Pu, Mintie et al. (2013) International Worm Meeting "Epigenetic Mechanism of Longevity Regulation in C. elegans."

Pu, Mintie, Ni, Zhuoyu, Wang, Xiujuan, Yu, Haiyuan, Lee, Siu Sylvia

[International Worm Meeting, 2013]

Recent studies demonstrate longevity is affected and controlled by histone modifications. The link between histone modification and aging was first demonstrated by the observation that RNAi knockdown or genetic inactivation of histone modification enzymes resulting in extended longevity in model organisms, including C. elegans. However how histone modification changes impact aging and how aging impacts histone modifications are not clear. To gain insights into the histone modification pattern during aging, we monitored the genomic locations and abundance of several histone modifications involved in gene transcriptional regulation at different ages using ChIP-seq. Here we surveyed the genomic H3K36me3 profile in C. elegans somatic cells during aging. We found H3K36me3 pattern is largely stably maintained during aging. Importantly, we found H3K36me3 level is negatively correlated with gene expression variation during aging. Our data indicate that genes that are lowly methylated are significantly more likely to show greater changes of mRNA levels with age. Our analyses indicate that this negative correlation between H3K36me3 modification level and gene expression variation is independent of gene expression level, gene length and tissue specificity, suggesting an interesting function of H3K36me3 in maintaining the stability of gene transcription during aging.

Pu, Mintie et al. (2017) International Worm Meeting "Dynamic changes of gene-body H3K4me3 with age in C. elegans somatic cells."

Wang, Minghui, Lee, Siu Sylvia, Pu, Mintie

[International Worm Meeting, 2017]

Recent studies demonstrate that altered histone modifications can profoundly impact longevity. However how histone modification changes modulate aging and how aging alters histone modifications are still largely unclear. To gain insights into the histone modification pattern during aging, we monitored the genomic locations and abundance of several histone modifications involved in gene transcriptional regulation in the somatic cells of C. elegans at different ages. Previously we found that the genome-wide pattern of H3K36me3 is largely stably maintained during aging and that H3K36me3 marking plays an important role in maintaining gene expression stability and lifespan. We have now surveyed the genome-wide pattern of H3K4me3 through similar aging time points. We identified specific regions of H3K4me3 marking that show dynamic change with age and that these H3K4me3 changes are highly correlated with gene expression alterations. Interestingly, the H3K4me3 regions that change with age and are accompanied by RNA expression alterations largely mark gene-bodies, contrary to the canonical marking of H3K4me3 around transcriptional start sites (TSS). Comparison with modENCODE data suggest that the gene-body marking of H3K4me3 that tend to change with age are not marked by H3K4me3 during early development but become apparent during late larval and/or adult stages, whereas the H3K4me3 marking around TSS are clearly established during early development and remain largely stable with age. Our study suggests an intriguing possibility of adult-specific H3K4me3 marking that are more susceptible to age-dependent change, which may have important implications for age-dependent RNA expression dynamics.

Fayolle M et al. (2021) Genes (Basel) "The Persistence of Staphylococcus aureus in Pressure Ulcers: A Colonising Role."

Sotto A, Chateauraynaud M, Morsli M, Fayolle M, Gelis A, Dunyach-Remy C, Lavigne JP, Yahiaoui-Martinez A

[Genes (Basel), 2021]

Decubitus pressure ulcers (PU) are a major complication of immobilised patients. <i>Staphylococcus aureus</i> is one of the most frequently detected microorganisms in PU samples; however, its persistence and role in the evolution of these wounds is unknown. In this study, we analysed <i>S. aureus</i> strains isolated from PU biopsies at inclusion and day 28. Eleven <i>S. aureus</i> (21.1%) were detected in 52 patients at inclusion. Only six PUs (11.5%) continued to harbour this bacterium at day 28. Using a whole genome sequencing approach (Miseq, Illumina), we confirmed that these six <i>S. aureus</i> samples isolated at D28 were the same strain as that isolated at inclusion, with less than 83 bp difference. Phenotypical studies evaluating the growth profiles (Infinite M Mano, Tecan) and biofilm formation (Biofilm Ring Test) did not detect any significant difference in the fitness of the pairs of <i>S. aureus</i>. However, using the <i>Caenorhabditis elegans</i> killing assay, a clear decrease of virulence was observed between strains isolated at D28 compared with those isolated at inclusion, regardless of the clinical evolution of the PU. Moreover, all strains at inclusion were less virulent than a control <i>S. aureus</i> strain, i.e., NSA739. An analysis of polymicrobial communities of PU (by metabarcoding approach), in which <i>S. aureus</i> persisted, demonstrated no impact of <i>Staphylococcus</i> genus on PU evolution. Our study suggested that <i>S. aureus</i> presented a colonising profile on PU with no influence on wound evolution.

Ding Y et al. (2015) PLoS One "Pu-erh tea down-regulates sterol regulatory element-binding protein and stearyol-CoA desaturase to reduce fat storage in Caenorhaditis elegans."

Chen D, Zou X, Liang B, Zhang Y, Ding Y, Wu J, Jiang X

[PLoS One, 2015]

Consumption of Pu-erh has been reported to result in numerous health benefits, but the mechanisms underlying purported weight-loss and lowering of lipid are poorly understood. Here, we used the nematode Caenorhaditis elegans to explore the water extract of Pu-erh tea (PTE) functions to reduce fat storage. We found that PTE down-regulates the expression of the master fat regulator SBP-1, a homologue of sterol regulatory element binding protein (SREBP) and its target stearoyl-CoA desaturase (SCD), a key enzyme in fat biosynthesis, leading to an increased ratio of stearic acid (C18:0) to oleic acid (C18:1n-9), and subsequently decreased fat storage. We also found that both the pharyngeal pumping rate and food uptake of C. elegans decreased with exposure to PTE. Collectively, these results provide an experimental basis for explaining the ability of Pu-erh tea in promoting inhibition of food uptake and the biosynthesis of fat via SBP-1 and SCD, thereby reducing fat storage.

Xiao RY et al. (2016) Molecules "Transcriptome Profile Reveals that Pu-Erh Tea Represses the Expression of Vitellogenin Family to Reduce Fat Accumulation in Caenorhabditis elegans."

Xiao RY, Zou XJ, Che YY, Hao J, Ding YH, Liang B

[Molecules, 2016]

Due to misbalanced energy surplus and expenditure, obesity has become a common chronic disorder that is highly associated with many metabolic diseases. Pu-erh tea, a traditional Chinese beverage, has been believed to have numerous health benefits, such as anti-obesity. However, the underlying mechanisms of its anti-obesity effect are yet to be understood. Here, we take the advantages of transcriptional profile by RNA sequencing (RNA-Seq) to view the global gene expression of Pu-erh tea. The model organism Caenorhabditis elegans was treated with different concentrations of Pu-erh tea water extract (PTE, 0 g/mL, 0.025 g/mL, and 0.05 g/mL). Compared with the control, PTE indeed decreases lipid droplets size and fat accumulation. The high-throughput RNA-Sequence technique detected 18073 and 18105 genes expressed in 0.025 g/mL and 0.05 g/mL PTE treated groups, respectively. Interestingly, the expression of the vitellogenin family (vit-1, vit-2, vit-3, vit-4 and vit-5) was significantly decreased by PTE, which was validated by qPCR analysis. Furthermore, vit-1(ok2616), vit-3(ok2348) and vit-5(ok3239) mutants are insensitive to PTE triggered fat reduction. In conclusion, our transcriptional profile by RNA-Sequence suggests that Pu-erh tea lowers the fat accumulation primarily through repression of the expression of vit(vitellogenin) family, in addition to our previously reported (sterol regulatory element binding protein) SREBP-SCD (stearoyl-CoA desaturase) axis.

Kuersten S et al. (1997) International C. elegans Meeting "AN INTERCISTRONIC SEQUENCE INVOLVED IN PROCESSING OF C. ELEGANS OPERON PRE-mRNAS"

MacMorris MA, Blumenthal TE, Spieth J, Kuersten S, Lea K

[International C. elegans Meeting, 1997]

Polycistronic pre-mRNAs in C. elegans are processed into monocistronic mRNAs by 3' end formation and SL2-specific trans-splicing. We are studying the mechanism of this type of RNA processing in vivo, using a heat-shock driven transgenic gpd-2/gpd-3 operon. We have identified a U-rich sequence (Ur) located within the intercistronic region, 75 bp upstream of the gpd-3 trans-splice site, but nevertheless required for trans-splicing of gpd-3. A Ur mutation (DELTAUr) weakly inhibits 3' end formation, resulting in some polycistronic RNA accumulation, but more dramatically prevents the accumulation of trans-spliced gpd-3 mRNA. In the absence of the Ur sequence and 3' end formation of gpd-2 , trans-spliced gpd-3 mRNA accumulates, but is only spliced to SL1. Insertion of a 10 base oligo(U) tract in place of the normal Ur sequence (DELTAUr/pU) partially rescues SL2 trans-splicing and completely restores 3' end formation. Under extreme heat-shock conditions that inhibit trans-splicing, we detect a primer extension product that extends from gpd-3 into the intercistronic region and ends just upstream of the Ur sequence. Accumulation of this product requires an intact Ur region and is strongly enhanced by 3' end formation. In the DELTAUr/pU mutant, multiple primer extension products that map within and just upstream of the inserted poly(U) accumulate. These products do not appear to represent a Y-branch intermediate (with the Ur sequence playing a role as a polypyrimidine tract) since it is stable to debranching treatment with S100 extract. Instead, we hypothesize that the Ur sequence may act to prevent transcription termination following 3' end formation of the upstream mRNA by protecting the uncapped downstream product from degradation

Bhat VD et al. (2019) Elife "Engineering a conserved RNA regulatory protein repurposes its biological function in vivo."

Alexander JC, Fonseca DR, Tanaka Hall TM, Bhat VD, Lo TW, Campbell ZT, Shukla T, Wang Y, McCann KL, Wickens M, Qiu C

[Elife, 2019]

PUF (<u>PU</u>milio/<u>F</u>BF) RNA-binding proteins recognize distinct elements. In <i>C. elegans</i>, PUF-8 binds to an 8-nt motif and restricts proliferation in the germline. Conversely, FBF-2 recognizes a 9-nt element and promotes mitosis. To understand how motif divergence relates to biological function, we determined a crystal structure of PUF-8. Comparison of this structure to that of FBF-2 revealed a major difference in a central repeat. We devised a modified yeast 3-hybrid screen to identify mutations that confer recognition of an 8-nt element to FBF-2. We identified several such mutants and validated structurally and biochemically their binding to 8-nt RNA elements. Using genome engineering, we generated a mutant animal with a substitution in FBF-2 that confers preferential binding to the PUF-8 element. The mutant largely rescued overproliferation in animals that spontaneously generate tumors in the absence of <i>puf-8</i>. This work highlights the critical role of motif length in the specification of biological function.

Conrad RC et al. (1991) International C. elegans Meeting "ANALYSIS OF SPLICE SITE DEFINITION IN C. ELEGANS"

Blumenthal TE, Conrad RC, Liou RF

[International C. elegans Meeting, 1991]

Both cis-splicing (intron removal) and trans-splicing occur in C. elegans transcripts. We are interested in the interplay between these two fundamentally similar reactions. A variety of data now supports the idea that the region of a trans-spliced gene transcript between the S' end and the trans-splice site, called the outron, is functionally equivalent to an intron without a functional S' splice site. This implies that the strength of any potential S' splice sites upstream determine whether or not a transcript will serve as a trans- splice acceptor. Our previous experiments have shown that insertion of an intron lacking a 5' splice site from one gene into the 5' untranslated region of another gene leads to splicing of the trans- spliced leader at the intron 3' splice, thus converting a conventional gene into a trans-spliced gene. We are interested in determining (a) what features are required for a functional trans-splice site (and by analogy, an intron 3' splice site); (b) what features are required for a functional S' splice site; and (c) whether differences exist between intron and outron 3' splice sites. These questions are being addressed through the use of transformation of C. elegans with altered genes that have an observable phenotype. Investigating these questions involves the use of competition between rival reactions, products of which are detected by PCR amplification of the RNA products. For the first question, a natural trans-splice site in a collagen gene, rol-6, is perturbed, causing the utilization of a cryptic splice site 20 nucleotides upstream. We have determined that replacement of some of the pyrimidines in the highly conserved UUUCAG/Pu 3' splice site with A's partially or fully inactivates the site. In addition, we have replaced the A+U-rich region upstream from this consensus sequence and downstream from the cryptic site with a non-A+U-rich region. This change results in partial switching to the cryptic site, supporting the concept that the A+Urich/non-A+U-rich boundary is a feature that directs splicing to the nearest good match to the UUUCAG/Pu consensus. To investigate the second question we have inserted a synthetic S' splice-site an appropriate distance upstream from the trans-splice site in rol-6, creating an exon out of part of the 173 nt outron. This creates a competition between trans- and cissplicing at the natural trans-splice site. We have determined that the insertion of the C. elegans consensus S' splice site, AAG/GUAAGWU, 131 nucleotides upstream from the trans-splice site is sufficient to generate a cis- splicing event, but not to the exclusion of trans-splicing: both cisspliced and trans-spliced mRNAs are formed. We are currently creating alternative arrangements of S' and 3' splice sites within the outron to try to shift the balance between the two splicing reactions.

Kolotuev, Irina et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "C. elegans prox-1 promotes excretory canal formation and amphid sheath cell tubule development"

Schwab, Yannick, Labouesse, Michel, Kolotuev, Irina

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The formation of tubular structures during organogenesis is a general process that remains poorly characterized at the molecular level. In C. elegans the excretory canal and amphid sheath tubules serve as models of tubulogenesis. We previously identified the mutation rdy-3(mc41) in a genetic screen for secretion and osmoregulatory defects. rdy-3(mc41) larvae die at the L1/L2 stage with no excretory canal arms and no DiI uptake in the amphids. We cloned rdy-3 and found that it corresponds to the C. elegans Prox1 homologue (aka ceh-26); we suggest renaming it prox-1. Prospero-related homeobox transcription factor (Prox1) is indispensable for the development of various tissues (notably D. melanogaster neurons, and mice lymphatic vessels), however its downstream targets are unknown. prox-1(mc41) is a 102bp deletion in the homeobox domain. We fully rescued mc41 phenotypes with a cosmid containing prox-1, and partially with a prox-1 cDNA driven by a heat-shock promoter. prox-1 expression is visible in a subset of head and body neurons and in the excretory canal. Using fluorescent reporter analysis and correlative TEM techniques we found that prox-1(mc41) does not cause cell fate transformation, but influences the process of excretory inner lumen formation and canal extension. Similarly, we found that the amphid sheath cell terminal tube is affected, while amphid neurons appear normal. To search for prox-1 downstream targets we used evolutionary bioinformatics based on the data of genome-wide analysis of fly Prospero binding sites. The search identified exc-5, an important factor in excretory canal extension and lumen formation, plus several other targets from the same pathway. Real time PCR showed upregulation of exc-5 in the prox-1 background and bioinformatic analysis revealed a pu tative Prox1 binding site. We propose that C.elegans can serve as a model to further characterize PROX-1 function in lumen formation and tubulogenesis.

Kagoshima H et al. (1998) Worm Breeder's Gazette "The (only?) C.elegans runt gene"

Burglin TR, Kagoshima H

[Worm Breeder's Gazette, 1998]

The Runt gene family is a new family of heteromeric DNA binding transcription factors. Recent work has revealed that members of this family play&nbsp; important roles in embryonic development of sea urchin, segmentation of Drosophila, and hematopoiesis and osteoblast differntiation in mouse and human. The heteromeric complex consists of two subunits, a DNA binding subunit aand its partner subunit b. The a subunit binds directly to a specific DNA sequence (PuCCPuCAG [Pu=A/G]) while theb subunit associates with ato form a heterodimer that has a greater affinity for DNA. The asubunit shares a region of high homology, termed the Runt domain (Fig. 1), with the products of the Drosophila segmentation gene runt and the human acute myeloid leukemia-related gene AML1. A series of biochemical analyses revealed that the Runt domain is sufficient for both functions of the asubunit: DNA binding and heterodimerization with the bsubunit. In mammals, there are 3 types of asubunits, aA,aB and aC. Targeted disruption of aA in mouse results in a complete lack of ossification in their skeletal systems. The human homolog of aB, AML1, is known as the most frequent target of chromosomal translocations associated with leukemia, and targeted mice lacking AML1 showed no fetal liver hematopoiesis. aB has thus proved to be essential for hematopoiesis of all lineages. We found a C. elegans homolog of runt by searching in the C. elegans databases, which we propose to call rnt-1. We made a promoter-gfp-lacZ reporter construct to examine the temporal and spatial expression pattern.&nbsp; We observed gfp expression in the hypodermis, the seam and probably their precursor cells starting from the 2-fold stage embryo to adult. This result implied that rnt-1 might have quite different functions in different organisms, which contrasts with the high sequence conservation. Moreover, so far&nbsp; no bsubunit gene has been found in C. elegans; this subunit has important regulatory functions for the Runt domain. To elucidate the function of rnt-1 in worms, we are going to do RNAi experiments and screen knock-out mutant libraries. This should give us new insights into the function of this gene family. &nbsp;