Bill Kelly (2001) International C. elegans Meeting "The Role of Chromatin Organization in Germ Line Function and Maintenance"

Bill Kelly

[International C. elegans Meeting, 2001]

The germ line in C. elegans is maintained by a variety of suppressive mechanisms throughout development and during adulthood. In the adult germ line both transcriptional and post-transcriptional mechanisms play roles in maintaining germ cell viability and function. Transcriptional suppression involves chromatin-based mechanisms that are products of a compromise reached by the competing goals of meiotic DNA: the chromosomes must be compacted along their lengths for synapsis and recombination, yet must also be transcriptionally active for maternal product synthesis. One might thus hypothesize that genes essential for germ line function have evolved mechanisms to escape aspects of chromatin silencing that likely accompany the compaction of meiotic DNA. Non-integrated transgenes are efficiently targeted for silencing in the germ line by chromatin-based mechanisms, since mutations that are defective in this silencing exhibit defects in chromatin organization. In addition, Valerie Reinke along with Stuart Kim and Anne Villeneuve, as well as my lab, have observed that the entire X chromosome appears to be silenced. Markers for transcriptionally active chromatin (e.g. histone acetylation) are uniquely excluded from the X chromosome in immature germ cells in both males and hermaphrodites. As germ cells mature past pachytene stage in hermaphrodites, however, the X chromosome becomes increasingly labeled by probes that correlate with transcriptional activation. This "activation" is not observed in male germ cells. Interestingly this correlates well with microarray data from Reinke et al. that indicates a paucity of sperm-specific or germ cell-intrinsic genes on the X, but a normal distribution of putative oocyte-specific genes (Mol. Cell. 6:605). Silenced transgenes strongly mimic the observed underacetylation of X chromosome histones in the early stages of meiotic prophase I, but fail to become "activated" in oocytes. Conversely, a transgene (for a normally autosomal gene) integrated onto the X exhibits a germ line expression that is always strongest in oocytes. This suggests the presence of cis-acting elements peculiar to the X that may be involved in post-pachytene activation of the X chromosome in oocytes. The nature of germ line X-regulation is unknown, but is unlikely to involve components of dosage compensation (which do not assemble onto the X until early embryogenesis). Indeed, an absence of X-linked genes essential for both male and female germ cells would appear to obviate a need for dosage compensation in germ cells, as well as permit X silencing in the immature germ cells of both sexes. This may in turn have played a role in the frequent appearance of hermaphroditism in nematode evolution. The effects of silencing mutants on transgene and X silencing, an analysis of X silencing in other hermaphroditic and gonochoristic nematode species, and the role of chromatin organization in germ line function will be presented and discussed.

B Kelly et al. (1999) International C. elegans Meeting "The Epigenetics of Germline Maintenance"

B Kelly, A Fire

[International C. elegans Meeting, 1999]

We are interested in characterizing the mechanism(s) by which the germline escapes the narrowing of developmental potential that is characteristic of somatic differentiation. There appear to be several processes specific to the germline that it likely employs to maintain its totipotency. These include the pie-1 mode of global transcriptional repression, which is effective in the early embryo 1 . pie-1 -mediated repression, however , is absent after the middle stages of embryogenesis; indeed, the larval germline is both proliferative and transcriptionally active. Therefore, the maintenance of totipotency in the larval and adult germline must allow for full transcription of numerous genes with concomitant repression of somatic differentiation. The mechanisms that restrict germline gene expression appear to coincide with mechanisms that prevent the efficient expression of many transgenes in the germ lineage. Mutations that disrupt transgene silencing in the germline disrupt germline viability; conversely, a subset of sterile mutations, including the mes (maternal effect sterile) mutants identified by the Strome lab, disrupt germline silencing. Other mutations that disrupt germline silencing include a subset of mutator (germline transposon activation) and him (chromosome segregation) mutants; these mutants also exhibit various degrees of germline defects. Our working hypothesis is that these mutations disrupt some aspect of germline chromatin organization. The processes by which germline silencing is maintained have an epigenetic nature-- that is, both the silenced and desilenced states are passaged, or "remembered" through multiple cell divisions and, in some cases, multiple generations. This "memory" is evidenced by a lag period between the disruption of components of the process, and manifestation of the phenotype. Hence, as in the case of the mes mutations, sterility is delayed for a full generation after homozygosity of the mutation. Acetylation of nucleosomal histones has been shown to play a role in epigenetic inheritance in many systems. We are currently investigating the functional significance of correlations between histone under-acetylation and germline silencing. 1 Seydoux, G., Mello, C. C., Pettitt, J., Wood, W. B., Priess, J. R., Fire, A. (1996). Nature 382: 713.

Kelly WG et al. (1995) International C. elegans Meeting "CLONING AND CHARACTERIZATION OF LET-858."

Fire A, Kelly WG

[International C. elegans Meeting, 1995]

As part of a screen for zygotic lethals on LG II (WBG 13.4:98), we have obtained three mutations in a novel gene, let-858, which exhibit a distinctive embryonic arrest phenotype. The two strongest alleles arrest between 1.5 and 2.5 fold elongation, frequently with the formation of prominent vacuoles in the head region. The embryonic lethality of these mutations can be rescued with a 6.8 kb fragment from cosmid F33A8, but the embryos grow up to be sterile or poorly fertile adults with abnormal germlines, suggesting a gonadal function in addition to the zygotic function in embryogenesis. Northern blot analysis reveals a ~2.8 kb mRNA that appears most abundant in embryos and adults, with minimally detectable amounts in early larvae. The transcript is detectable in fem-3 animals, but appears to be absent in germ-line deficient glp-4 (bn2) homozygotes, consistent with a role for let-858 in the developing germline.

Kelly KA et al. (1997) International C. elegans Meeting "Meiotic Recombination in C. elegans"

Villeneuve AM, Kelly KA

[International C. elegans Meeting, 1997]

Meiosis is a specialized form of division that diploid cells undergo to produce gametes containing a haploid chromosome number. During prophase of meiosis I, homologous chromosomes pair, synapse and undergo genetic recombination. Crossing over (reciprocal recombination) between homologs is required to ensure their proper disjunction at meiosis I, since it leads to the formation of chiasmata, temporary connections that hold homologs together until anaphase I and allow them to orient toward opposite poles on the meiosis I spindle. Our goal is to understand how these crucial crossovers are formed, and how crossing over is regulated. We are characterizing two mutations, me23 and me24, that cause defects in crossover formation during meiosis. Both mutants exhibit high frequencies of achiasmate chromosomes in oocyte nuclei, indicative of a severe reduction in crossing over for all chromosomes. The me24 mutation is allelic with him(me9), which is thought to be involved in regulating crossover formation. The other mutation, me23, is very tightly linked to a gene encoding a C. elegans homolog of the yeast MSH5 protein, a MutS family member involved in formation of a subset of meiotic crossovers in yeast. Further mapping, transformation rescue and sequence analysis will determine whether the me23 mutation is in this candidate C.e.MSH5 gene. Our lab has shown that another MutS homolog, HIM-14, is required for formation of meiotic crossovers in C. elegans (see Zalevsky and Villeneuve). To explore further the roles of MutS homologs in crossing over, we are expressing and purifying HIM-14 and C.e.MSH5 proteins to study their biochemical properties in vitro. In particular we will examine whether these proteins can physically interact with predicted recombination intermediates such as Holliday junctions, and whether they can affect the resolution of such structures.

Ginger Kelly et al. (2001) International C. elegans Meeting "Analysis of lin-1 Mutants and Screen for Suppressors of lin-1(n383)"

Ginger Kelly, Kerry Kornfeld

[International C. elegans Meeting, 2001]

A highly conserved RTK/Ras/MAP kinase pathway promotes the primary vulval cell fate by negatively regulating the ETS transcription factor LIN-1. Bob Horvitz and colleagues isolated 41 alleles of lin-1 in a variety of screens for vulval defects. These alleles include loss-of-function and gain-of-function mutations of different severity. To identify residues and domains that are important for LIN-1 structure and function, we used DNA sequencing to determine the molecular lesions of these alleles. The loss-of-function mutations include several missense mutations that affect the ETS domain and a series of nonsense mutations. The DNA-binding properties of the ETS domain mutants are being investigated. To identify genes that function downstream or in parallel to lin-1 to promote the primary cell fate, we are screening for mutations that suppress the multivulva (Muv) lin-1(n383) loss-of-function phenotype. We have screened about 5,500 haploid genomes and identified ten suppressors of lin-1 Muv. These ten mutations cause sterility. These suppressor mutations will being mapped relative to polymorphisms between the N2 strain and the Hawaiian CB 4856 strain. lin-1 is epistatic to the well characterized genes in the RTK/Ras/MAP kinase pathway, therefore these mutations may affect new genes that function downstream of lin-1 to regulate the primary cell fate.

Kelly Grant et al. (1999) International C. elegans Meeting "SEM-4 regulates vulval cell fate and expression of lin-39 hox"

Kelly Grant, Min Han, Wendy Hanna-Rose

[International C. elegans Meeting, 1999]

Both lin-39 hox gene and Ras pathway signaling activity are important components of vulval cell fate specification. Ras activity affects LIN-39 expression (Maloof and Kenyon, 1998 and Clandinin, et al, 1998) through a mechanism that has yet to be determined. We have identified a candidate transcriptional regulator of lin-39 and are investigating whether this factor may provide a link between Ras signaling and lin-39 regulation. In a screen for vulval defective mutants, we identified multiple mutants with abnormal vulval cell lineages, including 3 alleles of sem-4. sem-4 encodes a zinc-finger protein with recognized roles in fate specification of sex myoblasts, coelomyctes and several neurons (Basson and Horvitz, 1996), but sem-4 was not previously reported to affect vulval cell lineages. Approximately 60% of worms with sem-4 mutations exhibit an abnormal P7.p lineage and a less penetrant P5.p lineage defect. To examine the role of SEM-4 in vulval formation, we analyzed its interactions with other genes that affect vulval differentiation. Analysis of animals carrying mutations in both sem-4 and either let-60ras or lin-45raf suggested that sem-4 does not function directly in the Ras pathway. However, we observed a strong genetic interaction between sem-4 and lin-39. A sem-4; lin-39(n709ts) double mutant displays a significant decrease in induction of both P5.p and P7.p. Additionally, we have observed that expression of lin-39 ::lacZ (gift of Maloof and Kenyon) is reduced in the vulva in a sem-4 mutant background. Furthermore, over-expression of LIN-39 from a heat shock promoter can partially rescue a sem-4 mutant. These data suggest that lin-39 acts downstream of sem-4 to regulate vulval differentiation, and the molecular identity of SEM-4 suggests that it may regulate the transcription of lin-39. We have determined that a SEM-4::GFP translational fusion (gift of M. Koelle) is expressed in the vulva precursors cells just after the first cell division and persists until after cell division in the vulva lineage is complete. Expression is stronger in P7.p and its descendants, consistent with sem-4 mutations having a greater effect on P7.p. We are currently investigating whether SEM-4::GFP is regulated by signaling through the Ras pathway to determine if SEM-4 acts between Ras signaling and lin-39.

Kelly Colombo et al. (2001) International C. elegans Meeting "A novel Goloco motif protein is required for the asymmetric division of one cell stage embryos"

Stephan Grill, Kelly Colombo, Tony Hyman, Pierre Goenczy

[International C. elegans Meeting, 2001]

Kelly Harradine et al. (2001) International C. elegans Meeting "Molecular analysis of pel-2 and pel-3, two genes required for embryonic pharynx differentiation and morphogenesis"

Joel H Rothman, Kelly Harradine

[International C. elegans Meeting, 2001]

The pharynx is comprised of five different cell types, all of which derive from two blastomeres in the early embryo, ABa and MS. How are cells that arise from disparate lineages specified and patterned to form this single, coherently functioning organ? While some of the genes that impart pharynx-specific identity to the five pharynx cell types have been characterized, the mechanisms that regulate pharynx morphogenesis and differentiation are largely unknown. To address these problems, we are investigating two genes required for embryonic pharyngeal development, pel-2 IV and pel-3 II (p harynx and el ongation defective) . Both genes were identified from mutations isolated in a genome-wide screen for zygotic embryonic lethal mutants (JH Rothman, 8th International C. elegans Meeting). The pel-2 mutant shows a block in embryonic elongation, with arrest occurring at the comma to 1.5-fold stage. The pharynx neither elongates nor displays any signs of differentiation or morphogenesis (e.g., a lumen, grinder, buccal capsule, or muscle striations). The pel-3 mutant arrests at the two- to three-fold stage with a partially elongated pharynx that never attaches to the buccal opening and with variable defects in differentiation and morphogenesis (e.g., the grinder, lumen, and buccal capsule are variably absent). We are analyzing the expression of pharynx markers and the behavior of pharyngeal cells in an effort to assess whether the defects we observe arise from defective differentiation or morphogenesis of the pharynx. To test whether the pel genes act within the known pathway of regulators for pharynx development (e.g., pha-1, pha-4 , and ceh-22 ), we are examining expression of these genes in pel mutants and are investigating genetic interactions between them. We have narrowed the chromosomal positions of both pel genes using SNIP-SNP mapping in an effort to clone them. By dissecting the function of pel-2 and pel-3 genetically and molecularly, we hope to gain further insights into how organ development and assembly are molecularly orchestrated.

Ming Ming Huang et al. (2001) International C. elegans Meeting "Construction of GFP Tagged Chromosomes"

Don Moerman, Ming Ming Huang, Robert Barstead

[International C. elegans Meeting, 2001]

We have constructed 17 C elegans strains carrying GFP or YFP expressing transgenes integrated randomly in wild type chromosomes. The fluorescent transgenes will be useful as benign dominant markers in crosses to replace chromosomes and as weak balancers for mutations in closely linked essential genes. The integrated transgenes began life as an extrachromosomal array, with GFP fused to promoters for myo-2 and pes-10 or a gut-specific enhancer from F22B7.9. The arrays were integrated using standard methods (1). The resulting strains were outcrossed four times to wild type. Presently, we are mapping the location of each transgene. We will describe their map positions and phenotypes. 1. Mark Edgley, Jun Kelly Liu, Don Riddle, Andy Fire. Worm Breeder's Gazette 15(5): 20 (February 1, 1999).

Kelly WG (2000) East Coast Worm Meeting "Parent of Origin Effects in C. elegans: Worm Imprinting or an Insight Into Hybrid Dysgenesis?"

Kelly WG

[East Coast Worm Meeting, 2000]

Parent of origin effects in gene expression describe how the sex of a parent transmitting a particular allele affects the expression of that gene in its offspring. A gene that exhibits a differential expression in the offspring that depends on whether it was inherited from the mother or the father is considered to be imprinted. Imprinting has been shown in mammals to closely correlate with cytosine methylation, yet parent of origin effects have been frequently reported in Drosophila, an organism in which methylated cytosine is not observed (e.g. Lloyd et al, Genetics 151:1503). Imprinting in C. elegans has previously been investigated by looking at cross-progeny that were homozygous for a single chromosome from either parent, using him-6 to generate disomic or nullisomic gametes in each cross (Haack and Hodgkin, Mol.&Gen. Genet 228:482). No effects on viability were observed, leading to the conclusion that either chromosomal imprinting effects do not occur in the worm, or that their effects are minor. C. elegans also has no detectable methylcytosine. During crosses to build transgenic strains for testing germline desilencing, a high frequency of transient activation of silenced transgenes was observed in the germ cells of the offspring. Upon further investigation it was shown that this activation only occurs when the silenced transgene is inherited from the father, and the effect is temperature-sensitive. The activation is generally restricted to the F1 progeny, although a low frequency of heritable activation can be observed. Interestingly, the presence of a transgene in the recipient oocyte that bears partial homology to the incoming reporter suppresses the activation of the reporter. This suppression is still seen in animals arising from oocytes that have lost the suppressing DNA during meiosis. The effect of different mutations on this phenomenon, its applicability for germline transgene expression protocols, and its natural role in worm biology will be discussed.