Context-specific regulation of lysosomal lipolysis through network-level diverting of transcription factor interactions

Plasticity in multicellular organisms involves signaling pathways converting contexts—either natural environmental challenges or laboratory perturbations—into context-specific changes in gene expression. Congruently, the interactions between the signaling molecules and transcription factors (TF) regulating these responses are also context specific. However, when a target gene responds across contexts, the upstream TF identified in one context is often inferred to regulate it across contexts. Reconciling these stable TF–target gene pair inferences with the context-specific nature of homeostatic responses is therefore needed. The induction of the Caenorhabditis elegans genes lipl-3 and lipl-4 is observed in many genetic contexts and is essential to survival during fasting. We find DAF-16/FOXO mediating lipl-4 induction in all contexts tested; hence, lipl-4 regulation seems context independent and compatible with across-context inferences. In contrast, DAF-16–mediated regulation of lipl-3 is context specific. DAF-16 reduces the induction of lipl-3 during fasting, yet it promotes it during oxidative stress. Through discrete dynamic modeling and genetic epistasis, we define that DAF-16 represses HLH-30/TFEB—the main TF activating lipl-3 during fasting. Contrastingly, DAF-16 activates the stress-responsive TF HSF-1 during oxidative stress, which promotes C. elegans survival through induction of lipl-3. Furthermore, the TF MXL-3 contributes to the dominance of HSF-1 at the expense of HLH-30 during oxidative stress but not during fasting. This study shows how context-specific diverting of functional interactions within a molecular network allows cells to specifically respond to a large number of contexts with a limited number of molecular players, a mode of transcriptional regulation we name “contextualized transcription.”

Genetic analysis of Caenorhabditis elegans pry-1/Axin suppressors identifies genes involved in reproductive structure development, stress response, and aging

The Axin family of scaffolding proteins regulates a wide array of developmental and post-developmental processes in eukaryotes. Studies in the nematode, Caenorhabditis elegans, have shown that the Axin homolog, PRY-1, plays essential roles in multiple tissues. To understand the genetic network of pry-1, we focused on a set of genes that are differentially expressed in the pry-1-mutant transcriptome and are linked to reproductive structure development. Eight of the genes (ard-1, rpn-7, cpz-1, his-7, cdk-1, rnr-1, clsp-1, and spp-1), when knocked down by RNA interference, efficiently suppressed the plate-level multivulva phenotype of pry-1 mutants. In every case, other than clsp-1 and spp-1, the ectopic vulval precursor cell (VPC) induction was also inhibited. The suppressor genes are members of known gene families in eukaryotes and perform essential functions. Our genetic interaction experiments revealed that except for clsp-1, the genes participate in one or more pry-1-mediated biological events. While four of them (cpz-1, his-7, cdk-1, and rnr-1) function in VPC induction, stress response, and aging, the other three (spp-1, ard-1, and rpn-7) are specific to one or more of these processes. Further analysis of the genes involved in aging showed that his-7, cdk-1, and rnr-1 also interacted with daf-16/FOXO. The results of genetic epistasis experiments suggested that his-7 functions upstream of daf-16, whereas cdk-1and rnr-1 act downstream of the pry-1-daf-16 pathway. Altogether, these findings demonstrate the important role of pry-1 suppressors in C. elegans. Given that all of the genes described in this study are conserved, future investigations of their interactions with Axin and their functional specificity promises to uncover the genetic network of Axin under normal and disease states.

ANALYSIS OF CHD-7 DEFECTIVE DAUER NEMATODES IMPLICATES COLLAGEN MISREGULATION IN CHARGE SYNDROME FEATURES

ABSTRACTCHARGE syndrome is a complex developmental disorder caused by mutations in the chromodomain helicase DNA-binding protein7 (CHD7) and characterized by retarded growth and malformations in the heart and nervous system. However, despite the public health relevance of this disorder, relevant targets of CHD7 that relate to disease pathology are still poorly understood. Here we report thatchd-7, the nematode ortholog of CHD7, is required for dauer morphogenesis, lifespan determination, and stress response. Genetic epistasis placedchd-7in the TGF-β pathway. Consistent with our discoveries, we foundchd-7to be allelic toscd-3, a previously identified dauer suppressor from the TGF-β pathway. Interestingly, DAF-12 transcriptionally upregulatedchd-7, which is necessary to repressdaf-9for execution of the dauer program. Transcriptomic analysis comparingchd-7–defective and normal dauers showed enrichment of collagen genes, consistent with a conserved role for the TGF-β pathway in expression of the extracellular matrix. To validate a conserved function forchd-7in vertebrates, we usedXenopus laevisembryos, an established model to study craniofacial development. Morpholino mediated knockdown of Chd7 led to embryonic lethality, a reduction incol2a1mRNA levels and craniofacial defects in tadpoles. Both lethality and malformations were partially rescued in Chd7-depleted embryos by over-expression ofcol2a1. We suggest that pathogenic features of CHARGE syndrome caused by Chd7 mutations, such as craniofacial malformations, result from the reduction of collagen levels. These studies establishC. elegansas an amenable animal model to study the etiology of the developmental defects associated with pathogenic Chd7.

Nodal Modulator is required to sustain endoplasmic reticulum morphology

Nodal Modulator (NOMO) is a widely conserved type I transmembrane protein of unknown function, with three nearly identical orthologs specified in the human genome. We identified NOMO1 in a proteomics approach aimed at the identification of proteins that support the structural integrity of the endoplasmic reticulum (ER). Overexpression of NOMO1 imposes a sheet morphology on the ER, while depletion of NOMO1 and its orthologs causes a collapse of ER morphology concomitant with the formation of membrane-delineated holes in the ER network. These structures are positive for the autophagy marker LAMP1, and LC3 is profoundly upregulated upon NOMO depletion. In vitro reconstitution of NOMO1 revealed a dimeric state that is mediated by the cytosolic tail domain, with each monomer featuring a “beads on a string” structure likely representing bacterial Ig-like folds. Based on these observations and a genetic epistasis analysis including the known ER-shaping proteins Atlastin2 and Climp63, we propose a role for NOMO1 in the functional network of ER-shaping proteins.

Neuroglian regulates Drosophila intestinal stem cell proliferation through enhanced signaling via the Epidermal Growth Factor Receptor

SummaryThe Drosophila melanogaster intestine is an excellent system for elucidating mechanisms regulating stem cell behavior under homeostatic conditions or in response to injury, stress, or ageing. Here we show that the septate junction (SJ) protein Neuroglian (Nrg) is expressed in intestinal stem cells (ISCs) and daughter enteroblasts (EBs) within the fly midgut, the equivalent of the mammalian small intestine. Although Nrg localizes to the plasma membrane, SJs are not present between ISC/EBs, suggesting Nrg plays a different role in this tissue. Generation of ISCs homozygous for a null allele of Nrg revealed that Nrg is required for ISC proliferation in young flies, and depletion of Nrg from ISCs/EBs was able to suppress the increase in ISC proliferation with age. Conversely, overexpression of Nrg in ISC/EBs was sufficient to drive ISC proliferation, leading to an increase in cells expressing ISC/EB markers. In addition, we observed an increase in EGFR activation. Genetic epistasis experiments revealed that Nrg acts upstream of EGFR in the midgut to regulate ISC proliferation. As Nrg function is highly conserved in mammalian systems, our work characterizing the role of Nrg in the intestine has implications for the etiology and treatment of intestinal disorders due to altered ISC behavior.

S-adenosyl methionine synthetase SAMS-5 mediates dietary restriction-induced longevity in Caenorhabditis elegans

S-adenosyl methionine synthetase (SAMS) catalyzes the biosynthesis of S-adenosyl methionine (SAM), which serves as a universal methyl group donor for numerous biochemical reactions. Previous studies have clearly demonstrated that SAMS-1, a C. elegans homolog of mammalian SAMS, is critical for dietary restriction (DR)-induced longevity in Caenorhabditis elegans. In addition to SAMS-1, three other SAMS paralogs have been identified in C. elegans. However, their roles in longevity regulation have never been explored. Here, we show that depletion of sams-5, but not sams-3 or sams-4, can extend lifespan in worms. However, the phenotypes and expression pattern of sams-5 are distinct from sams-1, suggesting that these two SAMSs might regulate DR-induced longevity via different mechanisms. Through the genetic epistasis analysis, we have identified that sams-5 is required for DR-induced longevity in a pha-4/FOXA dependent manner.

Loss of dmrt1 restores zebrafish female fates in the absence of cyp19a1a but not rbpms2a/b

ABSTRACTSex determination and differentiation is a complex process regulated by multiple factors, including factors from the germline or surrounding somatic tissue. In zebrafish, sex-determination involves establishment of a bipotential ovary that undergoes sex-specific differentiation and maintenance to form the functional adult gonad. However, the relationships among these factors are not fully understood. Here, we identify potential Rbpms2 targets and apply genetic epistasis experiments to decipher the genetic hierarchy of regulators of sex-specific differentiation. We provide genetic evidence that the crucial female factor rbpms2 is epistatic to the male factor dmrt1 in terms of adult sex. Moreover, the role of Rbpms2 in promoting female fates extends beyond repression of Dmrt1, as Rbpms2 is essential for female differentiation even in the absence of Dmrt1. In contrast, female fates can be restored in mutants lacking both cyp19a1a and dmrt1, and prolonged in bmp15 mutants in the absence of dmrt1. Taken together, this work indicates that cyp19a1a-mediated suppression of dmrt1 establishes a bipotential ovary and initiates female fate acquisition. Then, after female fate specification, Cyp19a1a regulates subsequent oocyte maturation and sustains female fates independently of Dmrt1 repression.

Maintenance of mitochondrial integrity in midbrain dopaminergic neurons governed by a conserved developmental transcription factor

ABSTRACTThe degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson’s Disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, here we demonstrate that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. We further show that conditional ablation of Nato3, a mouse homolog of Fer2, in differentiated DA neurons results in locomotor impairments and mitochondrial abnormality in aged mice. Our results reveal the essential and conserved role of Fer2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modelling and treating PD.