Temporal Regulation of Foregut Development by HTZ-1/H2A.Z and PHA-4/FoxA

The histone variant H2A.Z is evolutionarily conserved and plays an essential role in mice, Drosophila, and Tetrahymena. The essential function of H2A.Z is unknown, with some studies suggesting a role in transcriptional repression and others in activation. Here we show that Caenorhabditis elegans HTZ-1/H2A.Z and the remodeling complex MYS-1/ESA1–SSL-1/SWR1 synergize with the FoxA transcription factor PHA-4 to coordinate temporal gene expression during foregut development. We observe dramatic genetic interactions between pha-4 and htz-1, mys-1, and ssl-1. A survey of transcription factors reveals that this interaction is specific, and thus pha-4 is acutely sensitive to reductions in these three proteins. Using a nuclear spot assay to visualize HTZ-1 in living embryos as organogenesis proceeds, we show that HTZ-1 is recruited to foregut promoters at the time of transcriptional onset, and this recruitment requires PHA-4. Loss of htz-1 by RNAi is lethal and leads to delayed expression of a subset of foregut genes. Thus, the effects of PHA-4 on temporal regulation can be explained in part by recruitment of HTZ-1 to target promoters. We suggest PHA-4 and HTZ-1 coordinate temporal gene expression by modulating the chromatin environment

Germline Maintenance Through the Multifaceted Activities of GLH/Vasa in

Vasa homologs are ATP-dependent DEAD-box helicases, multipotency factors, and critical components that specify and protect the germline. They regulate translation, amplify piwi-interacting RNAs (piRNAs), and act as RNA solvents; however, the limited availability of mutagenesis-derived alleles and their wide range of phenotypes have complicated their analysis. Now, with clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), these limitations can be mitigated to determine why protein domains have been lost or retained throughout evolution. Here, we define the functional motifs of GLH-1/Vasa i

P granules extend the nuclear pore complex environment in the C. elegans germ line

Like the nuclear pore complex, FG repeat–containing P-granule proteins interact to help establish a size-exclusion barrier

PhD

dissertationAs development progresses, cells become specialized and adopt a regional, cellular, tissue, or organ specific fate. This fate is directed by a specialized group of transcription factors called selector genes. Selector genes act at the top of a hierarchy and orchestrate expression of multiple target genes, thereby enacting developmental differentiation programs. One of these selector genes is the winged helix transcription factor FoxA. FoxA is essential for digestive tract development in all animals that it has been studied. For example, in the nematode Caenorhabditis elegans the FoxA homolog, pha-4, specifies pharyngeal, or foregut, cells. When it is disrupted, cells that would have become the pharynx develop as ectoderm. Despite the importance of FoxA proteins during foregut development, little is known about FoxA regulators and cofactors. To identify cofactors of FoxA, we performed a mutagenesis in C. elegans to screen for loci that interact genetically with pha-4. From this screen, 2 recessive and 11 dominant regulators of PHA-4 expression or activity were identified that suppress lethality associated with a partial loss of pha-4 function. Single nucleotide polymorphisms were used to map the 13 pha-4 suppressors, which showed that at least 9 genes were obtained from the screen. One of the dominant suppressors was identified as an allele of pha-4 itself, which demonstrated the effectiveness of the screen. Next, we looked in depth at a second pha-4 suppressor that was identified as a loss-of-function in the predicted DNA helicase ruvb-1. ruvb-1 is a known component of the Esa1/TIP60 and Swr1/p400 chromatin modifying and remodeling complex in eukaryotes. We investigated the role of chromatin remodeling during pharyngeal development and discovered that homologous components of the Esa1/TIP60 and Swr1/p400 remodeling complex act synergistically with pha-4 by associating the H2A histone variant, HTZ-1/H2AZ, to pharyngeal promoters. These findings are the first to demonstrate an in vivo requirement for chromatin remodeling during foregut development. Finally, we investigated additional phenotypes associated with the ruvb-1 mutant, which resemble mutations in the TOR kinase nutrient sensing pathway in C. elegans. The roles of ruvb-1 and pha-4 in nutrient sensing and metabolism during postembryonic development were examined

A Genomewide RNAi Screen for Genes That Affect the Stability, Distribution and Function of P Granules in Caenorhabditis elegans

P granules are non-membrane-bound organelles found in the germ-line cytoplasm throughout Caenorhabditis elegans development. Like their “germ granule” counterparts in other animals, P granules are thought to act as determinants of the identity and special properties of germ cells, properties that include the unique ability to give rise to all tissues of future generations of an organism. Therefore, understanding how P granules work is critical to understanding how cellular immortality and totipotency are retained, gained, and lost. Here we report on a genomewide RNAi screen in C. elegans, which identified 173 genes that affect the stability, localization, and function of P granules. Many of these genes fall into specific classes with shared P-granule phenotypes, allowing us to better understand how cellular processes such as protein degradation, translation, splicing, nuclear transport, and mRNA homeostasis converge on P-granule assembly and function. One of the more striking phenotypes is caused by the depletion of CSR-1, an Argonaute associated with an endogenous siRNA pathway that functions in the germ line. We show that CSR-1 and two other endo-siRNA pathway members, the RNA-dependent RNA polymerase EGO-1 and the helicase DRH-3, act to antagonize RNA and P-granule accumulation in the germ line. Our findings strengthen the emerging view that germ granules are involved in numerous aspects of RNA metabolism, including an endo-siRNA pathway in germ cells

Genetic Suppressors of Caenorhabditis elegans pha-4/FoxA Identify the Predicted AAA Helicase ruvb-1/RuvB

FoxA transcription factors are critical regulators of gut development and function. FoxA proteins specify gut fate during early embryogenesis, drive gut differentiation and morphogenesis at later stages, and affect gut function to mediate nutritional responses. The level of FoxA is critical for these roles, yet we know relatively little about regulators for this family of proteins. To address this issue, we conducted a genetic screen for mutants that suppress a partial loss of pha-4, the sole FoxA factor of Caenorhabditis elegans. We identified 55 mutants using either chemical or insertional mutagenesis. Forty-two of these were informational suppressors that affected nonsense-mediated decay, while the remaining 13 were pha-4 suppressors. These 13 alleles defined at least six different loci. On the basis of mutational frequencies for C. elegans and the genetic dominance of four of the suppressors, we predict that many of the suppressors are either unusual loss-of-function mutations in negative regulators or rare gain-of-function mutations in positive regulators. We characterized one dominant suppressor molecularly and discovered the mutation alters a likely cis-regulatory region within pha-4 itself. A second suppressor defined a new locus, the predicted AAA+ helicase ruvb-1. These results indicate that our screen successfully found cis- or trans-acting regulators of pha-4

The Target of Rapamycin pathway antagonizes pha-4/FoxA to control development and aging

FoxA factors are critical regulators of embryonic development and postembryonic life, but little is know about the upstream pathways that modulate their activity. C. elegans pha-4 encodes a FoxA transcription factor that is required to establish the foregut in embryos and to control growth and longevity after birth. We previously identified the AAA+ ATPase homolog ruvb-1 as a potent suppressor of pha-4 mutations.; Here we show that ruvb-1 is a component of the Target of Rapamycin (TOR) pathway in C. elegans (CeTOR). Both ruvb-1 and let-363/TOR control nucleolar size and promote localization of box C/D snoRNPs to nucleoli, suggesting a role in rRNA maturation. Inactivation of let-363/TOR or ruvb-1 suppresses the lethality associated with reduced pha-4 activity. The CeTOR pathway controls protein homeostasis and also contributes to adult longevity. We find that pha-4 is required to extend adult lifespan in response to reduced CeTOR signaling. Mutations in the predicted CeTOR target rsks-1/S6 kinase or in ife-2/eIF4E also reduce protein biosynthesis and extend lifespan, but only rsks-1 mutations require pha-4 for adult longevity. In addition, rsks-1, but not ife-2, can suppress the larval lethality associated with pha-4 loss-of-function mutations.; The data suggest that pha-4 and the CeTOR pathway antagonize one another to regulate postembryonic development and adult longevity. We suggest a model in which nutrients promote TOR and S6 kinase signaling, which represses pha-4/FoxA, leading to a shorter lifespan. A similar regulatory hierarchy may function in other animals to modulate metabolism, longevity, or disease

Enhancement of <i>pha-4(ts)</i> by <i>mys-1, ssl-1,</i> and <i>htz-1</i>

<div><p>(A) Feeding dsRNA to wild-type (WT) or <i>pha-4(ts)</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b018" target="_blank">18</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b025" target="_blank">25</a>] worms at the permissive temperature of 24 °C. WT worms generate viable progeny with <i>mys-1, ssl-1</i> or <i>htz-1</i> RNAi. In the <i>pha-4(ts)</i> background, L1 arrest increased with <i>mys-1, ssl-1,</i> or <i>htz-1</i> RNAi compared to control <i>GFP(RNAi)</i> (grey bars). Embryonic lethality remained unchanged (black bars). Effectiveness of RNAi feeding was manifest through viable, but sterile, progeny for <i>mys-1</i> and <i>ssl-1</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b014" target="_blank">14</a>], as well as repeated enhancement of L1 lethality for <i>pha-4(ts),</i> performed in parallel. <i>n</i> = 100 worms/plate, three plates per column. Error bars indicate the standard deviation.</p><p>(B) Alignment of <i>C. elegans htz-1</i> (R08C7.3) with human H2A.Z, yeast Htz1, and one of the core H2A genes from yeast, Hta1. Extended acid patch region essential for H2A.Z function is indicated by the bar [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b050" target="_blank">50</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b060" target="_blank">60</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020161#pgen-0020161-b067" target="_blank">67</a>].</p></div