Academic journal article Genetics

Genetic Suppressors of Caenorhabditis Elegans Pha-4/foxa Identify the Predicted AAA Helicase Ruvb-1/ruvb

Academic journal article Genetics

Genetic Suppressors of Caenorhabditis Elegans Pha-4/foxa Identify the Predicted AAA Helicase Ruvb-1/ruvb

Article excerpt

ABSTRACT

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.

DURING metazoan development, cells acquire specialized identities such as cell type, position, organ, or tissue. The developmental programs that establish these identities often depend on the activity of a subset of transcription factors called selector genes (Mann and Carroll 2002). Here we focus on the selector gene responsible for identity of cells of the foregut, which is the winged-helix transcription factor FoxA (Mango et al. 1994; Horner et al. 1998; Kalb et al. 1998).

FoxA, previously known as HNF3b (Kaestner et al. 2000), was identified as a protein enriched in liver nuclear extracts (Lai et al. 1990; Lai et al. 1991) and the gene defined by Drosophila fork head (Jurgens et al. 1984; Jurgens and Weigel 1988; Weigel et al. 1989). More recently, FoxA factors have been implicated in transcriptional control of breast cancer genes (Carroll et al. 2005; Laganiere et al. 2005), metabolic processes (Friedman and Kaestner 2006) and aging (Panowski et al. 2007). Loss of FoxA activity results in severe defects in foregut-derived organs such as liver and pancreas (Weigel et al. 1989; Ang and Rossant 1994; Mango et al. 1994; Weinstein et al. 1994; Dufort et al. 1998; reviewed in Zaret 2002; Lee et al. 2005). Conversely, ubiquitous expression of pha-4/FoxA in Caenorhabditis elegans is sufficient to induce ectopic foregut cells (Horner et al. 1998). These data reveal that appropriately regulated expression of pha-4/FoxA is critical for its normal functions.

Microarray studies have identified .300 potential targets of PHA-4 in C. elegans, and promoter analysis has revealed that many of these genes are direct PHA-4 targets (Gaudet and Mango 2002). The targets are expressed at varying times during the development, differentiation, or functioning of the pharynx. The appropriate timing of expression depends on combinatorial mechanisms among different cis-regulatory sites (Kalb et al. 2002; Ao et al. 2004; Gaudet et al. 2004; Vilimas et al. 2004; Raharjo and Gaudet 2007). In addition, the affinity of PHA-4 for its binding sites is an important contributor to temporal control (Gaudet and Mango 2002; Ao et al. 2004; Gaudet et al. 2004). FoxA proteins recognize the consensus TRTTKRY (R=A/G, K=T/G, and Y=T/C) in the cis-regulatory regions of gut-specific targets (Overdier et al. 1994; Kalb et al. 1998; Gaudet and Mango 2002). Target genes with high affinity sites are competent to fire early in embryogenesis, whereas genes with lower affinity sites are typically expressed late (Gaudet and Mango 2002; Gaudet et al. 2004). This regulatory configuration implies that the level and activity of PHA-4 are carefully controlled to achieve proper timing for pharyngeally expressed genes. …

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