Yanowitz, Judith et al. (2009) International Worm Meeting "him-5 regulates meiotic break formation."

Favani, Nathan, Yanowitz, Judith, Wagner, Cynthia, Heinis, Frazer, Meneely, Philip, DiRienzi, Sara

[International Worm Meeting, 2009]

Chromosome segregation in meiosis requires the proper number and position of crossovers. Yet chromosomes show characteristic distributions and numbers of crossover events suggesting that chromosome-specific factors are shaping the crossover landscape. A growing body of evidence implicates chromatin structure as a major determinant of crossover distribution. Nevertheless, the genes involved in this process have remained largely unknown. Many meiotic mutants, known as precondition mutants, alter the crossover landscape. It is likely that analysis of these mutants will reveal the underlying state of chromatin along the chromosomes. We have been analyzing the C. elegans precondition mutant him-5. him-5 was identified in Hodgkin''s original HIM screen and was shown to have strong specificity for the X chromosome. We have analyzed his alleles e1467 and e1490, as well as a null allele, ok1896. All three mutants cause an increase in male self-progeny ranging from 20-40% caused by a failure of recombination on the X chromosomes. In addition, all three alleles show a slight increase in autosomal nondisjunction, with age-related changes in the spectrum of severity. We show that him-5 is defective in making the double strand breaks that initiate recombination. We can completely rescue the X and autosomal nondisjunction phenotypes by creating breaks with ionizing radiation. In the mutant, chromosomes are completely paired and synapsed, but they prematurely disassemble the synaptonemal complex on the X chromosome, a phenotype that is also rescued by irradiation. These results are striking because it was previously thought that chromosome-specific meiotic functions were confined to homologue recognition and pairing. Together with our work on xnd-1 (which has a similar phenotype), we have defined a novel step at which chromosome-specific identities are essential for proper disjunction. him-5 corresponds to D1086.4 and encodes a protein with no known homologues outside of C. elegans. Two-hybrid analysis suggests that HIM-5 acts in a protein complex but the precise function of this complex is unknown. HIM-5 and XND-1 act independently of one another suggesting that further analysis of HIM-5 will elucidate novel aspects of chromosome-specific crossover control.

Judith Yanowitz (2007) International Worm Meeting "Genome stability and telomere maintenance are regulated by him-15/rfs-1."

Judith Yanowitz

[International Worm Meeting, 2007]

I have identified him-15 as the rad-51 paralogue gene, rfs-1. rfs-1 has a 2% HIM phenotype and is sensitive to ionizing radiation. rfs-1 displays a classic MRT phenotype with populations of worms going sterile after 20 passages from starved plates, with evidence of telomere shortening, and with end-to-end fusions in the generation preceding sterility. However, rfs-1 has several characteristics that make it a unique MRT gene and have important implications for telomere maintenance. Specifically, although shorter telomeres can be seen in rfs-1 mutants, lengthening events are also observed. Similarly, rfs-1 can enhance the MRT phenotype of telomerase mutant, trt-1; but it also partially suppresses the phenotype. Consistent with these results, rfs-1 trt-1 double mutants show a slower rate of telomere attrition than the trt-1 signle mutants. The rfs-1 trt-1 "survivors" have stabilized karyotypes with fewer chromosomes- in one case, with only 2 chromosomes observed in all diakinetic nuclei. These results suggest that in rfs-1 mutants, an alternative pathway for telomere regulation has been uncovered. Simon Boulton''s lab has shown that rfs-1 regulates replication fork progression through stalled forks. My results are consistent with a model in which replication fork progression is the sole function of rfs-1. Defects in replicating telomeric repeats lead to stalled forks that need to be repaired by recombination, leading to both lengthening and shortening events. Rescue in the rfs-1 trt-1 double mutant can result from interchromosomal recombination. Additional genomic instability arises when telomeres recombine with internal telomeric repeats leading to inversions, deletions, and traslocations or when replication of other repetitive elements leads to stalling and subsequent recombination repair.

Judith Yanowitz et al. (2001) International C. elegans Meeting "Genetic Analysis of Coelomocyte Specification and Patterning"

Judith Yanowitz, Andrew Fire, Ed Hedgecock

[International C. elegans Meeting, 2001]

We are interested in studying the patterning of mesodermal lineages during late embryogenesis in C. elegans. To this end, we are examining the processes which affect the specification of the coelomocytes. Coelomocytes are highly endocytic mesodermal cells that reside in the pseudocoelomic space. Four of the six coelomocytes arise late in embryogenesis from MS progeny; the other two arise from the post-embryonic divisions of the M mesoblast. The lineages and timing of coelomocyte specification present several questions about cell fate decisions. In particular, we are interested in understanding how cells choose between muscle and non-muscle fates, how mesodermal cell fate choices are linked to the cell cycle, and how two unqiue lineages can give rise to cells with the same terminal phenotypes. W have screened for defects in coelomocyte specification by assaying for changes in tissue-specific GFP expression. We have isolated over 20 mutations and are in the process of characterizing three of these in more detail. The first mutant may be a cell cycle mutant which prevents the coelomocyte mother from dividing. The second appears to be a lineage mutant affecting only the pair of coelomocytes from the MSpp blastomere. The third mutant gives a variable reduction in coelomocyte number and has an associated withered tail defect. Further genetic and phenotypic characterization of these mutants will be presented. During the course of these analyses, we observed that unc-39 mutants also have coelomocyte specification defects. We have set out to clone the unc-39 gene, and progress on its cloning and characterization will be presented.

Judith Yanowitz et al. (2003) International Worm Meeting "UNC-39, a Six-Class Homeodomain Transcription Factor, Affects Mesoderm Development"

Andrew Fire, Erik Lundquist, Judith Yanowitz, Ed Hedgecock, Afaq Shakir

[International Worm Meeting, 2003]

The unc-39 gene has previously been shown to have pleiotropic effects with uncoordinated, egg-laying defective, and withered tail phenotypes. At the cellular level, unc-39 causes defects in neuronal (CAN) and mesodermal cell (M and coelomocyte) migrations, as well as misspecification of the mesodermal cell hmcR to a Z5/ somatic gonad fate. We have extended the characterization of the mesodermal defects and show that unc-39 affects the specification of most mesodermal subtypes. unc-39 mutants can have aberrant numbers of coelomocytes, body wall muscles, sex myoblasts, vulval muscles, and head mesodermal cells. Each of these defects is partially penetrant and does not appear to co-segregate, suggesting that the effects of unc-39 are variable and cell autonomous. unc-39 is unique in C. elegans because it affects the specification of a broad range of mesodermal tissues but does not appear to act as a mesodermal identity gene. One possibility for the role of unc-39 is as a regulator of choice between migration and differentiation. Consistent with this idea, all of the specification defects seen in unc-39 mutants are in cells which undergo migration prior to differentiation. The accompanying abstract by Yanowitz et al. describes the cloning and developmental expression pattern of unc-39. UNC-39 belongs to the Six family of homeodomain transcription factors and is most similar to human Six5. hSix5 is implicated in myotonic dystrophy type I (DMI) as patients with this disease have reduced levels of Six5 protein. DMI is a multisystemic disorder with widely variable clinical presentation. Patients can exhibit any a variety of defects including myotonia, cataracts, gonadal atrophy, cardiac conduction defects, and mental impairment. Although the Six5 and UNC-39 proteins are only around 35% identical in the Six and homeodomains, the similar phenotypic spectrum with respect to mesodermal and neuronal differentiation suggested that the proteins might be functional homologues. To test this hypothesis, we swapped the conserved domains from the human protein into a full-length unc-39::GFP construct and assayed for rescue of the unc, egl, and coelomocyte specification defects. Each of these defects is at least partially rescued by the fusion construct. This result suggests that UNC-39 and hSix5 share DNA binding specificity and possibly protein:protein interaction specificity. Further analysis of upstream regulators and downstream targets of unc-39 is underway and may be valuable for understanding the nature of DMI.

Judith L Yanowitz et al. (2003) International Worm Meeting "Genetic Analysis of Coelomocyte Specification"

Andrew Z Fire, Judith L Yanowitz

[International Worm Meeting, 2003]

We are interested in understanding the molecular pathways which regulate mesodermal fate specification. In C. elegans there are a small number of mesodermal tissues: striated muscles of the body wall; non-striated muscles of the pharynx, uterus, vulva and enteric system; the somatic component of the gonad (Z1 and Z4, and their descendants); six coelomocytes; six GLRs; and the two head mesodermal cells. With the exception of some body wall and pharyngeal muscle, all mesodermal tissues arise from divisions of the MS blast cell. We are focusing on the coelomocytes of C.elegans as our model system. Coelomocytes are highly endocytic cells that reside in the pseudocoelomic space. Four of the six coelomocytes arise late in embryogenesis from MS progeny; the other two arise from the post-embryonic divisions of the M mesoblast. We are taking a two pronged approach to understanding coelomocyte specification. First, we have screened EMS- generated alleles and candidate genes for changes in the number of coelomocytes by assaying for changes in tissue-specific GFP expression. We have isolated over 20 mutations. These fall into several phenotypic categories based on (1) which cells are affected, (2) the number of cells, and (3) the timing of changes in cell number. We have isolated a novel allele of cyclin D, cyd-1(cc600) which is homozygous viable and fertile and appears to only be defective in the coelomocyte mother cell division. Another mutation gives a lineage specific defect, the loss of the MSpp derived coelomocytes, and also have defects in distal tip cell migration. A third mutation that we have been characterizing gives a variable decrease in coelomocyte number and appears to have additional pleiotropic effects. The second approach we are using is promoter and enhancer bashing. Using two coelomocyte specific reporters, we have been able to identify 70bp elements that are sufficient for coelomocyte-specific expression. We are in the process of determining whether specific motifs within these fragments are critical for expression and hope to use this information to identify additional coelomocyte specific genes.

Judith Yanowitz et al. (2003) International Worm Meeting "UNC-39 is Similar to the Six-Class Homeodomain Transcription Factor Six5 and Affects Anterior Neuronal Development"

Ed Hedgecock, Erik Lundquist, Judith Yanowitz, Andrew Fire, Afaq Shakir

[International Worm Meeting, 2003]

unc-39 mutants were previously shown to have defects in CAN cell migration and mesodermal specification (i.e. a third somatic gonad precursor dubbed Z5). The accompanying abstract by Yanowitz et al. describes mesodermal defects in unc-39 mutants. Here we describe that unc-39 mutations also cause axon pathfinding defects of neurons born in or that reside in the anterior region of the animal. Mid-body and posterior neurons were unaffected by unc-39, suggesting that unc-39 might affect the development of anterior-derived neurons specifically.We defined a genetic and physical interval that includes unc-39 and contains the predicted gene ceh-35, which can encode a Six-class homeodomain transcription factor similar to human Six5. We reasoned that unc-39 mutations might affect ceh-35, as Six-class molecules have been shown to affect anterior ectodermal development in many species. Indeed, ceh-35 DNA rescued unc-39 mutants, ceh-35 RNAi mimicked the unc-39 phenotype, and lesions in the ceh-35 gene were associated with each of the three unc-39 alleles. A full-length unc-39::gfp fusion construct rescued unc-39 mutants. Expression of this fusion was first seen at gastrulation in 10-12 anterior nuclei whose positions were consistent with anterior AB and/or MS descendants. Expression in anterior nuclei (likely neuroblasts and neurons) continued to the 2-fold stage. At 240 minutes, expression was observed in posterior nuclei likely to be mesodermal cells, including gonad precursors Z1 and Z4 and M and possibly the embryonic coelomocytes and muIntR. Expression in Z1 and Z4 persisted into the L1 larva. The unc-39 promoter also drove gfp expression in the CAN neurons and in all body wall muscle cells beginning in late embryogenesis.Type I myotonic dystrophy (DM1) is an inherited disease that mighe be due to perturbation of the human Six5 gene. DM1 is characterized by anterior ectoderm defects (e.g. mental impairment and cataracts) and mesoderm defects (e.g. gonad dysgenesis and fragile musculature). The similar phenotypic spectrum of DM1 in humans and unc-39 in worms suggests that unc-39 might serve as a useful genetic model for DM1.

Leydig, Zachary et al. (2021) International Worm Meeting "An Exploration of the protein FIGL-1 in the Caenorhabditis elegans Germline and Insights into its role in Homologous Recombination"

Yanowitz, Judith, Leydig, Zachary

[International Worm Meeting, 2021]

Repair of damaged DNA is crucial for the survival of both the individual organism and species. Double-strand breaks (DSB) are the most cytotoxic lesions. Homologous recombination (HR) is a high fidelity DSB repair pathway that relies on an undamaged homologous template. HR is especially important during meiosis prophase I, as it is a pathway that can repair programmed DSBs as crossovers (COs). CO homeostasis is a process that prevents either too few or too many COs between homologous chromosomes and without it, genome instability may ensue. In part, CO homeostasis is achieved by the mutual antagonism between HR/CO-promoting and HR/CO-inhibiting factors. The recombinase RAD-51 is central to HR as it coats the ssDNA filament to promote strand exchange. The anti-recombinase, FIGL-1 (Fidgetin-like protein 1), was shown in humans, mice, and plants to antagonize HR by directly interacting with RAD-51. Studies of how figl-1 functions in the germ line of Caenorhabditis elegans have been lacking in part because knockout of figl-1 is embryonic lethal. To determine if the interactions between RAD-51 and FIGL-1 are conserved in worms, I have been performing a yeast two-hybrid (Y2H) with the full-length proteins. If an interaction exists, interaction motifs will be mapped through a series of amino acid deletions and substitutions. It will be particularly interesting to determine whether the partially conserved FIGL1-RAD51 binding domain (FRBD) functions similarly in figl-1. CRISPR-Cas9 genome editing will be employed to introduce the mapped mutations into the figl-1 locus to observe the phenotypic effects in vivo. Additionally, plans to introduce targeted missense mutations into the highly conserved Walker A and B motifs, which are known to be important for ATP hydrolysis in FIGL-1, are underway. Interestingly, previous results have suggested that FIGL-1's ATP hydrolysis activity is independent of its anti-recombinase activity, suggesting that these mutations may result in separation-of-function mutants. Through these studies, specific domains within worm figl-1 will be scrutinized to shed more light on its function in the germ line as well its role in HR.

Rana, Mainpal et al. (2013) International Worm Meeting "Multiple Aspects of C. elegans Germ Cell Development are Regulated by XND-1."

Rana, Mainpal, Yanowitz, Judith

[International Worm Meeting, 2013]

Despite the central importance of germ cells for the transmission of genetic material between generations, very little is known about the molecular programs that regulate their development. Defects during germ cell formation and differentiation can lead to infertility, birth defects, and formation of germ cell cancers. Work in our lab has identified xnd-1 (X nondisjunction factor-1) as a key regulator of germ cell development in the nematode, Caenorhabditis elegans. Our analysis has revealed that xnd-1 is one of the first genes turned on in the primordial germ cells (PGC) suggesting it may be a key regulator of PGC development. Consistent with this hypothesis, xnd-1 mutant embryos exhibit multiple defects in PGC development including aberrant PGCs number and size, mislocalization, premature proliferation and missegregation of P-granules. Furthermore, co-depletion of XND-1 and NOS-2, a C. elegans Nanos homolog important for PGC development, reveals a synthetic sterile phenotype. The sterility is specific to nos-2, as it is not seen in nos-1 or nos-3 double mutants. These results suggest that xnd-1 and nos-2 function in parallel pathways for germline specification. Using time lapse imaging, we are exploring the causes and consequences of the PGC defects. We have also identified key embryonic regulations affecting XND-1 expression in the PGC.

Wagner, Cynthia et al. (2009) International Worm Meeting "Germline Chromatin Structure is Regulated by XND-1."

Yanowitz, Judith, Wagner, Cynthia

[International Worm Meeting, 2009]

We are investigating the role of chromatin modifications on meiotic recombination and germline development. Mutations in xnd-1(X nondisjunction factor-1) lead to defects in crossover formation on the X as well as a change in the global distribution of crossovers. We show that xnd-1 directly regulates the formation of double strand breaks (which are required for the first step in recombination) since crossovers can be rescued by exogenous breaks caused by ionizing radiation and since repair proteins fail to localize properly to the X. Surprisingly XND-1 protein is found on autosomes, suggesting that it affects the X chromosome in trans. We will present data that suggests that XND-1 shapes the genomic crossover landscape by inhibiting crossover formation near active genes on the autosomes and allowing for crossovers to occur on the heterochromatic-like X chromosome. Supporting a direct role of XND-1 in germline chromatin organization, the lack of crossovers on the X is strongly suppressed by removing MES protein function (which is required for X chromosome silencing). Thus it appears that XND-1 and the MES proteins are acting antagonistically in the germline to coordinate X chromosome behaviors. We also show that loss of xnd-1 leads to progressive sterility-both as animals age and as populations are propagated, suggesting this gene is required to maintain germline immortality. We have identified a heterochromatic histone mark that is upregulated in xnd-1 mutants, suggesting that XND-1 directly controls X chromosome behavior, recombination, and germline maintenance through a unique set of chromatin modifications.

McClendon, Brooke et al. (2013) International Worm Meeting "Acetylation of H2AK5 and genome instability in xnd-1 mutants."

McClendon, Brooke, Yanowitz, Judith

[International Worm Meeting, 2013]

Maintenance of genome integrity is important for both the survival of the individual and the propagation of the species. Homologous recombination (HR) is a conserved process that is required for the error-free repair of double-strand breaks (DSB) and the restarting of stalled replication forks. During meiosis, DSBs are purposefully created and must be repaired by HR to form crossovers (CO) between homologous chromosomes, linking them together and ensuring faithful separation during meiosis I. Failure to create COs results in nondisjunction and subsequent aneuploidy. In humans, chromosomal aneuploidies account for over half of spontaneous abortions during the first trimester of pregnancy. Thus, competent DNA repair by HR is essential for the formation of viable eggs and sperm. In a screen for meiotic recombination regulatory proteins in C. elegans, we identified X chromosome nondisjunction-1 (xnd-1) and found that it is required for programmed DSB formation on the X chromosome, the initial step of CO recombination. Additionally, xnd-1 mutants exhibit low survival, low brood size, progressive sterility, and the appearance of spontaneous mutations - phenotypes indicative of genome instability. Indeed, xnd-1 mutants display sensitivity to ionizing radiation (IR) compared to wild type animals. Microarray analyses of xnd-1 mutant germlines do not implicate misregulation of genes involved in DSB repair, suggesting XND-1 may regulate genome stability either directly or through its effect on chromatin structure. xnd-1 mutant germlines have increased levels of acetylated H2AK5 (H2AK5ac); RNAi against mys-1 reduced H2AK5ac and the incidence of seven DAPI-staining bodies at diakinesis. We are now exploring if/how increased H2AK5ac contributes to other xnd-1 phenotypes. To this end we have been characterizing xnd-1;mys-1(n3681) double mutants. Preliminary results suggest that knockdown of mys-1 in xnd-1 mutants may partially rescue the IR sensitivity. Further studies are ongoing to determine how XND-1 regulates H2AK5ac through MYS-1 and the developmental consequences of this regulation.