GRAS-1 is a novel regulator of early meiotic chromosome dynamics in C. elegans.

Chromosome movements and licensing of synapsis must be tightly regulated during early meiosis to ensure accurate chromosome segregation and avoid aneuploidy, although how these steps are coordinated is not fully understood. Here we show that GRAS-1, the worm homolog of mammalian GRASP/Tamalin and CYTIP, coordinates early meiotic events with cytoskeletal forces outside the nucleus. GRAS-1 localizes close to the nuclear envelope (NE) in early prophase I and interacts with NE and cytoskeleton proteins. Delayed homologous chromosome pairing, synaptonemal complex (SC) assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in gras-1 mutants, supporting functional conservation. However, Tamalin, Cytip double knockout mice do not exhibit obvious fertility or meiotic defects, suggesting evolutionary differences between mammals. gras-1 mutants show accelerated chromosome movement during early prophase I, implicating GRAS-1 in regulating chromosome dynamics. GRAS-1-mediated regulation of chromosome movement is DHC-1-dependent, placing it acting within the LINC-controlled pathway, and depends on GRAS-1 phosphorylation at a C-terminal S/T cluster. We propose that GRAS-1 coordinates the early steps of homology search and licensing of SC assembly by regulating the pace of chromosome movement in early prophase I.

Histone demethylase AMX-1 is necessary for proper sensitivity to interstrand crosslink DNA damage.

Histone methylation is dynamically regulated to shape the epigenome and adjust central nuclear processes including transcription, cell cycle control and DNA repair. Lysine-specific histone demethylase 2 (LSD2) has been implicated in multiple types of human cancers. However, its functions remain poorly understood. This study investigated the histone demethylase LSD2 homolog AMX-1 in C. elegans and uncovered a potential link between H3K4me2 modulation and DNA interstrand crosslink (ICL) repair. AMX-1 is a histone demethylase and mainly localizes to embryonic cells, the mitotic gut and sheath cells. Lack of AMX-1 expression resulted in embryonic lethality, a decreased brood size and disorganized premeiotic tip germline nuclei. Expression of AMX-1 and of the histone H3K4 demethylase SPR-5 is reciprocally up-regulated upon lack of each other and the mutants show increased H3K4me2 levels in the germline, indicating that AMX-1 and SPR-5 regulate H3K4me2 demethylation. Loss of AMX-1 function activates the CHK-1 kinase acting downstream of ATR and leads to the accumulation of RAD-51 foci and increased DNA damage-dependent apoptosis in the germline. AMX-1 is required for the proper expression of mismatch repair component MutL/MLH-1 and sensitivity against ICLs. Interestingly, formation of ICLs lead to ubiquitination-dependent subcellular relocalization of AMX-1. Taken together, our data suggest that AMX-1 functions in ICL repair in the germline.

Fanconi Anemia FANCM/FNCM-1 and FANCD2/FCD-2 Are Required for Maintaining Histone Methylation Levels and Interact with the Histone Demethylase LSD1/SPR-5 in Caenorhabditis elegans.

The histone demethylase LSD1 was originally discovered by removing methyl groups from di- and monomethylated histone H3 lysine 4 (H3K4me2/1). Several studies suggest that LSD1 plays roles in meiosis as well as in the epigenetic regulation of fertility given that, in its absence, there is evidence of a progressive accumulation of H3K4me2 and increased sterility through generations. In addition to the progressive sterility phenotype observed in the mutants, growing evidence for the importance of histone methylation in the regulation of DNA damage repair has attracted more attention to the field in recent years. However, we are still far from understanding the mechanisms by which histone methylation is involved in DNA damage repair, and only a few studies have focused on the roles of histone demethylases in germline maintenance. Here, we show that the histone demethylase LSD1/CeSPR-5 interacts with the Fanconi anemia (FA) protein FANCM/CeFNCM-1 using biochemical, cytological, and genetic analyses. LSD1/CeSPR-5 is required for replication stress-induced S phase-checkpoint activation, and its absence suppresses the embryonic lethality and larval arrest observed in fncm-1 mutants. FANCM/CeFNCM-1 relocalizes upon hydroxyurea exposure and colocalizes with FANCD2/CeFCD-2 and LSD1/CeSPR-5, suggesting coordination between this histone demethylase and FA components to resolve replication stress. Surprisingly, the FA pathway is required for H3K4me2 maintenance, regardless of the presence of replication stress. Our study reveals a connection between FA and epigenetic maintenance and therefore provides new mechanistic insight into the regulation of histone methylation in DNA repair.

A histone methylation network regulates transgenerational epigenetic memory in C. elegans.

How epigenetic information is transmitted from generation to generation remains largely unknown. Deletion of the C. elegans histone H3 lysine 4 dimethyl (H3K4me2) demethylase spr-5 leads to inherited accumulation of the euchromatic H3K4me2 mark and progressive decline in fertility. Here, we identified multiple chromatin-modifying factors, including H3K4me1/me2 and H3K9me3 methyltransferases, an H3K9me3 demethylase, and an H3K9me reader, which either suppress or accelerate the progressive transgenerational phenotypes of spr-5 mutant worms. Our findings uncover a network of chromatin regulators that control the transgenerational flow of epigenetic information and suggest that the balance between euchromatic H3K4 and heterochromatic H3K9 methylation regulates transgenerational effects on fertility.

MAPK Hog1 closes the S. cerevisiae glycerol channel Fps1 by phosphorylating and displacing its positive regulators.

The aquaglyceroprin Fps1 is responsible for glycerol transport in yeast in response to changes in extracellular osmolarity. Control of Fps1 channel activity in response to hyperosmotic shock involves a redundant pair of regulators, Rgc1 (regulator of the glycerol channel 1) and Rgc2, and the MAPK Hog1 (high-osmolarity glycerol response 1). However, the mechanism by which these factors influence channel activity is unknown. We show that Rgc2 maintains Fps1 in the open channel state in the absence of osmotic stress by binding to its C-terminal cytoplasmic domain. This interaction involves a tripartite pleckstrin homology (PH) domain within Rgc2 and a partial PH domain within Fps1. Activation of Hog1 in response to hyperosmotic shock induces the rapid eviction of Rgc2 from Fps1 and consequent channel closure. Hog1 was recruited to the N-terminal cytoplasmic domain of Fps1, which it uses as a platform from which to multiply phosphorylate Rgc2. Thus, these results reveal the mechanism by which Hog1 regulates Fps1 in response to hyperosmotic shock.

Mutants in the Candida glabrata glycerol channels are sensitized to cell wall stress.

Many fungal species use glycerol as a compatible solute with which to maintain osmotic homeostasis in response to changes in external osmolarity. In Saccharomyces cerevisiae, intracellular glycerol concentrations are regulated largely by the high osmolarity glycerol (HOG) response pathway, both through induction of glycerol biosynthesis and control of its flux through the plasma membrane Fps1 glycerol channel. The channel activity of Fps1 is also controlled by a pair of positive regulators, Rgc1 and Rgc2. In this study, we demonstrate that Candida glabrata, a fungal pathogen that possesses two Fps1 orthologs and two Rgc1/-2 orthologs, accumulates glycerol in response to hyperosmotic stress. We present an initial characterization of mutants with deletions in the C. glabrata FPS1 (CAGL0C03267 [www.candidagenome.org]) and FPS2 (CAGL0E03894) genes and find that a double mutant accumulates glycerol, experiences constitutive cell wall stress, and is hypersensitive to treatment by caspofungin, an antifungal agent that targets the cell wall. This mutant is cleared more efficiently in mouse infections than is wild-type C. glabrata by caspofungin treatment. Finally, we demonstrate that one of the C. glabrata RGC orthologs complements an S. cerevisiae rgc1 rgc2 null mutant, supporting the conclusion that this regulatory assembly is conserved between these species.

Yeast Fps1 glycerol facilitator functions as a homotetramer.

The Saccharomyces cerevisiae Fps1 glycerol channel is a member of the major intrinsic protein (MIP) family of plasma membrane channel proteins that functions in osmoregulatory pathways to transport glycerol passively out of the cell. The MIP family is subdivided into members that are selectively permeable to water (aquaporins) and those permeated by glycerol (aquaglyceroporins or glycerol facilitators). Although aquaporins function as homo-tetramers with each monomer possessing its own channel, previous studies have suggested that aquaglyceroporins may function as monomers. Here we provide both genetic and biochemical evidence that Fps1 functions as a homotetramer to regulate glycerol transport in yeast.

SPR-5 is a histone H3K4 demethylase with a role in meiotic double-strand break repair.

Regulation of histone methylation levels has long been implicated in multiple cellular processes, many of which involve transcription. Here, however, we report a unique role for the Caenorhabditis elegans histone demethylase SPR-5 in meiotic DNA double-strand break repair (DSBR). SPR-5 shows enzymatic activity toward H3K4me2 both in vitro and in the nematode germline, and spr-5 mutants show several phenotypes indicating a perturbation of DSBR, including increased p53-dependent germ cell apoptosis, increased levels of the DSBR marker RAD-51, and sensitivity toward DSB-inducing treatments. spr-5 mutants show no transcriptional misregulation of known DSBR involved genes. Instead, SPR-5 shows a rapid subcellular relocalization upon DSB-inducing treatment, which suggests that SPR-5 may function directly in DSBR.