Gao J et al. (2015) PLoS Genet "NatB domain-containing CRA-1 antagonizes hydrolase ACER-1 linking acetyl-CoA metabolism to the initiation of recombination during C. elegans meiosis."

Gao J, Elia AE, Colaiacovo MP, Kim HM, Elledge SJ

[PLoS Genet, 2015]

The formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

Xu L et al. (2003) Nature "BTB proteins are substrate-specific adaptors in an SCF-like modular ubiquitin ligase containing CUL-3."

Vidal M, Harper JW, Reboul J, Wei Y, Xu L, Elledge SJ, Vaglio P, Shin TH

[Nature, 2003]

Programmed destruction of regulatory proteins through the ubiquitin-proteasome system is a widely used mechanism for controlling signalling pathways(1,2). Cullins(3) are proteins that function as scaffolds for modular ubiquitin ligases typified by the SCF (Skp1-Cul1-F-box) complex(4-6). The substrate selectivity of these E3 ligases is dictated by a specificity module that binds cullins. In the SCF complex, this module is composed of Skp1, which binds directly to Cul1, and a member of the F-box family of proteins(4-7). F-box proteins bind Skp1 through the F-box motif(7), and substrates by means of carboxy-terminal protein interaction domains(1,2,5). Similarly, Cul2 and Cul5 interact with BC-box-containing specificity factors through the Skp1-like protein elongin C-2. Cul3 is required for embryonic development in mammals and Caenorhabditis elegans(8-10) but its specificity module is unknown. Here we report the identification of a large family of BTB-domain proteins as substrate-specific adaptors for C. elegans CUL-3. Biochemical studies using the BTB protein MEL-26 and its genetic target MEI-1 (refs 12, 13) indicate that BTB proteins merge the functional properties of Skp1 and F-box proteins into a single polypeptide.

Chou DM et al. (2010) Proc Natl Acad Sci U S A "A chromatin localization screen reveals poly (ADP ribose)-regulated recruitment of the repressive polycomb and NuRD complexes to sites of DNA damage."

Nottke AC, Gygi SP, Colaiacovo MP, Elledge SJ, Chou DM, Hurov KE, Adamson B, Dephoure NE, Tan X

[Proc Natl Acad Sci U S A, 2010]

Many proteins that respond to DNA damage are recruited to DNA lesions. We used a proteomics approach that coupled isotopic labeling with chromatin fractionation and mass spectrometry to uncover proteins that associate with damaged DNA, many of which are involved in DNA repair or nucleolar function. We show that polycomb group members are recruited by poly(ADP ribose) polymerase (PARP) to DNA lesions following UV laser microirradiation. Loss of polycomb components results in IR sensitivity of mammalian cells and Caenorhabditis elegans. PARP also recruits two components of the repressive nucleosome remodeling and deacetylase (NuRD) complex, chromodomain helicase DNA-binding protein 4 (CHD4) and metastasis associated 1 (MTA1), to DNA lesions. PARP plays a role in removing nascent RNA and elongating RNA polymerase II from sites of DNA damage. We propose that PARP sets up a transient repressive chromatin structure at sites of DNA damage to block transcription and facilitate DNA repair.

Yoon JC et al. (2014) Proc Natl Acad Sci U S A "GLTSCR2/PICT1 links mitochondrial stress and Myc signaling."

Lee DY, Yoon JC, Sack LM, Steinbaugh MJ, Ling AJ, Isik M, Elledge SJ, Sinclair DA, Blackwell TK, Boduch AN

[Proc Natl Acad Sci U S A, 2014]

Mitochondrial defects underlie a multitude of human diseases. Genetic manipulation of mitochondrial regulatory pathways represents a potential therapeutic approach. We have carried out a high-throughput overexpression screen for genes that affect mitochondrial abundance or activity using flow-cytometry-based enrichment of a cell population expressing a high-complexity, concentration-normalized pool of human ORFs. The screen identified 94 candidate mitochondrial regulators including the nuclear protein GLTSCR2, also known as PICT1. GLTSCR2 enhances mitochondrial function and is required for the maintenance of oxygen consumption, consistent with a pivotal role in the control of cellular respiration. RNAi inactivation of the Caenorhabditis elegans ortholog of GLTSCR2 reduces respiration in worms, indicating functional conservation across species. GLTSCR2 controls cellular proliferation and metabolism via the transcription factor Myc, and is induced by mitochondrial stress, suggesting it may constitute a significant component of the mitochondrial signaling pathway.

Smogorzewska A et al. (2010) Mol Cell "A genetic screen identifies FAN1, a Fanconi anemia-associated nuclease necessary for DNA interstrand crosslink repair."

Smogorzewska A, Schlabach M, Elledge SJ, Kunkel TA, Lach FP, Colaiacovo MP, Harper JW, Saito TT, Desetty R, Sowa ME, Clark AB

[Mol Cell, 2010]

The Fanconi anemia (FA) pathway is responsible for interstrand crosslink repair. At the heart of this pathway is the FANCI-FAND2 (ID) complex, which, upon ubiquitination by the FA core complex, travels to sites of damage to coordinate repair that includes nucleolytic modification of the DNA surrounding the lesion and translesion synthesis. How the ID complex regulates these events is unknown. Here we describe a shRNA screen that led to the identification of two nucleases necessary for crosslink repair, FAN1 (KIAA1018) and EXDL2. FAN1 colocalizes at sites of DNA damage with the ID complex in a manner dependent on FAN1's ubiquitin-binding domain (UBZ), the ID complex, and monoubiquitination of FANCD2. FAN1 possesses intrinsic 5'-3' exonuclease activity and endonuclease activity that cleaves nicked and branched structures. We propose that FAN1 is a repair nuclease that is recruited to sites of crosslink damage in part through binding the ubiquitinated ID complex through its UBZ domain.

Kamura T et al. (1999) Science "Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase."

Koepp DM, Lane WS, Conaway RC, Iliopoulos O, Conrad MN, Conaway JW, Elledge SJ, Moreland RJ, Kaelin WG, Kamura T, Harper JW, Skowyra D

[Science, 1999]

The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in most human kidney cancers. The VHL protein is part of a complex that includes Elongin B, Elongin C, and Cullin-2, proteins associated with transcriptional elongation and ubiquitination. Here it is shown that the endogenous VHL complex in rat liver also includes Rbx1, an evolutionarily conserved protein that contains a RING-H2 fingerlike motif and that interacts with Cullins. The yeast homolog of Rbx1 is a subunit and potent activator of the Cdc53-containing SCFCdc4 ubiquitin ligase required for ubiquitination of the cyclin-dependent kinase inhibitor Sic1 and for the G1 to S cell cycle transition. These findings provide a further link between VHL and the cellular ubiquitination machinery.

Glock C et al. (2015) Methods Mol Biol "Microbial Rhodopsin Optogenetic Tools: Application for Analyses of Synaptic Transmission and of Neuronal Network Activity in Behavior."

Glock C, Gottschalk A, Nagpal J

[Methods Mol Biol, 2015]

Optogenetics was introduced as a new technology in the neurosciences about a decade ago (Zemelman et al., Neuron 33:15-22, 2002; Boyden et al., Nat Neurosci 8:1263-1268, 2005; Nagel et al., Curr Biol 15:2279-2284, 2005; Zemelman et al., Proc Natl Acad Sci USA 100:1352-1357, 2003). It combines optics, genetics, and bioengineering to render neurons sensitive to light, in order to achieve a precise, exogenous, and noninvasive control of membrane potential, intracellular signaling, network activity, or behavior (Rein and Deussing, Mol Genet Genomics 287:95-109, 2012; Yizhar et al., Neuron 71:9-34, 2011). As C. elegans is transparent, genetically amenable, has a small nervous system mapped with synapse resolution, and exhibits a rich behavioral repertoire, it is especially open to optogenetic methods (White et al., Philos Trans R Soc Lond B Biol Sci 314:1-340, 1986; De Bono et al., Optogenetic actuation, inhibition, modulation and readout for neuronal networks generating behavior in the nematode Caenorhabditis elegans, In: Hegemann P, Sigrist SJ (eds) Optogenetics, De Gruyter, Berlin, 2013; Husson et al., Biol Cell 105:235-250, 2013; Xu and Kim, Nat Rev Genet 12:793-801, 2011). Optogenetics, by now an "exploding" field, comprises a repertoire of different tools ranging from transgenically expressed photo-sensor proteins (Boyden et al., Nat Neurosci 8:1263-1268, 2005; Nagel et al., Curr Biol 15:2279-2284, 2005) or cascades (Zemelman et al., Neuron 33:15-22, 2002) to chemical biology approaches, using photochromic ligands of endogenous channels (Szobota et al., Neuron 54:535-545, 2007). Here, we will focus only on optogenetics utilizing microbial rhodopsins, as these are most easily and most widely applied in C. elegans. For other optogenetic tools, for example the photoactivated adenylyl cyclases (PACs, that drive neuronal activity by increasing synaptic vesicle priming, thus exaggerating rather than overriding the intrinsic activity of a neuron, as occurs with rhodopsins), we refer to other literature (Weissenberger et al., J Neurochem 116:616-625, 2011; Steuer Costa et al., Photoactivated adenylyl cyclases as optogenetic modulators of neuronal activity, In: Cambridge S (ed) Photswitching proteins, Springer, New York, 2014). In this chapter, we will give an overview of rhodopsin-based optogenetic tools, their properties and function, as well as their combination with genetically encoded indicators of neuronal activity. As there is not "the" single optogenetic experiment we could describe here, we will focus more on general concepts and "dos and don'ts" when designing an optogenetic experiment. We will also give some guidelines on which hardware to use, and then describe a typical example of an optogenetic experiment to analyze the function of the neuromuscular junction, and another application, which is Ca(2+) imaging in body wall muscle, with upstream neuronal excitation using optogenetic stimulation. To obtain a more general overview of optogenetics and optogenetic tools, we refer the reader to an extensive collection of review articles, and in particular to volume 1148 of this book series, "Photoswitching Proteins."