<i>C</i>. <i>elegans</i> ZHP-4 is required at multiple distinct steps in the formation of crossovers and their transition to segregation competent chiasmata

Correct segregation of meiotic chromosomes depends on DNA crossovers (COs) between homologs that culminate into visible physical linkages called chiasmata. COs emerge from a larger population of joint molecules (JM), the remainder of which are repaired as noncrossovers (NCOs) to restore genomic integrity. We present evidence that the RNF212-like C. elegans protein ZHP-4 cooperates with its paralog ZHP-3 to enforce crossover formation at distinct steps during meiotic prophase: in the formation of early JMs and in transition of late CO intermediates into chiasmata. ZHP-3/4 localize to the synaptonemal complex (SC) co-dependently followed by their restriction to sites of designated COs. RING domain mutants revealed a critical function for ZHP-4 in localization of both proteins to the SC and for CO formation. While recombination initiates in zhp-4 mutants, they fail to appropriately acquire pro-crossover factors at abundant early JMs, indicating a function for ZHP-4 in an early step of the CO/NCO decision. At late pachytene stages, hypomorphic mutants exhibit significant levels of crossing over that are accompanied by defects in localization of pro-crossover RMH-1, MSH-5 and COSA-1 to designated crossover sites, and by the appearance of bivalents defective in chromosome remodelling required for segregation. These results reveal a ZHP-4 function at designated CO sites where it is required to stabilize pro-crossover factors at the late crossover intermediate, which in turn are required for the transition to a chiasma that is required for bivalent remodelling. Our study reveals an essential requirement for ZHP-4 in negotiating both the formation of COs and their ability to transition to structures capable of directing accurate chromosome segregation. We propose that ZHP-4 acts in concert with ZHP-3 to propel interhomolog JMs along the crossover pathway by stabilizing pro-CO factors that associate with early and late intermediates, thereby protecting designated crossovers as they transition into the chiasmata required for disjunction.减数分裂染色体的精准分离依赖于同源染色体间的DNA交叉互换（crossovers, COs），后者最终形成可见的物理联结——交叉结（chiasmata）。交叉互换起源于庞大的联合分子（joint molecules, JM）群体，其余联合分子则以非交叉互换（noncrossovers, NCOs）的途径完成修复，以维持基因组的完整性。本研究提供证据表明，秀丽隐杆线虫中类RNF212蛋白ZHP-4与其旁系同源蛋白ZHP-3协同作用，在减数分裂前期的不同阶段促进交叉互换的形成：一是早期联合分子的形成阶段，二是晚期交叉互换中间体向交叉结的转化阶段。ZHP-3与ZHP-4先以共依赖的方式定位至联会复合体（synaptonemal complex, SC），随后被局限于已指定的交叉互换位点。RING结构域突变实验证实，ZHP-4对于两种蛋白定位至联会复合体以及交叉互换的形成均发挥关键功能。尽管zhp-4突变体能够起始重组，但无法在大量早期联合分子处正常获取促交叉互换因子，这表明ZHP-4在交叉互换/非交叉互换的早期决策步骤中发挥作用。在粗线期晚期，功能减退突变体仍可检测到显著水平的染色体交叉互换，但同时存在促交叉互换因子RMH-1、MSH-5与COSA-1在已指定交叉互换位点的定位缺陷，且出现了染色体分离所需的染色体重塑缺陷的二价体。上述结果揭示了ZHP-4在已指定交叉互换位点的功能：它可稳定晚期交叉互换中间体中的促交叉互换因子，而这些因子是转化为二价体重塑所需的交叉结所必需的。本研究证实，ZHP-4在调控交叉互换的形成以及其转化为可指导精准染色体分离的结构两方面均具有不可或缺的作用。我们提出，ZHP-4与ZHP-3协同作用，通过稳定结合于早期与晚期中间体的促交叉互换因子，推动同源联合分子沿交叉互换通路进展，从而在已指定的交叉互换转化为分离所需的交叉结的过程中对其起到保护作用。