites (39, 41, 42). In the absence of COSA-1/CNTD1, MutSγ components persist as numerous foci in pachytene, and crossover formation is eliminated or severely compromised (33, 40), demonstrating a crucial role of COSA-1/CNTD1 in converting early recombination intermediates into crossovers. Because both COSA-1 and CNTD1 are members of the cyclin family, it is plausible that they form a complex with a cyclin-dependent kinase (CDK) and regulate the recombination process through phosphorylation.Several lines of evidence have suggested that CDK2 might be a relevant kinase partner for CNTD1. CDK2 interacts with CNTD1 in yeast-two hybrid assays (41, 42) and localizes to interstitial chromosome sites (43, 44) in a CNTD1-dependent manner (40). Reduced CDK2 activity leads to a failure in crossover formation, while a hyperactive form of CDK2 causes an increased number of MLH1 foci (45). However, due to its requirement at telomeres in tethering chromosomes to the nuclear envelope, deletion of CDK2 leads to severe defects in SC assembly between paired homologs (synapsis) and pachytene arrest (46–49). Further, while a full-length CNTD1-specific protein of the excepted size was detected (using CNTD1 antibodies) in one study (42), a short CNTD1 isoform that cannot interact with CDK2 was the predominant isoform detected in another study (using hemagglutinin antibodies in Cntd1^FH/FH mice with an epitope tag sequence inserted into the endogenous Cntd1 locus) (41), raising questions regarding the extent to which CDK2 and CNTD1 might act as functional partners. Thus, it has been difficult to determine the role of CDK2 in crossover recombination. Moreover, key meiotic targets of CDK2 have not yet been identified.We reasoned that CDK-2, the C. elegans homolog of CDK2, might also localize and function at crossover sites. However, global knockdown of C. elegans CDK-2 by RNA interference leads to cell cycle arrest of mitotically proliferating germ cells (50), thereby precluding the analysis of its requirement during meiotic prophase. To overcome this limitation and establish the meiotic function of CDK-2, we use the auxin-inducible degradation system to deplete CDK-2 from the adult germline, demonstrating that CDK-2 partners with COSA-1 to promote crossover formation during C. elegans meiosis. Moreover, we identify MSH-5 as a key substrate for CDK-2 and provide evidence that CDK-2 and COSA-1 partner to promote crossover designation through phosphorylation and activation of the MutSγ complex.     

The utility of peripheral thyrotropin mRNA in the diagnosis of follicular neoplasms and surveillance of thyroid cancers

BACKGROUND: Thyroid cells in peripheral circulation express uniquely thyrotropin receptor (TSHR) mRNA, and their detection may aid thyroid cancer management. METHODS: Since 2002, 258 patients had prospective TSHR mRNA measurement by quantitative RT-PCR from peripheral blood before and/or after thyroidectomy. Thyroid cancer detection was assessed from known clinical diagnostic criteria and mRNA for patients with follicular neoplasms (n = 64) and long-term cancer follow-up (n = 13). RESULTS: Adding TSHR mRNA to fine-needle aspiration biopsy (FNAB) maintained high sensitivity (90%) but improved specificity (73%) for thyroid cancer diagnosis. When FNAB specimens indicated follicular neoplasm, a decision algorithm combining TSHR mRNA and abnormal thyroid ultrasound features correctly diagnosed all cancer patients (100% sensitivity) and would have spared operation for benign disease in 38%. Elevated TSHR mRNA on postoperative day 1 predicted persistent/recurrent cancer. During long-term thyroid cancer surveillance, TSHR mRNA had a 91% concordance with radioactive iodine whole body scan (WBS)-detectable disease, agreed with thyroglobulin (Tg) levels in 64% of patents, missed disease in 5%, but was more sensitive to detecting disease than Tg levels in 31% of patients, including all patients with Tg antibodies. CONCLUSIONS: Detecting circulating thyroid cancer cells is useful for initial thyroid cancer diagnosis and postoperatively predicts recurrent cancer. This novel test promises to enhance thyroid cancer patient care by management algorithms that combine histologic, genomic, and clinical criteria.     

Secondary developmental glaucoma in eyes with congenital aphakia

Purpose:To describe the clinical spectrum and management of glaucoma in congenital aphakia.Methods:The demographics and clinical spectrum of eyes with congenital aphakia with and without glaucoma were compared, and management outcomes of congenital aphakia cases with glaucoma were studied retrospectively between April 2000 and June 2020.Results:There were a total of 168 eyes (84 subjects) with a diagnosis of congenital aphakia, of which 29 eyes of 18 subjects were diagnosed with glaucoma. Corneal opacity was the presenting complaint in 26/29 eyes with glaucoma and 139/139 eyes without glaucoma. The (interquartile range (IQR)) horizontal corneal diameter was 10.5mm (IQR, 9.0-12.5) and 8mm (IQR, 5-10) in eyes with and without glaucoma (P = 0.01), respectively. The median (IQR) axial length was 17.5mm (IQR, 13.5-19.5) and 15mm (IQR, 14-16) mm in eyes with and without glaucoma (P = 0.03), respectively. Nineteen eyes with glaucoma had adequate intraocular pressure (IOP) control with one medication. Three eyes underwent transscleral diode cyclophotocoagulation and maintained IOP without medications. Three eyes underwent trabeculectomy and trabeculotomy, trabeculectomy followed by penetrating keratoplasty, and trabeculectomy, respectively, of which two eyes became phthisical. At the last follow-up, the median (IQR) IOP was 14 mm Hg (IQR, 14-17) Hg. The median (IQR) follow-up duration was 4.53 months (IQR, 2.03- 48.06).Conclusion:One-fifth of the eyes with congenital aphakia had secondary developmental glaucoma. The corneal diameter and axial lengths were higher in the eyes with glaucoma compared to eyes without glaucoma. Medical management is the preferred short-term mode of IOP control. Transscleral cyclophotocoagulation may be preferred over surgical intervention.     

Sex,gender, and retinoblastoma: analysis of 4351 patients from 153 countries

ObjectiveTo investigate in a large global sample of patients with retinoblastoma whether sex predilection exists for this childhood eye cancer.MethodsA cross-sectional analysis including 4351 treatment-naive retinoblastoma patients from 153 countries who presented to 278 treatment centers across the world in 2017. The sex ratio (male/female) in the sample was compared to the sex ratio at birth by means of a two-sided proportions test at global level, country economic grouping, continent, and for selected countries.ResultsFor the entire sample, the mean retinoblastoma sex ratio, 1.20, was higher than the weighted global sex ratio at birth, 1.07 (p < 0.001). Analysis at economic grouping, continent, and country-level demonstrated differences in the sex ratio in the sample compared to the ratio at birth in lower-middle-income countries (n = 1940), 1.23 vs. 1.07 (p = 0.019); Asia (n = 2276), 1.28 vs. 1.06 (p < 0.001); and India (n = 558), 1.52 vs. 1.11 (p = 0.008). Sensitivity analysis, excluding data from India, showed that differences remained significant for the remaining sample (χ² = 6.925, corrected p = 0.025) and for Asia (χ² = 5.084, corrected p = 0.036). Excluding data from Asia, differences for the remaining sample were nonsignificant (χ² = 2.205, p = 0.14).ConclusionsNo proof of sex predilection in retinoblastoma was found in the present study, which is estimated to include over half of new retinoblastoma patients worldwide in 2017. A high male to female ratio in Asian countries, India in specific, which may have had an impact on global-level analysis, is likely due to gender discrimination in access to care in these countries, rather than a biological difference between sexes.Subject terms: Eye cancer, Risk factors     

A short-term chick embryo in vivo xenograft model to study retinoblastoma cancer stem cells

Purpose:Cancer stem cells (CSCs) reported in various tumors play a crucial role in tumorigenesis and metastasis of retinoblastoma (Rb). Following the efforts to reduce, replace, and refine the use of mammalian models, we aimed to establish a short-term xenograft for Rb to evaluate the CSC properties of CD133^- Rb Y79 cells, using the well-established chick embryo chorioallantoic membrane (CE-CAM) assay.Methods:Y79 cells were cultured, labeled with two different dyes (CM-Dil Y79 and enhanced green fluorescent protein (eGFP)) and sorted for CD133^- and CD133 + subsets. Two million cells from each of the labeled groups were transplanted onto the abraded CAM on embryonic day 7 (E7). On E14, the tumor nodule formation on CAM and spontaneous metastasis to the embryos were evaluated by confocal microscopy, in vivo imaging, and histology.Results:Y79 cells formed pink–white raised perivascular nodules with feeder vessels on the CAM with both the types of labeled CD133^- cells. CD133^- cells, when compared to CD133 + cells, demonstrated significantly larger tumor volume (40.45 ± 7.744 mm³ vs 3.478 ± 0.69 mm³, P = 0.0014) and higher fluorescence intensity (CM-Dil: AUF = 6.37 × 10⁷ ± 7.7 × 10⁶ vs 1.08 × 10⁷ ± 1.6 × 10⁶; P < 0.0001; eGFP: AUF = 13.94 × 10⁴ ± 2.54 × 10⁴ vs AUF = 1.39 × 10⁴ ± 0.4 × 10⁴; P = 0.0003). The metastatic potential of CD133^- cells was also observed to be higher as noted by in vivo imaging and histopathology.Conclusion:This study highlights that CE-CAM is a feasible alternative nonmammalian model for evaluating tumorigenicity and metastatic potential of Y79 CSCs. Increased tumorigenicity and metastatic potential of CD133^- subset of tumor cells substantiate their CSC properties.