Das, Moushumi et al. (2019) International Worm Meeting "3D Genomic Architecture and Transcriptional Regulation."

Semple, Jennifer, Ridder, Jeroen de, Meister, Peter, Allahyar, Amin, Das, Moushumi, Straver, Roy

[International Worm Meeting, 2019]

Understanding how our genes are regulated is a central question in biology. Studies have highlighted multiple levels of regulation for genomic expression, ranging from transcription factors binding to nuclear organization of the genome. Recent technical optimization of chromosome conformation capture (3C) analysis has revealed new levels of chromosome compartmentalization. Interphase chromosomes are arranged into specific kilo- to mega-base sized domains, known as Topologically Associated Domains (TADs). Functionally, most enhancer-promoter interactions occur within the same TAD. Moreover, during differentiation, genes clustered within the same TAD display a similar expression pattern, and alteration of these domains result in aberrant expression of the genes, suggesting their involvement in transcriptional regulation. TADs appear remarkably stable between cell types, but can differ in the level of compaction inside individual TADs. TADs encompassing inactive genes are more compact than TADs comprising active ones. This raises the question whether TAD compaction directly regulates or somehow limits gene expression. This project aims at answering this question using a model system, dosage compensation (DC) in the nematode, Caenorhabditis elegans. It has been previously shown that on the X chromosome, more compact TADs correlate with the down-regulation of X-linked genes in hermaphrodites compared to males. This provides an ideal system to understand the mechanism of transcriptional regulation by TAD formation. I have setup a chromatin conformation capture technique coupled with long molecule sequencing (Oxford Nanopore Technology) to capture gene structures at high resolution, in DC and non-DC environment. In contrast to standard HiC that captures pair-wise contacts, this approach can directly identify multi-way chromosomal contacts from within single cells. My aim is to understand how chromatin structure is modified at the gene level by TAD formation and whether and how this regulates gene expression, thereby deciphering the importance of genome folding in transcriptional regulation.

Das, Ritika et al. (2011) International Worm Meeting "Role of endocytosis and ER proteostasis in C. elegans longevity."

Stolzenburg, Lindsay, Rath, Sneha, Samuelson, Andrew V., Das, Ritika, Lee, Nam

[International Worm Meeting, 2011]

Insulin signaling is a major pathway controlling lifespan in multiple organisms, such as worms, flies and mammals. The insulin signaling pathway initiated by the activation of the insulin receptor (DAF-2) brings about the repression of the downstream transcription factor DAF-16. DAF-16 is one of the key transcription factors that regulate the expression of genes involved in lifespan as well as other functions such as development, immunity and stress resistance. We have previously conducted a genome wide RNAi screen to identify genes that are essential for enhanced longevity in daf-2 mutant worms {Samuelson, 2007}1. This screen led to the identification of 103 gene inactivations which cause premature aging (the "progeric gene panel", PGP) . Interestingly, 19 genes in the PGP are annotated to be involved in endosomal protein sorting or vesicular trafficking. Similar studies in yeast have also identified genes involved in vacuolar protein sorting to be essential for longevity suggesting a conserved role for proper endosomal function in regulating aging {Fabrizio, 2010}2. However, the mechanism by which endocytosis influences longevity is still not clear. To explore this further, our lab has undertaken a systems level analysis to identify the members of the PGP that are involved in endocytosis, the ER unfolded protein response (ERUPR), and protein homeostasis. To this end, transgenic worms for different endocytosis markers such as the Rab GTPases and GFP fusions to those proteins whose proper localization within the worm depends on functional endocytic machinery have been screened. While the endocytosis machinery is critical to protein degradation and proper protein sorting, ER serves as a primary site for protein synthesis and folding and daf-2 mutant animals exhibit lower levels of the ERUPR marker hsp-4::GFP indicating improved ER homeostasis {Korenblit, 2010}3 . To identify the members of the PGP that influence longevity via the ER stress response pathway, hsp-4::GFP transgenic worms have been screened to identify gene inactivations that either induce the ERUPR in the absence of stress or conversely impair the ERUPR after stress. Lastly, we have identified the members of the PGP that cause the premature collapse of protein homeostasis, as measured by foci formation of Q35::YFP. Highlights of this comprehensive analysis will be presented. References 1. Samuelson et al (2007) Genes Dev 21(22):2976-94 2. Fabrizio et al (2010) PLOS Genet. 6 (7):e1001024 3. Korenblit et al (2010) PNAS 107(21):9730-5.

Das, Moushumi et al. (2021) International Worm Meeting "Condensin I organizes the C. elegans interphase genome"

Meister, Peter, Statzer, Cyril, Campos, Julie, Ewald, Collin, Semple, Jennifer, Das, Moushumi, Gitchev, Todor

[International Worm Meeting, 2021]

Recent data in mammalian cells have shown the role of cohesin, a Structural Maintenance of chromosomes (SMC) complex, in interphase genome organization into loops and domains. Since these complexes are involved in cell division, it is difficult to evaluate the functional consequences of their absence in animals. Here we use Caenorhabditis elegans, an animal, 90% of whose cells are post-mitotic at birth. Similarly to mammals, nematodes have three SMC complexes: cohesin, condensin I and condensin II. Additionally, condensin IDC, a variant of condensin I, is involved in dosage compensation (DC). To uncover which SMC complex(es) organize the C. elegans interphase genome and evaluate functional consequences of genome unfolding, we constructed strains in which individual SMC complexes - cohesin, condensin I/IDC and II - are acutely inactivated in vivo in fully differentiated animals. We then assessed their phenotype and carried out chromatin conformation capture (Hi-C) and RNA-seq. Our data shows that in contrast to mammalian cells, the major determinant of genome folding in C. elegans is condensin I/IDC and not cohesin. Its cleavage reduces short-range contact probabilities and causes genome-wide de-compaction on all chromosomes. In contrast, cohesin cleavage has marginal impact while condensin II cleavage has no consequence on genome folding. RNA-seq data show that about a third of expressed genes are significantly differently expressed upon cohesin and condensin II inactivation, however the effect sizes were very small, and a similar number of genes were up and down regulated. In contrast, cleavage of condensin I/IDC leads to up-regulation of 93% of all expressed X-linked genes versus only 1% down regulated, indicating the necessity of the constant presence of condensin IDC for maintenance of DC. Cleavage of cohesin and condensin II has little effect on animal post-embryonic survival, as their lifespan are similar to control animals. In contrast, inactivation of condensin I/IDC causes drastic reduction in life expectancy; yet additional experiments show that lifespan reduction is due to lack of DC rather than genome unfolding. Taken together, we discovered that condensin I is the main SMC complex folding the nematode interphase genome, yet genome unfolding has no major effect in C. elegans in standard laboratory conditions, apart from X-linked gene up-regulation and its consequences.

Das, Debabrata et al. (2021) International Worm Meeting "Regulation of oocyte number in C.elegans: Counting on RAS/ERK pathway"

Chen, Shin-Yu, Das, Debabrata, Arur, Swathi

[International Worm Meeting, 2021]

Oocyte numbers, a critical determinant of female reproductive fitness, are highly regulated, yet the mechanisms underlying this regulation remain largely undefined. In the Caenorhabditis elegans gonad, RAS/extracellular signal-regulated kinase (ERK) signaling regulates oocyte numbers; mechanisms are unknown. We show that the RAS/ERK pathway phosphorylates meiotic chromosome axis protein HTP-1 at serine-325 to control chromosome dynamics and regulate oocyte number. Phosphorylated HTP-1(S325) accumulates in vivo in an ERK-dependent manner in early-mid pachytene stage germ cells and is necessary for synaptonemal complex extension and/or maintenance. Lack of HTP-1 phosphorylation leads to asynapsis and persistence of meiotic double-strand breaks, causing delayed meiotic progression and reduced oocyte number. In contrast, early onset of ERK activation causes precocious meiotic progression, resulting in increased oocyte number, which is reversed by removal of HTP-1 phosphorylation. The RAS/ERK/HTP-1 signaling cascade thus functions to monitor formation and maintenance of synapsis for timely resolution of double-strand breaks, oocyte production, and reproductive fitness.

Das P et al. (1995) International C. elegans Meeting "VERTEBRATE HOMOLOGS IN THE DWARFIN FAMILY OF TGF-b SIGNAL TRANSDUCERS"

Padgett RW, Townsend SR, Savage C, Das P

[International C. elegans Meeting, 1995]

The TGF-b signalling pathway is important in various aspects of development and yet its components are relatively unknown. In trying to elucidate the pathway, we are studying three homologous genes, sma-2, -3 and -4 (see abstract by Savage et al.) that are believed to be in the pathway. These genes are members of the DWARFIN family that encode a novel set of proteins characterised by two highly conserved regions with a proline-rich region in between. The functions of these regions are not known. We were interested in isolating sma homologs in vertebrates. Using the conserved sequences in the three sma genes in C. elegans, we designed degenerate primers and used genomic DNA from the mouse for PCR. We have sequenced several clones from the mouse and have identified three possible homologs that are part of the DWARFIN family. All three homologs show more similarity to sma-2 and the Drosophila Mad gene than to any of the other members. Using these as probes, we have screened a mouse cDNA library and are currently sequencing the cDNAs. Efforts are ongoing to identify additional members of the family from C. elegans and the mouse. In addition, we are using the yeast two-hybrid system in an effort to evolve a model of potential interactions among these members and other proteins in the pathway.

Partha Pratim Das et al. (2006) European Worm Meeting "Role of Short RNAs in Caenorhabditis elegans Germline Development"

Eric Miska, Partha Pratim Das, Heeran Buhecha

[European Worm Meeting, 2006]

Several hundreds of endogenous small RNAs, namely microRNAs (miRNAs), repeat- associated small interfering RNAs (rasiRNAs), small interfering RNAs (siRNAs) and tiny non-coding RNAs (tncRNAs) have been discovered in diverse organisms. These small RNAs are derived from different sources and they cause transcriptional gene silencing, translational repression and mRNA cleavage through effector complexes. Several functionally distinct RNA silencing effector complexes have already been isolated from different organisms. Effector complexes are composed of members of the Argonaute (AGO) protein family and single stranded small RNAs, along with other associated proteins. Members of the piwi subfamily Argonaute proteins have evolutionary conserved roles in germline development and stem cell maintenance. In C.elegans, PRG-1 and PRG-2 are the two members of piwi subfamily of Argonaute proteins. In order to study the role of PRG-1 and PRG-2 in C.elegans germline development, we generated prg-1; prg-2 double mutant worms, which show severe germline defects. Additionally, microarray analysis showed several genes are up-regulated in the double mutant worms compared to wild type. Interestingly, these up-regulated genes are the predicted targets of some of newly cloned short RNAs. To understand the detailed mechanism of PRG-1 and PRG-2 and their role in germline development, we are currently isolating PRG-1 containing complexes and their components. Cloning of short RNAs associated with PRG-1/2. Potential components of the complexes will be investigated by in vitro (protein-protein interaction) as well as in vivo (RNAi) studies.

Das, Ravi et al. (2021) International Worm Meeting "Local compression mechanosensing by DVA proprioceptors curbs body bends during locomotion"

Sanfeliu-Cerdan, Neus, Porta-de-la-Riva, Montserrat, Catala-Castro, Frederic, Krieg, Michael, Pidde, Aleksandra, Das, Ravi, Lin, Li-Chun

[International Worm Meeting, 2021]

Coordinated cell shape changes is the basis for a myriad of dynamic processes in animals, from limb movement to locomotion, but also for visceral morphodynamics such as lung expansion. These processes are supervised by specialized, proprioceptive mechanosensors, which are capable to accurately transduce mechanical information from organ deformation into biochemical signals. However, we still have little knowledge about the physiologically relevant mechanical stresses and deformations that lead to the activation of mechanosensitive neurons during proprioception or visceral mechanosensation. The stereotypical locomotion phenotype of C elegans can help us elucidate several mechanotransduction pathways coming into play for proprioception. We used a conditional knock-out strategy and found that unc-70 beta-spectrin has cell-specific roles in the DVA neuron in limiting body bending amplitudes during locomotion. By means of calcium imaging, microfluidic manipulation and genetically encoded tension sensors we found that the spectrin cytoskeleton is able to bear compressive stresses that are transduced by the trp-4 ion channel of the TRPN/NOMPC family. In conjunction with optical tweezer based force spectroscopy assays of cultured DVA neurons, we found that the potassium leak channel twk-16 is responsible for inhibiting depolarization during axonal stretch, which adjusts the proprioceptive mechanism of DVA within compression. Finally, we formulate our observations within a neuromechanical framework that shows that compartmentalized, compressive mechanosensitivity triggers local muscle contraction that is critical for animal locomotion.

Das, Ritika et al. (2015) International Worm Meeting "The homeodomain protein kinase, HPK-1 influences aging and is a central component of the heat shock response ."

Samuelson, Andrew, Das, Ritika, Kim, Richard, Schwartz, Elliot

[International Worm Meeting, 2015]

The homeodomain protein kinase, HPK-1 influences aging and is a central component of the heat shock response Ritika Das1,2, Richard Kim2, Elliot Schwartz3, and Andrew V. Samuelson21.Department of Biology, University of Rochester, Rochester NY2.Department of Biomedical Genetics, University of Rochester Medical Center, Rochester NY3.Program for Neuroscience, University of California, Los Angeles, Los Angeles CAAging, stress resistance, and the maintenance of protein homeostasis are dependent on the activity of the heat shock transcription factor HSF-1, which in turn regulates the expression of cellular chaperones. Yet the underlying signaling mechanisms that activate HSF-1 remain poorly understood. We find that the homeodomain protein kinase, HPK-1, is both necessary and sufficient to alter aging in Caenorhabditis elegans, as measured by changes in lifespan and protein homeostasis. Interestingly, we provide genetic, molecular, and cellular evidence that HPK-1 and HSF-1 function in a common genetic pathway. Furthermore, HPK-1 is induced by thermal stress, implying that HPK-1 is a bona fide heat shock response gene. Consistent with this notion hpk-1 null mutant animals have compromised thermotolerance, which is accompanied with reduced induction of heat shock responsive chaperones hsp-16.2 and hsp-70 after thermal stress. Additionally, hpk-1 mutants fail to the obtain the hormetic benefit conferred by a transient heat stress, which in wild-type animals manifests as increased lifespan, implying that the roles of HPK-1 in the heat shock response, proteostasis, and aging are intimately connected. Collectively, we propose that HPK-1 is an essential component of the heat shock response whose function ultimately influences both protein homeostasis and aging.

Waring DA (1996) West Coast Worm Meeting "A C. ELEGANS HOMOLOG OF NEUROD"

Waring DA

[West Coast Worm Meeting, 1996]

The neuroD gene was identified in our lab as a bHLH transcription factor controlling neuronal differentiation in mouse and Xenopus. Extopic expression in Xenopus embryos leads to production of ectopic neurons within the epidermis. It is proposed that neuroD acts downstream of the proneural genes and negative regulators of the Notch/Delta pathway. I am characterizing a C. elegans homolog of neuroD cnd-1. In order understand the function of the gene I am in the process of knocking the gene out. I have identified an insertion of a transposable element within an intron of the gene. This has no phenotype and is apparently spliced out without effecting the gene. I am presently screening for imprecise excision of the transposable element that will knock out cnd-1. In addition I am characterizing larger deficiencies and lethal mutation in the region. I have generated antibodies to cnd-1 and I am beginning to characterize the expression pattern. Neurogenesis in C. elegans is still poorly characterized. Several bHLH proteins related to the Achaete/Scute family, have been found in C. elegans, but as yet there is functional data for only one of these, lin-32 which was identified by mutations lacking a subset of neuroblasts, suggesting that as in other species bHLH proteins may regulate neurogenesis. If cnd-1 functions in C. elegans as its homologs do in vertebrates, the single cell resolution that C. elegans affords should be very useful in understanding its role in neurogenesis, and its relationship to the proneural genes.

Das, Alakananda et al. (2021) International Worm Meeting "Conserved extracellular proteins determine mechanoelectrical transduction channel localization and function in C. elegans touch receptor neurons"

Franco, Joy A., Pruitt, Beth L., Wang, Lingxin, Kuhl, Ellen, Wang, Lucy M., Das, Alakananda, Goodman, Miriam B., Chapman, Dail

[International Worm Meeting, 2021]

The sense of touch is made possible by neurons that use ion channels to convert mechanical stimuli into electrical signals. Many other proteins affect mechanosensation, either through direct force transfer by acting as physical tethers, or indirectly by modulating cell stiffness and morphology, protein trafficking, and channel positioning. Subcellular localization of mechanosensory ion channels is especially important for localized force sensation, since channels further away from the point of stimulation are recruited as stimulus intensity and speed increase. Here we examine the micro-environment of ion channels to investigate the molecular basis of channel positioning, leveraging the well-characterized MEC-4 mechanoelectrical transduction channel in C. elegans touch receptor neurons (TRNs). We show that MEC-4 channels localize to punctae distributed along TRN neurites in vivo but not in cultured TRNs in vitro, suggesting that in vivo channel localization depends on factors that are absent in vitro. Both time-lapse imaging and fluorescence recovery after photobleaching shows that most in vivo MEC-4 puncta are immobile over several minutes, suggesting that they are anchored to stable structures. To identify these structures, we screened mutants affecting plasma membrane proteins, cytoskeletal proteins, and extracellular matrix (ECM) proteins to investigate what factors regulate the positioning and stability of the MEC-4 puncta. In doing so, we discovered that the ECM protein nidogen regulates MEC-4 puncta distribution and both behavioral and electrical responses to touch. Nidogen associates with laminin networks in basement membranes and we show that both nidogen and laminin co-localize with MEC-4 puncta, in a manner that depends upon another ECM protein, MEC-1. To learn more about the physiological significance of channel puncta, we modeled mechanical strain distribution along a neurite in silico and found that physical attachment of the neurite to the ECM at puncta generates localized regions of increased strain. We speculate that this amplifies and focuses the mechanical stimulus for ion channel opening. Future experiments will determine whether these ECM proteins physically anchor the MEC-4 channels, accounting for the immobility of the MEC-4 puncta, and whether such a physical tether is responsible for ion channel opening in response to a mechanical stimulus.