Fredrique Gay et al. (2002) East Coast Worm Meeting "Acetylation regulates the subcellular localization of POP-1, a C. elegans cell fate-determining transcription factor"

Fredrique Gay, Rueyling Lin, Yang Shi, Dominica Calvo, Miao-Chia Lo, Julian Ceron

[East Coast Worm Meeting, 2002]

LEF/TCF family of HMG-box transcription factors are conserved from C. elegans to human, and are nuclear effectors of the Wnt signaling pathway. This pathway not only controls embryonic patterning and cell fate decisions in various species, but is also involved in carcinogenesis. During early C. elegans embryogenesis, the Wnt signaling controls anterior/posterior polarity of the EMS blastomere, the common precursor of MS (mesodermal) and E (endodermal) cells. POP-1, the C. elegans ortholog of LEF/TCF factors, plays a crucial role in the specification of the anterior MS blastomere, at least in part through supressing the expression an early E cell fate determining gene, end-1. In the posterior E cell, POP-1 nuclear level is downregulated by the Wnt signaling, leading to the activation of end-1 and the initiation of the endoderm differentiation programs. Although phosphorylation appears to be involved in the Wnt signaling-mediated downregulation of POP-1, the molecular mechanisms regulating POP-1 biological activity remain largely unknown. We investigated acetylation in the regulation of POP-1 functions, and showed that the human histone acetyltransferase CBP, as well as its C. elegans ortholog CBP-1, interact and acetylate POP-1 in vitro and in vivo. Mutational analysis identified three acetylation sites (lysines K185, K187 and K188) adjacent to the HMG-box DNA binding domain of POP-1. Although CBP also acetylates the neighboring K187 and K188 residues, K185 appears to be the major site targeted by CBP in vitro. We provide evidence that K185 is also acetylated by CBP-1 in worm embryos. To study the functional consequences of POP-1 acetylation, we analyzed the subcellular distribution of wild-type (wt) POP-1 and acetylation-defective mutants (triple, double, or individual mutations of the 3 K to A or R) in mammalians cells. We found that wt POP-1 is subjected to an active nuclear export mechanism, and shuttles between nuclear and cytoplasmic compartments although it predominantly localizes in the nucleus of most of the cells. In contrast, acetylation-defective mutants are exclusively cytoplasmically localized. Consistant with a role for acetylation in regulating the subcelluler distribution of POP-1, inhibition of histone deacetylase activities promotes nuclear accumulation of wt POP-1. Distinct sets of experiments allowed us to demonstrate that POP-1 mutants are not only more efficiently exported than wt POP-1 but also display a defect in nuclear entry, and that all 3 K are required for proper nuclear localization of POP-1 in mammalian cells. Consistantly, we found that the acetylation-defective POP-1 mutants (3K to A or R) are also mislocalized in C. elegans embryos, and fail to rescue the pharyngeal defects and embryonic lethal phenotype of the pop-1 (zu189) genetic mutant. Taken together, these results identify a novel physical and functional interaction between an histone acetyltransferase and the cell fate-determining transcription factor POP-1, and highlight a crucial role for acetylation in the regulation of POP-1 biological activity during C. elegans embryogenesis.

Martin Victor et al. (2001) International C. elegans Meeting "A POP-1 repressor complex restricts inappropriate cell type-specific gene transcription during C. elegans embryogenesis"

Dominica Calvo, Morris Maduro, Margaret Po-Shan Luke, Yang Shi, Guangchao Sui, Joel Rothman, Martin Victor, Gengyun Wen

[International C. elegans Meeting, 2001]

During animal development, cell fate determination is a critical decision process that sets the stage for future differentiation of specific cell types. In C. elegans , previous studies have shown that histone acetyltransferase CBP-1 counteracts the repressive activity of the histone deacetylase HDA-1 to allow endoderm differentiation, which is specified by the E cell. In the sister MS cell, the endoderm fate is prevented by the action of an HMG box-containing protein POP-1 through an unknown mechanism. In this study, we show that the actions of CBP-1, HDA-1 and POP-1 converge on end-1 , an initial endoderm-determining gene. In the E lineage, CBP-1 is required to overcome inhibition by both HDA-1 and POP-1 in order to activate end-1 . We further identify a molecular mechanism for the endoderm-suppressive effect of POP-1 by demonstrating that POP-1 functions as a transcriptional repressor that inhibits inappropriate end-1 transcription. We provide biochemical and functional evidence that POP-1 represses transcription via the recruitment of HDA-1 and UNC-37, the C. elegans homolog of the co-repressor Groucho. These findings demonstrate the importance of the interplay between acetyltransferases and deacetylases in the regulation of a critical cell fate-determining gene during development. Furthermore, these results identify a strategy by which concerted actions of histone deacetylases and other co-repressors ensure maximal repression of inappropriate cell type-specific gene transcription. This combinatorial scheme appears to be conserved in multiple organisms.

Schlipalius DI et al. (2012) Science "A core metabolic enzyme mediates resistance to phosphine gas."

Jagadeesan R, Nayak MK, Cheng Q, Anderson C, Collins PJ, Hilliard MA, Tuck AG, Mau YS, Schlipalius DI, Zuryn S, Kaur R, Ma L, Ebert PR, Goldinger A, Schirra HJ, Valmas N, Kuang J

[Science, 2012]

Phosphine is a small redox-active gas that is used to protect global grain reserves, which are threatened by the emergence of phosphine resistance in pest insects. We find that polymorphisms responsible for genetic resistance cluster around the redox-active catalytic disulfide or the dimerization interface of dihydrolipoamide dehydrogenase (DLD) in insects (Rhyzopertha dominica and Tribolium castaneum) and nematodes (Caenorhabditis elegans). DLD is a core metabolic enzyme representing a new class of resistance factor for a redox-active metabolic toxin. It participates in four key steps of core metabolism, and metabolite profiles indicate that phosphine exposure in mutant and wild-type animals affects these steps differently. Mutation of DLD in C. elegans increases arsenite sensitivity. This specific vulnerability may be exploited to control phosphine-resistant insects and safeguard food security.

Calvo AC et al. (2008) FASEB J "Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans."

Loer CM, Calvo AC, Ying M, Martinez A, Pey AL

[FASEB J, 2008]

In humans, liver phenylalanine hydroxylase (PAH) has an established catabolic function, and mutations in PAH cause phenylketonuria, a genetic disease characterized by neurological damage, if not treated. To obtain novel evolutionary insights and information on molecular mechanisms operating in phenylketonuria, we investigated PAH in the nematode Caenorhabditis elegans (cePAH), where the enzyme is coded by the pah-1 gene, expressed in the hypodermis. CePAH presents similar molecular and kinetic properties to human PAH [S0.5(L-Phe) approximately 150 microM; Km for tetrahydrobiopterin (BH4) approximately 35 microM and comparable Vmax], but cePAH is devoid of positive cooperativity for L-Phe, an important regulatory mechanism of mammalian PAH that protects the nervous system from excess L-Phe. Pah-1 knockout worms show no obvious neurological defects, but in combination with a second cuticle synthesis mutation, they display serious cuticle abnormalities. We found that pah-1 knockouts lack a yellow-orange pigment in the cuticle, identified as melanin by spectroscopic techniques, and which is detected in C. elegans for the first time. Pah-1 mutants show stimulation of superoxide dismutase activity, suggesting that cuticle melanin functions as oxygen radical scavenger. Our results uncover both an important anabolic function of PAH and the change in regulation of the enzyme along evolution.-Calvo, A. C., Pey, A. L., Ying, M., Loer, C. M., Martinez, A. Anabolic function of phenylalanine hydroxylase in Caenorhabditis elegans.

Choi, Hailey H. et al. (2013) International Worm Meeting "An RNAi screen for maternal factors influencing endoderm specification."

Choi, Hailey H., Maduro, Morris F.

[International Worm Meeting, 2013]

Department of Cell, Molecular, and Developmental Biology, University of California, Riverside1 Department of Biology, University of California, Riverside2 Gene expression between otherwise identical cells can vary due to stochastic factors, such as differences in the number of transcription factor molecules available to bind a promoter. We have developed a system to examine how gene networks buffer noise using the well-studied endoderm specification network. In this network, input from maternal and zygotic genes ultimately influences activation of the embryonic E specification genes end-1 and end-3. Activation of these genes leads to activation of elt-2 and elt-7, which direct endoderm differentiation. Activation of elt-2 has been hypothesized to occur as a result of accumulation of threshold amounts of end-1 mRNA (Raj et al., 2010). To examine the phenotypic outcomes from near-threshold activation of gut specification, we have generated "partial gut specification" strains in which gut is specified by single-copy transgenes of end-1 and/or end-3 in which the binding sites for the MED-1,2 factors have been mutated. One of these strains, carrying a MED site (-) end-1 in a double mutant end-1,3(-) background, makes gut in ~25% of embryos. Using an integrated elt-2::GFP reporter to identify fully committed gut cells (even in arrested embryos), we are using RNAi to screen for maternal factors that may influence the ability of this strain to specify gut. We hypothesize that such genes might act generally in gene expression, or may be more restricted to endoderm specification. Using the Vidal feeding library, we have selected a subset of genes that are known to be expressed in the germline. In a pilot screen of ~10% of these clones, we have found several genes that, when knocked down, cause an increase in the proportion of embryos making gut over controls. One of these, hda-1, has been previously shown to cause an increase in gut specification when the endoderm network is compromised (Calvo et al., 2001). We will report on further progress with the screen.