Martin Victor et al. (2001) International C. elegans Meeting "The HAT activity of CBP-1 is indispensable for its biological function during C. elegans embryogenesis"

Craig C Mello, Martin Victor, Yang Shi

[International C. elegans Meeting, 2001]

CBP/p300 represent a conserved family of transcriptional cofactors possessing histone acetyltransferase (HAT) activity. This group of proteins has been shown to play critical roles in differentiation and cell proliferation in C. elegans , Drosophila , mouse and human. Despite their importance, the mechanisms by which they regulate transcription and thus exert their biological functions are poorly understood. A central question is whether the HAT activity is essential for the biological functions of these proteins. In this study, we investigated the importance of the HAT activity for CBP-1 in C. elegans . We show that a truncated CBP-1 protein lacking the HAT domain fails to promote differentiation, consistent with the notion that the HAT activity of CBP-1 is essential for its biological functions. To further address this question, we used a chemical that has been shown to be a specific inhibitor of the enzymatic activity of CBP/p300 HAT. Injection of this chemical into the gonads of the hermaphrodite mothers resulted in dead embryos arrested at different stages of development. Significantly, some of these embryos appear identical to cbp-1(RNAi) embryos in every aspect, as judged by the excess cell number, lack of endoderm and mesoderm tissues but excess neuronal differentiation (Shi and Mello, 1998). Taken together, our findings provide in vivo evidence that the HAT activity is not only critical, but also may be the only biochemical activity that is necessary for the biological functions of CBP-1. Shi, Y. and Mello,C. (1998), 'A CBP/p300 homolog specifies multiple differentiation pathways in Caenorhabditis elegans .' Genes Dev. 12(7), 943-55.

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.

M Victor et al. (1999) International C. elegans Meeting "CBP-1 in C. elegans embryonic development and differentiation"

M Victor, Y Shi, C Mello

[International C. elegans Meeting, 1999]

Mammalian p300 and CBP are closely related transcriptional coactivators that possess histone acetyltransferase (HAT) activity and function as integrators of transcription and signaling events. Recently, we have shown that a C. elegans gene closely related to p300/CBP, cbp-1 , is required to specify multiple differentiation events during embryogenesis. In endoderm differentiation, cbp-1 appears to promote differentiation by antagonizing the repressive activity of histone deacetylases, suggesting that a balance between histone acetyltransferase and deacetylase may be important for endodermal differentiation (1). To further investigate the mechanisms underlying the differentiation-promoting activities of CBP-1, we isolated a cbp-1 deletion mutant using a PCR-based approach. Antibody staining shows that this deletion resulted in a cbp-1 null allele. The majority of embryos homozygous for the cbp-1 deletion die around the 2-fold stage, suggesting that maternally provided CBP-1 is sufficient for the initiation of most differentiation events. We have begun to rescue the cbp-1 deletion mutant using a YAC that carries a wildtype cbp-1 gene. Preliminary results suggest that both maternal and zygotic defects can be rescued. Dead embryos segregated from the rescued animals morphologically resemble the cbp-1 (RNAi) embryos. A detailed analysis of these embryos as compared to the cbp-1 (RNAi) embryos and embryos derived from the cbp-1 deletion mutant will be presented. Work is in progress to use this YAC-based approach to determine the role of various functional domains of CBP-1 (i.e. the HAT domain of CBP-1) in differentiation. (1) Shi, Y. and Mello, C. (1998) A CBP/p300 homolog specifies multiple differentiation pathways in Caenorhabditis elegans , Genes Dev. 12 , 943-55. Martin Victor is supported by a postdoctoral fellowship from the DAAD, Germany.

Victor M et al. (1998) East Coast Worm Meeting "The role of Histone Acetylase and Deacetylase in C.elegans differentiation and development"

Victor M, Shi Y

[East Coast Worm Meeting, 1998]

Protein modification by acetylation/deacetylation has been shown to be an important step in the regulation of transcription. While hyperacetylation is correlated with increased transcription, hypoacetylation is generally correlated with transcriptional repression. The level of histone acetylation is determined by the action of both histone acetylases (HATs) and histone deacetylases (HDACs). We are interested in understanding the role of these regulators in the context of C.elegans differentiation and development. Recently, in collaboration with Craig Mello, we showed that CBP-1, the C.elegans homolog of the mammalian transcriptional coactivator proteins CBP and p300, is involved in specifying multiple differentiation pathways during C.elegans embryogenesis. In contrast, histone deacetylases appear to play antagonistic, repressive roles. In C.elegans, we identifieded at least 6 putative histone deacetylase genes, but their precise roles in development and differentiation remain to be defined. To further study these two classes of transcriptional regulators, we developed specific antibodies that recognize CBP-1 as well as three of the most conserved HDAC proteins. The expression pattern of these proteins will be presented. Furthermore, to investigate the mechanism of action of these proteins, we aimed at isolating genetic mutants for the corresponding genes. We therefore constructed a DNA-library from chemically mutagenized worms. Using a PCR-based screening approach, we could isolate deletion mutants for two of the histone deacetylase genes as well as for cbp-1. Sequence analysis of the mutant cpb-1 DNA revealed an internal deletion resulting in the lack of a 2.7kb fragment in the most conserved region (with respect to its mammalian counterpart) of the gene. This deletion takes out several critical functional domains, including the bromodomain, the HAT domain as well as the CH3 domain. The bromo- and CH3-domains have been shown to be important for interacting with a multitude of regulatory proteins including the viral oncoprotein E1A. Preliminary data suggest that these mutants are homozygous inviable due to embryonic death at the 2-fold stage. The initial characterization of the isolated deletion mutants will be discussed.   Martin Victor is supported by a postdoctoral fellowship from the 'Deutscher Akademischer Austauschdienst' (DAAD), Germany.

Sze JY (2000) West Coast Worm Meeting "Transcriptional regulation of the tryptophan hydroxylase gene tph-1"

Sze JY

[West Coast Worm Meeting, 2000]

tph-1 encodes a tryptophan hydroxylase, the key enzyme for serotonin biosynthesis. When I was in Gary Ruvkun's lab, I collaborated with Martin Victor in Yang Shi's lab and isolated a tph-1 deletion mutation, tph-1(mg280). tph-1(mg280) shows no detectable serotonin based on anti-serotonin antibody staining and exhibits several behavioral and metabolic defects (Sze et al., 2000). It has been proposed that in mammals changes in the level of TPH mRNA correlate with long-term changes in the cellular serotonin level and that the regulation of TPH mRNA tends to be "inducer"- and region-specific (Semple-Rowland et al., 1996; Clark and Russo, 1997; Siuciak et al., 1998. Bethea, et al., 2000). Thus, the regulation of TPH expression may be a key step by which the nervous system adjusts its long-term synaptic serotonin levels. To identify genes regulating tph-1, I have started a genetic screen for mutations that alter tph-1::gfp expression in specific neurons. Thus far, mutations identified can be classified into three classes. (1) I have previously reported that mutations in the POU-homeodomain transcription factor UNC-86 abolish tph-1::gfp expression in NSM and HSN, but the expression in ADF is not affected. UNC-86 is expressed in HSN and NSM, but not in ADF. (2) I have identified 18 mutations where tph-1::gfp expression in ADF is reduced or eliminated. Laser ablation of ADF and ASI causes animals to form dauers (Bargmann and Horvitz, 1993; Schackwitz et al., 1996) and the tph-1(mg280) mutation downregulates daf-7::gfp expression and enhances daf-7(e1372) dauer formation at 15oC. To test the role of ADF-produced serotonin is important for a non-dauer growth, I am testing if these mutations enhance daf-7(e1372) dauer formation at 15oC. (3) I have identified one mutation that has tph-1::gfp expressed in an extra neuron. These mutants provide me as useful means to characterize the role of serotonin produced in specific neurons.

Emily Bates et al. (2002) East Coast Worm Meeting "Potential Roles of CBP, CREB, and HDAC in Polyglutamine Induced Neurodegeneration in C. elegans"

Emily Bates, Martin Victor, Anne C Hart, Cindy Voisine, Yang Shi

[East Coast Worm Meeting, 2002]

Huntington's disease and at least eight other neurodegenerative diseases result from expansions of polyglutamine tracts. Though it is clear that these expansions are toxic in many cell types, the molecular basis of polyglutamine induced cell death in the nervous system is unknown. Inhibitors of histone deacetylases(HDAC) reduce polyglutamine induced cell death in cell culture and Drosophila.(McCampbell, 2001; Steffan, 2001). A transcription activator, cAMP response element binding protein (CREB), and a histone acetyltransferase, CREB binding protein (CBP) have been found in mutant polyglutamine aggregates. (McCampbell, 2000) These data suggest that expanded polyglutamine may alter transcription by sequestering glutamine rich proteins such as CBP and CREB perturbing their function. This hypothesis predicts that 1) overexpression of CBP and CREB protects cells from polyglutamine induced degeneration and 2) CBP or CREB loss of function mutations enhance polyglutamine induced neurodegeneration in C. elegans. We have established a C. elegans model to address the molecular mechanism of polyglutamine toxicity. N-terminal fragments of human huntingtin with varying lengths of glutamine stretches were expressed in C. elegans sensory neurons. (Faber, 1999) Up to 30% of the neurons expressing mutant huntingtin fragment degenerate in 8 day old animals. A putative role of CREB, CBP, and HDAC in polyglutamine induced neurodegeneration is being examined using loss of function alleles. Additional experiments are underway to look at overexpression of these genes. These experiments will further define the role of the CREB/CBP pathway in polyglutamine expansion diseases.

Emily A Bates et al. (2004) East Coast Worm Meeting "C. elegans HDACs, CBP, and CREB play roles in polyglutamine neurotoxicity"

Emily A Bates, Martin Victor, Cindy Voisine, Yang Shi, Anne Hart

[East Coast Worm Meeting, 2004]

The expansion of a polyglutamine (polyQ) tract in the huntingtin protein causes neuronal death in Huntington's disease, but the molecular basis of cell death is unknown. Transcriptional regulation via histone acetylase (HAT) activity and histone deacetylase (HDAC) activity is critical to many cellular processes. Some results from Drosophila and vertebrate systems suggest that expanded polyQ tracts may alter transcription by sequestering glutamine rich transcriptional regulatory proteins. CREB binding protein (CBP), a glutamine rich histone acetyltransferase, has been found in expanded polyQ aggregates in patient tissue and cell culture models. HDAC inhibitors reduce polyQ induced death in cell culture and Drosophila. This transcriptional hypothesis for polyQ toxicity suggests that 1) overexpression of CBP and CREB or loss of HDAC function will protect cells from polyQ-induced degeneration and 2) CBP (cbp-1) or CREB (crh-1) loss of function mutations will enhance polyQ-induced neurodegeneration. We are testing this hypothesis in a C. elegans model of polyQ toxicity. N-terminal fragments of human huntingtin with varying lengths of glutamine tracts are expressed in C. elegans ASH sensory neurons. At least 30% of the ASH neurons expressing mutant huntingtin fragment degenerate in 8 day old animals. Putative roles of crh-1 (Alkema & Horvitz), CBP, and HDAC in polyQ-induced neurodegeneration were examined using RNA interference and loss of function alleles. Deletion of CREB enhances polyQ toxicity in ASH neurons. Similarly, deletion of one copy of cbp-1 enhances polyQ toxicity. Loss of function alleles and RNA interference were used to systematically reduce function of each C. elegans HDAC in our model of polyQ toxicity. Knockdown of most CeHDACs enhanced polyglutamine toxicity; only one CeHDAC knockdown resulted in suppressed polyQ toxicity. Additional experiments are underway to determine if overexpression of these proteins modulates polyQ toxicity. These studies will further define the role of the CREB/CBP pathway in polyQ expansion diseases.

Emily A Bates et al. (2005) International Worm Meeting "Differential contributions of C. elegans histone deacetylases to huntingtin polyglutamine toxicity"

Johnathan Whetstine, Martin Victor, Emily A Bates, Anne Hart, Yang Shi

[International Worm Meeting, 2005]

Expansion of a polyglutamine (polyQ) tract in the huntingtin protein causes neuronal degeneration and death in Huntington's disease, but the molecular mechanisms underlying polyQ mediated cell death are unclear. Previous data suggest that expanded polyQ tracts alter transcription by sequestering glutamine rich transcriptional regulatory proteins, thereby perturbing their function. We tested this hypothesis in a C. elegans model of polyQ toxicity. Loss of function alleles and RNAi were used to examine contributions of C. elegans CREB, CBP, and HDACs to polyQ-induced neurodegeneration. Deletion of CREB (crh-1)(Mark Alkema and Robert Horvitz) enhances polyQ toxicity in ASH sensory neurons. Loss of one copy of CBP (cbp-1) increases polyQ toxicity. Loss of function alleles and RNAi were used to reduce function of each C. elegans HDAC in a neuronal model of polyQ toxicity. Knockdown of most C. elegans HDACs increased Htn-Q150 toxicity; knockdown of hda-3 decreased polyQ toxicity. Increased expression of hda-1 in Htn-Q150 expressing neurons protects against Htn-Q150 toxicity. Neuronal expression of hda-3 restores Htn-Q150 induced degeneration and suggests that HDA-3 acts in the neurons to promote Htn-Q150 neurotoxicity. hda-3 loss of function suppresses degeneration in crh-1;Htn-Q150 animals suggesting that HDA-3 and CRH-1 directly or indirectly oppose each other in regulating the transcription of genes important for polyQ toxicity. Target genes may provide a link to previously implicated pathways underlying polyQ toxicity such as protein degradation, apoptosis, metabolic pathway, or axonal transport. hda-3 loss of function does not decrease neurodegeneration in hda-1;Htn-Q150 animals suggesting that HDA-1 and HDA-3 have different targets. These studies support the role of the CREB/CBP pathway in polyQ expansion diseases and suggest that specific HDAC inhibition as an interesting therapeutic approach.

Emily A Bates et al. (2003) International Worm Meeting "Candidate modifiers of polyglutamine toxicity"

Mark Alkema, Cindy Voisine, Yang Shi, Martin Victor, Emily A Bates, Anne Hart

[International Worm Meeting, 2003]

Expanded polyglutamine (polyQ) tracts in proteins can induce neuronal death leading to diseases including Huntington's, and several ataxias. The expansion of a polyQ tract in the huntingtin protein has been identified as the culprit for Huntington's disease pathology, but the molecular basis of cell death in the nervous system is unknown. Recent data suggest that expanded polyQ tracts may alter transcription by sequestering glutamine rich proteins thereby perturbing their function. Inhibitors of histone deacetylases (HDACs) reduce polyQ induced death in cell culture and Drosophila (McCampbell, 2001; Steffan, 2001). A histone acetyltransferase, CREB binding protein (CBP), has been found in expanded polyQ aggregates (McCampbell, 2000). This transcriptional hypothesis predicts that 1) overexpression of CBP and CREB or loss of HDAC function will protect cells from polyQ-induced degeneration and 2) CBP or CREB loss of function mutations will enhance polyQ-induced neurodegeneration.N-terminal fragments of human huntingtin with varying lengths of glutamine tracts were expressed in C. elegans ASH sensory neurons to address the molecular mechanism of polyQ toxicity (Faber, 1999). Up to 30% of the ASH neurons expressing mutant huntingtin fragment degenerate in 8 day old animals. Putative roles of CREB, CBP, and HDAC in polyQ-induced neurodegeneration are being examined using RNA interference, loss of function alleles, and one putative gain of function allele. Deletion of CREB enhances polyQ toxicity in ASH neurons. Loss of gon-10, the C. elegans gene most closely related to human type 1 HDACs, enhances neurodegeneration. Deletion of one copy of cbp-1 enhances polyQ toxicity. RNA interference is being used to define roles of other HDAC genes. Additional experiments are underway to determine if overexpression of these proteins modulates polyQ toxicity. These studies will further define the role of the CREB/CBP pathway in polyQ expansion diseases.

Melissa M Rolls et al. (2001) International C. elegans Meeting "Targeting of rough endoplasmic reticulum membrane proteins and ribosomes to the cell body of C. elegans neurons"

Melissa M Rolls, Ernst H K Stelzer, Tom A Rapoport, Martin Victor, David H Hall, Yang Shi

[International C. elegans Meeting, 2001]

The endoplasmic reticulum (ER) is a continuous membrane system that contains specialized domains. The rough endoplasmic reticulum (RER) shares most proteins with the smooth endoplasmic reticulum (SER), but is enriched in a set of proteins that translocate secretory and membrane proteins across the lipid bilayer. We studied targeting of RER membrane proteins within the ER in C. elegans . In several cell types fluorescent protein fusions to RER and general ER proteins colocalized, but in neurons RER markers were concentrated in the cell body while general ER markers were found in the neurites as well as the cell body. To determine whether RER membrane proteins were localized to the cell body by immobilization we compared the diffusion of RER and general ER membrane proteins. Surprisingly both types of proteins were equally mobile. We also examined whether the distribution of ribosomes was as restricted as RER membrane proteins. By light and electron microscopy, ribosomes were extremely concentrated in the cell body and rare in neurites. This indicates that, like mammalian axons, C. elegans neurites largely exclude protein synthetic machinery.