Bui, T. — Page 2 | Search bioRxiv

bioRxiv2023

The T1150A cancer mutant of the protein lysine dimethyltransferase NSD2 can introduce H3K36 trimethylation

Somatic mutations in protein lysine methyltransferases are frequently observed in cancer cells. We show here that the NSD1 mutations Y1971C, R2017Q and R2017L observed mostly in solid cancers are catalytically inactive suggesting that NSD1 acts as tumor suppressor gene in these tumors. In contrast, the frequent T1150A in NSD2 and its T2029A counterpart in NSD1, both observed in leukemia, are hyperactive and introduce up to H3K36me3 in biochemical and cellular assays, while wildtype NSD2 and NSD1 only generate up to H3K36me2. MD simulations with NSD2 revealed that H3K36me3 formation is possible due to an enlarged active site pocket of T1150A and loss of direct contacts of T1150 to critical residues which regulate the product specificity of NSD2. Bioinformatic analyses of published data suggest that the NSD2 T1150A mutation in lymphocytic leukemia could alter gene regulation by antagonizing H3K27me3 finally leading to the upregulation of oncogenes.

molecular biology↗

bioRxiv2023

Multiple pals gene modules control a balance between immunity and development in Caenorhabditis elegans

The immune system continually battles against pathogen-induced pressures, which often leads to the evolutionary expansion of immune gene families in a species-specific manner. For example, the pals gene family expanded to 39 members in the Caenorhabditis elegans genome, in comparison to a single mammalian pals ortholog. Our previous studies have revealed that two members of this family, pals-22 and pals-25, act as antagonistic paralogs to control the Intracellular Pathogen Response (IPR). The IPR is a protective transcriptional response, which is activated upon infection by two molecularly distinct natural intracellular pathogens of C. elegans - the Orsay virus and the fungus Nematocida parisii from the microsporidia phylum. In this study, we identify a previously uncharacterized member of the pals family, pals-17, as a newly described negative regulator of the IPR. pals-17 mutants show constitutive upregulation of IPR gene expression, increased immunity against intracellular pathogens, as well as impaired development and reproduction. We also find that two other previously uncharacterized pals genes, pals-20 and pals-16, are positive regulators of the IPR, acting downstream of pals-17. These positive regulators reverse the effects caused by the loss of pals-17 on IPR gene expression, immunity and development. We show that the negative IPR regulator protein PALS-17 and the positive IPR regulator protein PALS-20 colocalize inside intestinal epithelial cells, which are the sites of infection for IPR-inducing pathogens. In summary, our study demonstrates that several pals genes from the expanded pals gene family act as ON/OFF switch modules to regulate a balance between organismal development and immunity against natural intracellular pathogens in C. elegans. AUTHOR SUMMARYImmune responses to pathogens induce extensive rewiring of host physiology. In the short term, these changes are generally beneficial as they can promote resistance against infection. However, prolonged activation of immune responses can have serious negative consequences on host health, including impaired organismal development and fitness. Therefore, the balance between activating the immune system and promoting development must be precisely regulated. In this study, we used genetics to identify a gene in the roundworm Caenorhabditis elegans called pals-17 that acts as a repressor of the Intracellular Pathogen Response (IPR), a defense response against viral and microsporidian infections. We also found that pals-17 is required for the normal development of these animals. Furthermore, we identified two other pals genes, pals-20 and pals-16, as suppressors of pals-17 mutant phenotypes. Finally, we found that PALS-17 and PALS-20 proteins colocalize inside intestinal cells, where viruses and microsporidia invade and replicate in the host. Taken together, our study demonstrates a balance between organismal development and immunity that is regulated by several genetic ON/OFF switch modules in C. elegans.

immunology↗

bioRxiv2019

Nanoluciferase-based Method for Detecting Gene Expression in C. elegans

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, GFP and other fluorophores have limited sensitivity, particularly in tissues that autofluoresce like the intestine of the nematode Caenorhabditis elegans. Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in multi-well format to detect constitutive and inducible gene expression in C. elegans. We optimize detection of bioluminescent signal from NanoLuc in C. elegans and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult \"hidden\" in a pool of 5,000 N2 wild-type animals. We also optimized various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof of concept, we used NanoLuc to monitor promoter activity of the pals-5 stress/immune reporter and we were able to measure 300 and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring gene expression in C. elegans.

genetics↗