Bian, Qian et al. (2013) International Worm Meeting "X-Chromosome Restructuring Imposed by the Dosage Compensation Complex and Its Relationship to Nuclear Pores."

Crane, Emily, Meyer, Barbara J., Bian, Qian, Uzawa, Satoru

[International Worm Meeting, 2013]

In C. elegans, dosage compensation equalizes X-chromosome gene expression between males (XO) and hermaphrodites (XX) by reducing transcription from both hermaphrodite X chromosomes by half. Dosage compensation is implemented by a dosage compensation complex (DCC) that contains 5 subunits homologous to canonical subunits of condensin, a complex that restructures mitotic and meiotic chromosomes in preparation for their segregation. The similarity between the DCC and condensin suggests the DCC may modulate X-linked gene expression by remodeling X-chromosome structure. To test this hypothesis, we first examined the relative nuclear location of DCC binding sites using 3D FISH. We found that pairs of rex sites (DCC recruitment sites on X) between 500 kb and 8 Mbp apart colocalize at a higher frequency in wild-type XX embryos and XO mutant embryos with a DCC-bound X than wild-type XO embryos lacking DCC binding on X. Both 5C and Hi-C experiments confirmed the DCC-dependent interactions between rex sites and revealed significant differences in X-chromosome architecture between wild-type embryos and DCC-deficient embryos. We also examined the 3D location of non-DCC binding sites on X using FISH. We found that several pairs of non-DCC binding sites 500 kbp apart are in physical proximity more frequently when the DCC is bound to X. This result suggests that regions of X distal to DCC binding sites become more compacted upon DCC binding, likely a result of direct interactions mediated by DCC binding at distal rex sites. We also found that distant rex sites preferentially co-localize at the nuclear periphery in embryos with DCC binding to X, but not in wild-type XO embryos. Using 3D structured illumination microscopy, we performed sub-diffraction imaging of the nuclear periphery in embryos and observed that the pairs of rex sites in close proximity are associated with nuclear pore complexes (NPC) and not nuclear lamin. Furthermore, we show that the DCC also associates with NPCs at the nuclear periphery. Our results show that the DCC imposes a structure on X and imply that nuclear pores may assist in restructuring X chromosomes through physical interactions with the DCC.

Anderson, Erika et al. (2015) International Worm Meeting "Remodeling X chromosome topology during dosage compensation."

Meyer, Barbara J., Bian, Qian, Anderson, Erika

[International Worm Meeting, 2015]

In C. elegans, dosage compensation reduces the expression of X-linked genes by half in hermaphrodites (XX) to equal their expression level in males (X0). During dosage compensation, a condensin-like dosage compensation complex (DCC) actively remodels X chromosomes of hermaphrodites into a unique, sex-specific spatial conformation, distinct from that of autosomes, using its highest-affinity recruitment sites on X (rex) to facilitate long-range interactions across X. The dosage-compensated X chromosome consists of self-interacting domains resembling mammalian topologically associating domains (TADs). TADs are found in all metazoans examined, but neither the mechanism by which they are formed nor their functional significance for transcriptional regulation is well understood. Because dosage compensation involves global changes in chromosome structure accompanied by chromosome-wide gene repression, we can use this system to investigate the mechanisms of TAD boundary establishment and the relationship between TAD structure and transcription. Each DCC-dependent TAD boundary contains a strong rex site. To test the model that rex interactions establish TAD boundaries, we are deleting rex sites from X and inserting new rex sites on X and autosomes. We deleted three strong rex sites that interact with each other and are located at adjacent DCC-dependent TAD boundaries using CRISPR/Cas9-mediated homology-directed repair. For one of these sites, we have shown that the deletion causes a loss of DCC binding and significantly diminishes the TAD boundary. We are assessing the impact on TAD structure and gene expression caused by deleting all three pivotal rex sites.

Anderson, Erika et al. (2017) International Worm Meeting "X chromosome topology driven by condensin binding to eight high-affinity sites."

Bian, Qian, Anderson, Erika, Schartner, Caitlin, Meyer, Barbara J.

[International Worm Meeting, 2017]

Interphase chromosome structure is regulated at multiple length scales, and each level of organization controls nuclear functions such as transcription, replication, and recombination. To learn how these structures are formed, we dissected the mechanism by which a condensin complex imposes a distinct, higher-order structure across X chromosomes of C. elegans hermaphrodites. Here our deletion analysis of the endogenous X revealed that architectural proteins can remodel chromosome-wide topology by binding to a small number of sites. The dosage compensation complex (DCC), a specialized condensin complex, is recruited to dozens of specific sites (rex sites) on both hermaphrodite X chromosomes and represses X-linked gene expression by half. The DCC establishes a higher-order structure composed of megabase-scale topologically associating domains (TADs) that is distinct from the structure of autosomes and male X chromosomes. The DCC-dependent TAD boundaries all contain a strong rex site, and the DCC promotes long-range interactions both between rex sites at TAD boundaries and between rex sites within TADs. To discern the contributions of these different rex-site interactions to TAD boundary formation, we deleted the eight rex sites at DCC-dependent TAD boundaries and examined X structure using an updated in-nucleus Hi-C protocol that detects distant interactions efficiently. In the rex-delete strain, the specific loops between adjacent DCC-dependent TAD boundaries were eliminated. All eight DCC-dependent boundaries were lost or significantly weakened, producing a structure that recapitulates the X structure of a DCC mutant. Therefore, though the DCC binds dozens of sites on X, the TAD boundaries are established by binding to these eight high-affinity sites. Disruption of TAD structure by deleting a series of cis elements uniquely allows us to assess the effects of TAD boundaries on gene expression. The rex-deleted worms lack strong dosage compensation phenotypes, indicating that TAD boundaries alone are insufficient to enact full repression of X gene expression. Ongoing sensitive gene expression assays are determining whether TAD boundary disruption causes local or subtle transcriptional changes. Intra-TAD interactions present in wild-type and rex-delete animals but absent in DCC mutants likely underlie mechanisms that control X gene expression. Furthermore, Hi-C results showed that in C. briggsae, which also uses a condensin DCC, X chromosomes have a unique TAD structure compared to that of autosomes, even though the rex sites are in different locations relative to C. elegans' sites. Hi-C also revealed errors in C. briggsae's genome assembly, which are now being corrected based on Hi-C data. This improved genome assembly facilitates studies of how condensin's role in shaping higher-order chromosome structure is maintained as its binding sites evolve.

Wheeler, Bayly et al. (2015) International Worm Meeting "Regulation of nuclear organization and long-range gene expression by condensin."

Meyer, Barbara J., Frokjaer-Jensen, Christian, Anderson, Erika, Jorgensen, Erik, Wheeler, Bayly, Bian, Qian

[International Worm Meeting, 2015]

The relationship between chromosome structure, nuclear positioning, and long-range gene regulation is poorly understood. To explore this relationship, we dissected X-chromosome-wide gene regulation enacted by a dosage compensation complex (DCC), which represses X transcription in hermaphrodites to balance gene expression between sexes. We inserted transgenes throughout the genome and queried their expression to determine whether different transcriptional environments exist on X and autosomes. Transgenes integrated on X were dosage compensated regardless of position, meaning their expression was equal in wild-type males and hermaphrodites but elevated in dosage-compensation-defective hermaphrodites. This result indicates the X chromosome is broadly permissive for repression, and endogenous genes that escape have special features enabling them to overcome this repression. In contrast, we found no chromosome-wide mechanism to balance X expression with that of autosomes, given that transgenes on X were expressed at half the level of transgenes on autosomes. Repression of X transgenes was independent of their proximity to DCC recruitment sites (rex), highlighting the long-range mechanism of regulation employed by the DCC. We already showed that changes in higher order X-chromosome structure accompany repression of X-linked genes, so we next explored whether spatial positioning of X influences dosage compensation. We first addressed a model of others that rex sites target X to the nuclear periphery in males to increase gene expression, and DCC binding to rex sites in hermaphrodites helps relocate X to the interior, thereby repressing X. Using FISH, we found for both sexes that neither endogenous rex sites on X nor ectopically inserted rex sites on autosomes were preferentially located at the nuclear periphery. Furthermore, though rex insertions on autosomes recruit the DCC, the expression of adjacent genes was not elevated in DCC-depleted animals. These observations disfavor the proposed model. Instead, we found that pairs of distant rex sites interact in a DCC-dependent manner, and interacting rex sites are preferentially located at the nuclear periphery compared to non-interacting sites. Interacting rex pairs associate with nuclear pores, not the lamina. We propose the nuclear pore might act as a scaffold to promote rex site interactions, which in turn influence gene expression by remodeling higher order chromosome structure.

Zhu, Di et al. (2021) International Worm Meeting "NuRD mediates mitochondrial stress-induced longevity via chromatin remodeling in response to acetyl-CoA level"

Zhu, Di, Tian, Ye, Li, Xinyu, Wu, Junbo, Wu, Xueying, Li, Jiasheng, Bian , Qian, Zhou, Jun, Huang, Xiahe, Wang, Yingchun

[International Worm Meeting, 2021]

Mild mitochondrial stress experienced early in life can have beneficial effects on the life span of organisms through epigenetic regulations. Here, we report that acetyl-coenzyme A (CoA) represents a critical mitochondrial signal to regulate aging through the chromatin remodeling and histone deacetylase complex (NuRD) in Caenorhabditis elegans. Upon mitochondrial stress, the impaired tricarboxylic acid cycle results in a decreased level of citrate, which accounts for reduced production of acetyl-CoA and consequently induces nuclear accumulation of the NuRD and a homeodomain-containing transcription factor DVE-1, thereby enabling decreased histone acetylation and chromatin reorganization. The metabolic stress response is thus established during early life and propagated into adulthood to allow transcriptional regulation for life-span extension. Furthermore, adding nutrients to restore acetyl-CoA production is sufficient to counteract the chromatin changes and diminish the longevity upon mitochondrial stress. Our findings uncover the molecular mechanism of the metabolite-mediated epigenome for the regulation of organismal aging.

Boeck, Max E. et al. (2009) International Worm Meeting "Predicted 3D Structures of the DNA-Binding Domain for the Ten C. elegans GATA Transcription Factors and Their Preferred DNA Sequence."

Waterston, Robert, Thompson, James, Bradley, Phil, Boeck, Max E.

[International Worm Meeting, 2009]

Based on the paradigm that structure dictates function we used a computational approach to determine the preferred DNA motif bound by each of the GATA transcription factors found in C. elegans. GATA transcription factors have been shown to be important for organogenesis and cell fate specification in every metazoan from C. elegans to humans. The ability of GATA factors to specify cell-fate decisions is dictated by their ability to bind a DNA motif, WGATAR, with high fidelity. GATA factors have been shown to bind the motif through the actions of a conserved DNA-binding structure, CXXC(X)17-19CXXC [1]. While the basic DNA-binding structure is conserved between members of the GATA family,numerous specificity-determining residues vary between GATA genes. Using a maximum-likelihood phylogenetic approach [2] we estimated a phylogenetic tree in order to model the evolutionary relationships between GATA factors in the sequenced Caenorhabditis species. We then used Rosetta homology modeling [3] to construct structural models of the DNA-binding structures of all the GATA factors found within C. elegans. By tracking the specificity-determining residues that have changed along the phylogenetic tree of these GATA factors, we have defined a prediction of which GATA factors are likely to bind very similar segments of DNA. [1] Lowry, Atchley J Mol Evol. 2000 50:103-115 [2] Guindon S, Gascuel O. Syst Bio. 2003 52(5):696-704. [3] Qian et al, Nature. 2007 Nov 8;450(7167):259-64.

Doug Braun et al. (2001) International C. elegans Meeting "Identification of New Components in the C. Elegans PKD Pathway"

Doug Braun, Maureen M Barr

[International C. elegans Meeting, 2001]

Autosomal Dominant Polycystic Kidney Disease (ADPKD) strikes about 1 in every thousand people. This systemic disease can cause the growth of kidney cysts and affect the liver, pancreas and cardiovascular system. Half of affected individuals will suffer from end stage renal failure by the time they are 60. Approximately 95% of all cases of ADPKD are caused by mutations in either PKD1 or PKD2. The C. elegans homologs of PKD1 and PKD2 ( lov-1 and pkd-2 ) are expressed in male sensory neurons in the head, hook and rays(1). Mutations in lov-1 and pkd-2 result in defects in two male mating behaviors: response and vulva location (1). Localization and mutant phenotypes suggest the C. elegans polycystins LOV-1 and PKD-2 act in the same pathway. The cellular role of the polycystins is still unknown. The cytoplasmic tails of polycystin-1 and polycystin-2 (encoded by PKD1 and PKD2 respectively) have been shown to interact using the yeast two-hybrid system (2,3). Polycystin-2 has also been shown to interact with itself through its probable coiled-coil domain. Recent studies indicate polycystin-1 and polycystin-2 form a cation channel. We are using the two-hybrid system to examine interactions between LOV-1 and PKD-2 and to identify new proteins in the polycystin pathway. We have created two cDNA libraries (one directionally cloned, the other random) using mRNA from a male-enriched him-5(e1490) population. Initially we will use as bait the intracellular carboxy termini of LOV-1 and PKD-2. These regions are of interest because of the demonstrated interaction between the human polycystins and because a 58 amino acid c-terminal truncation of LOV-1 results in a nonfunctional protein. Another bait to be used in screens is the PLAT domain in LOV-1 because this region is conserved in all polycystin-1 family members. At the meeting we will present potential candidates isolated and characterized thus far. 1. Barr, M., Sternberg, P. Nature . 23 Sep 1999; 401:386-389 2. Qian, F., et al. Nature Genetics . June 1997; 16:179-183 3. Tsiakas, L., et al. Proc. Natl. Acad. Sci . June 1997; 94:6965-6970

Mark Alkema et al. (2001) International C. elegans Meeting "A C. elegans CREB PROTEIN MODULATES TGF-BETA SIGNALING"

Bob Horvitz, Mark Alkema

[International C. elegans Meeting, 2001]

The cyclic AMP-response element binding protein CREB plays a central role in long-term memory in Aplysia , Drosophila and mice. Analysis of the genome sequence showed that C. elegans has a single CR EB - h omologous gene ( crh-1 ). We characterized the C. elegans and C. briggsae crh-1 genes and found that they have a similar gene structure and that their encoded proteins are 85% identical. The DNA-binding bZIP domain and cAMP-dependent kinase site, as defined in the mammalian and Drosophila CREB family members, are highly conserved in the nematode proteins. The C. elegans crh-1 gene has several alternatively-spliced isoforms. A crh-1::gfp transgene is ubiquitously expressed during early embryogenesis and is specifically expressed in several neurons from the L1 stage to adulthood. CRH-1 can bind to cyclic AMP-response element (CRE) sites and can be phosophorylated by cAMP-dependent protein kinase (PKA) and Calmodulin-dependent protein kinase II (CaMKII) in vitro . To determine the function of crh-1 , we isolated three crh-1 deletion alleles from a deletion library. Two deletions remove the second exon and are predicted to cause early truncations; the third deletes part of the bZIP domain. No CRH-1 protein is detected by western blot analyses of these three mutant strains, suggesting that all three are null alleles. crh-1 mutants are viable and show no obvious abnormalities in brood size, locomotion, mechanosensation, chemotaxis or thermotaxis. We found that mutations in crh-1 confer a dauer-constitutive phenotype (Daf-c). However, like unc-3, unc-31, and unc-64 1 mutants, crh-1 mutants form dauers at 27C but not at 25C. In addition, like the Daf-c mutants daf-1, daf-7, daf-8, and daf-14 , crh-1 mutants tend to accumulate at the edge of a bacterial lawn (bordering) and form clumps of animals. Mutations in many genes that cause a Daf-c phenotype at 25C have been isolated. Characterization of these mutants has shown that an insulin-like and a DAF-7 transforming growth factor (TGF)-beta signaling pathway act in parallel to regulate dauer formation. Dauer pheromone, temperature, and food signals modulate the dauer decision in part by regulating the expression of the TGF-beta homolog, DAF-7 in the ASI chemosensory neurons. Although unc-3, unc-31 , unc-64 1 , and crh-1 single mutants do not have strong defects in dauer formation at lower temperatures, double mutant combinations of unc-3, unc-31, and unc-64 1 are strongly Daf-c at 25C. Double mutants between crh-1 and either unc-31 or unc-64, but not unc-3, show a strongly enhanced Daf-c phenotype at 25C. This observation suggests that crh-1 and unc-3 affect similar aspects of dauer formation. unc-3 encodes a transcription factor that is expressed in the ASI neurons and may regulate the expression of daf-7 2 . Consistent with this notion we found that the expression of a daf-7::gfp reporter is strongly reduced in crh-1 mutants. Our data suggest that crh-1 mutants incorrectly integrate environmental cues that induce dauer formation and that crh-1 is part of a chemosensory cascade that regulates TGF-beta signaling. 1. Ailion and Thomas. (2000) Genetics 156 , 1047-1067. 2. Ren, Qian, McCron, and Riddle. (1998) Midwest Worm Meeting abstract, p. 30.