Carolyn M. Phillips

HIM-8 Binds to the X Chromosome Pairing Center and Mediates Chromosome-Specific Meiotic Synapsis

The him-8 gene is essential for proper meiotic segregation of the X chromosomes in C. elegans. Here we show that loss of him-8 function causes profound X chromosome-specific defects in homolog pairing and synapsis. him-8 encodes a C2H2 zinc-finger protein that is expressed during meiosis and concentrates at a site on the X chromosome known as the meiotic pairing center (PC). A role for HIM-8 in PC function is supported by genetic interactions between PC lesions and him-8 mutations. HIM-8 bound chromosome sites associate with the nuclear envelope (NE) throughout meiotic prophase. Surprisingly, a point mutation in him-8 that retains both chromosome binding and NE localization fails to stabilize pairing or promote synapsis. These observations indicate that stabilization of homolog pairing is an active process in which the tethering of chromosome sites to the NE may be necessary but is not sufficient.

Cytoskeletal forces span the nuclear envelope to coordinate meiotic chromosome pairing and synapsis

During meiosis, each chromosome must pair with its unique homologous partner, a process that usually culminates with the formation of the synaptonemal complex (SC). In the nematode Caenorhabditis elegans, special regions on each chromosome known as pairing centers are essential for both homologous pairing and synapsis. We report that during early meiosis, pairing centers establish transient connections to the cytoplasmic microtubule network. These connections through the intact nuclear envelope require the SUN/KASH domain protein pair SUN-1 and ZYG-12. Disruption of microtubules inhibits chromosome pairing, indicating that these connections promote interhomolog interactions. Dynein activity is essential to license formation of the SC once pairing has been accomplished, most likely by overcoming a barrier imposed by the chromosome-nuclear envelope connection. Our findings thus provide insight into how homolog pairing is accomplished in meiosis and into the mechanisms regulating synapsis so that it occurs selectively between homologs. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

Ethnic-Difference Markers for Use in Mapping by Admixture Linkage Disequilibrium

Mapping by admixture linkage disequilibrium (MALD) is a potentially powerful technique for the mapping of complex genetic diseases. The practical requirements of this method include (a) a set of markers spanning the genome that have large allele-frequency differences between the parental ethnicities contributing to the admixed population and (b) an understanding of the extent of admixture in the study population. To this end, a DNA-pooling technique was used to screen microsatellite and diallelic insertion/deletion markers for allele-frequency differences between putative representatives of the parental populations of the admixed Mexican American (MA) and African American (AA) populations. Markers with promising pooled differences were then confirmed by individual genotyping in both the parental and admixed populations. For the MA population, screening of >600 markers identified 151 ethnic-difference markers (EDMs) with delta>0.30 (where delta is the absolute value of each allele-frequency difference between two populations, summed over all marker alleles and divided by two) that are likely to be useful for MALD analysis. For the AA population, analysis of >400 markers identified 97 EDMs. In addition, individual genotyping of these markers in Pima Amerindians, Yavapai Amerindians, European American (EA) individuals, Africans from Zimbabwe, MA individuals, and AA individuals, as well as comparison to the CEPH genotyping set, suggests that the differences between subpopulations of an ethnicity are small for many markers with large interethnic differences. Estimates of admixture that are based on individual genotyping of these markers are consistent with a 60% EA:40% Amerindian contribution to MA populations and with a 20% EA:80% African contribution to AA populations. Taken together, these data suggest that EDMs with large interpopulation and small intrapopulation differences can be readily identified for MALD studies in both AA and MA populations.

Identification of chromosome sequence motifs that mediate meiotic pairing and synapsis in C. elegans.

Caenorhabditis elegans chromosomes contain specialized regions called pairing centres, which mediate homologous pairing and synapsis during meiosis. Four related proteins, ZIM-1, 2, 3 and HIM-8, associate with these sites and are required for their essential functions. Here we show that short sequence elements enriched in the corresponding chromosome regions selectively recruit these proteins in vivo. In vitro analysis using SELEX indicates that the binding specificity of each protein arises from a combination of two zinc fingers and an adjacent domain. Insertion of a cluster of recruiting motifs into a chromosome lacking its endogenous pairing centre is sufficient to restore homologous pairing, synapsis, crossover recombination and segregation. These findings help to illuminate how chromosome sites mediate essential aspects of meiotic chromosome dynamics.

PIWI associated siRNAs and piRNAs specifically require the Caenorhabditis elegans HEN1 ortholog henn-1.

Small RNAs—including piRNAs, miRNAs, and endogenous siRNAs—bind Argonaute proteins to form RNA silencing complexes that target coding genes, transposons, and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in Caenorhabditis elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the chromatin factors mes-4 and gfl-1, the Argonaute ergo-1, and the 3′ methyltransferase henn-1. Quantitative RT–PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans.

mut-16 and other mutator class genes modulate 22G and 26G siRNA pathways in Caenorhabditis elegans

Argonaute-associated siRNAs and Piwi-associated piRNAs have overlapping roles in silencing mobile genetic elements in animals. In Caenorhabditis elegans, mutator (mut) class genes mediate siRNA-guided repression of transposons as well as exogenous RNAi, but their roles in endogenous RNA silencing pathways are not well-understood. To characterize the endogenous small RNAs dependent on mut class genes, small RNA populations from a null allele of mut-16 as well as a regulatory mut-16(mg461) allele that disables only somatic RNAi were subjected to deep sequencing. Additionally, each of the mut class genes was tested for a requirement in 26G siRNA pathways. The results indicate that mut-16 is an essential factor in multiple endogenous germline and somatic siRNA pathways involving several distinct Argonautes and RNA-dependent RNA polymerases. The results also reveal essential roles for mut-2 and mut-7 in the ERGO-1 class 26G siRNA pathway and less critical roles for mut-8, mut-14, and mut-15. We show that transposons are hypersusceptible to mut-16–dependent silencing and identify a requirement for the siRNA machinery in piRNA biogenesis from Tc1 transposons. We also show that the soma-specific mut-16(mg461) mutant allele is present in multiple C. elegans laboratory strains.

MUT-16 promotes formation of perinuclear Mutator foci required for RNA silencing in the C. elegans germline

RNA silencing can be initiated by endogenous or exogenously delivered siRNAs. In Caenorhabditis elegans, RNA silencing guided by primary siRNAs is inefficient and therefore requires an siRNA amplification step involving RNA-dependent RNA polymerases (RdRPs). Many factors involved in RNA silencing localize to protein- and RNA-rich nuclear pore-associated P granules in the germline, where they are thought to surveil mRNAs as they exit the nucleus. Mutator class genes are required for siRNA-mediated RNA silencing in both germline and somatic cells, but their specific roles and relationship to other siRNA factors are unclear. Here we show that each of the six mutator proteins localizes to punctate foci at the periphery of germline nuclei. The Mutator foci are adjacent to P granules but are not dependent on core P-granule components or other RNAi pathway factors for their formation or stability. The glutamine/asparagine (Q/N)-rich protein MUT-16 is specifically required for the formation of a protein complex containing the mutator proteins, and in its absence, Mutator foci fail to form at the nuclear periphery. The RdRP RRF-1 colocalizes with MUT-16 at Mutator foci, suggesting a role for Mutator foci in siRNA amplification. Furthermore, we demonstrate that genes that yield high levels of siRNAs, indicative of multiple rounds of siRNA amplification, are disproportionally affected in mut-16 mutants compared with genes that yield low levels of siRNAs. We propose that the mutator proteins and RRF-1 constitute an RNA processing compartment required for siRNA amplification and RNA silencing.

Cytological Analysis of Meiosis in Caenorhabditis elegans

The nematode Caenorhabditis elegans has emerged as an informative experimental system for analysis of meiosis, in large part because of the advantageous physical organization of meiotic nuclei as a gradient of stages within the germline. Here we provide tools for detailed observational studies of cells within the worm gonad, including techniques for light and electron microscopy.

piRNAs and piRNA-Dependent siRNAs Protect Conserved and Essential C. elegans Genes from Misrouting into the RNAi Pathway

piRNAs silence foreign genes, such as transposons, to preserve genome integrity, but they also target endogenous mRNAs by mechanisms that are poorly understood. Caenorhabditis elegans piRNAs interact with both transposon and nontransposon mRNAs to initiate sustained silencing via the RNAi pathway. To assess the dysregulation of gene silencing caused by lack of piRNAs, we restored RNA silencing in RNAi-defective animals in the presence or absence of piRNAs. In the absence of piRNAs and a cellular memory of piRNA activity, essential and conserved genes are misrouted into the RNAi pathway to produce siRNAs that bind the nuclear Argonaute HRDE-1, resulting in dramatic defects in germ cell proliferation and function such that the animals are sterile. Inactivation of RNAi suppresses sterility, indicating that aberrant siRNAs produced in the absence of piRNAs target essential genes for silencing. Thus, by reanimating RNAi, we uncovered a role for piRNAs in protecting essential genes from RNA silencing.

MUT-14 and SMUT-1 DEAD Box RNA Helicases Have Overlapping Roles in Germline RNAi and Endogenous siRNA Formation

More than 2,000 C. elegans genes are targeted for RNA silencing by the mutator complex, a specialized small interfering RNA (siRNA) amplification module which is nucleated by the Q/N-rich protein MUT-16. The mutator complex localizes to Mutator foci adjacent to P granules at the nuclear periphery in germ cells. Here, we show that the DEAD box RNA helicase smut-1 functions redundantly in the mutator pathway with its paralog mut-14 during RNAi. Mutations in both smut-1 and mut-14 also cause widespread loss of endogenous siRNAs. The targets of mut-14 and smut-1 largely overlap with the targets of other mutator class genes; however, the mut-14 smut-1 double mutant and the mut-16 mutant display the most dramatic depletion of siRNAs, suggesting that they act at a similarly early step in siRNA formation. mut-14 and smut-1 are predominantly expressed in the germline and, unlike other mutator class genes, are specifically required for RNAi targeting germline genes. A catalytically inactive, dominant-negative missense mutant of MUT-14 is RNAi defective in vivo; however, mutator complexes containing the mutant protein retain the ability to synthesize siRNAs in vitro. The results point to a role for mut-14 and smut-1 in initiating siRNA amplification in germ cell Mutator foci, possibly through the recruitment or retention of target mRNAs.