Ozdemir, Isa et al. (2021) International Worm Meeting "Regulation of transgenerational epigenetic H3K27me3 inheritance"

Steiner, Florian, Delaney, Kamila, Wenda, Joanna, Ozdemir, Isa

[International Worm Meeting, 2021]

A crucial process in life is the ability of cells to pass on information to their descendants. This transmission can occur at several levels from genetics to epigenetics. Epigenetic inheritance refers to the heritable phenotypes affected by gene expression without altering the DNA sequence and occurs through various factors including histone modifications. Interruption of this transmission process can lead to severe developmental defects and fertility diseases. Methylation of histone H3 at position 27 (H3K27me3) is an epigenetic mark that is associated with heterochromatin and repressed gene expression. Several studies have demonstrated that the genomic patterns of H3K27me3 can be inherited from one generation to the next by depositing H3K27me3 histones and PRC21. However, the limits of this inheritance are difficult to test because PRC2 mutants are maternal-effect sterile in C. elegans. We have recently shown that the expression of an H3.3K27M mutant acts in a dominant-negative manner and alters genomic H3K27me3 patterns and distribution of PRC2 and results in infertility phenotypes in C. elegans2. We used this strain to follow the trans-generational inheritance of the H3.3K27M-induced phenotypes. Strikingly, we observed that the altered patterning of H3K27me3 and the fertility defects are heritable for multiple generations after the H3.3K27M mutation is lost. These findings were also validated in a tetracycline-inducible system where we can abruptly switch on and off the H3.3K27M expression. We performed a targeted RNAi screen in our tetracycline-inducible system and detected several enhancer and suppressor modifiers of H3.3K27M defects such as chromatin remodeling complexes, the nuclear RNAi pathway, and histone writer and reader complexes. Overall, our results revealed that the transmission of H3K27me3 is an epigenetically programmed event that can last for generations and that several biological pathways seem to be involved in the regulation of transmission of H3K27me3 patterns across generations. 1. L. J. Gaydos, W. Wang, S. Strome. H3K27me and PRC2 transmit a memory of repression across generations and during development. Science 345, 1515-1518 American Association for the Advancement of Science (AAAS), 2014. 2. Kamila Delaney, Maude Strobino, Joanna M. Wenda, Andrzej Pankowski, Florian A. Steiner. H3.3K27M-induced chromatin changes drive ectopic replication through misregulation of the JNK pathway in C. elegans. Nature Communications 10 Springer Science and Business Media LLC, 2019.

Ozdemir, Isa et al. (2015) International Worm Meeting "Biochemical and computational characterization of miRNA biosynthesis protein PASH-1 and its mutant PASH-1 C515Y."

Gulseren-Cakmakci, Nihal, Sar, Funda, Ozdemir, Isa

[International Worm Meeting, 2015]

The characterization of PASH-1 and PASH-1(C515Y) mutationNihal Gulseren-Cakmakci, isa Ozdemir, Funda SarMolecular Biology and Genetics Department, Koc University, Istanbul, TurkeyC. elegans PASH-1, partner of Drosha, is a 790 amino acid long RNA binding protein. PASH-1 is orthologous to Drosophila melanogaster Pasha and human DGCR8. PASH-1/DGCR8 and Drosha form the microprocessor complex and function in the first step of miRNA biosynthesis. PASH-1/DGCR8 binds to primary miRNA (pri-miRNA) transcribed by Polymerase II. PASH-1/DGCR8 acts as a molecular anchor by mediating the binding of DROSHA to correct sequences and secondary structures on pri-miRNA. Drosha crops pri-miRNA and generates 70 nucleotide long hairpin structured precursor miRNA (pre-miRNA). Exportin-5 transports pre-miRNA into cytoplasm where Dicer cleaves pre-miRNA and produces 22 nucleotide long mature miRNA. Then miRNA bind the target mRNA by partial base-pairing and inhibits protein expression. That inhibition is achieved by 2 main mechanisms: 1) target mRNA digestion by RNAse III enzymes 2) repression of varies steps of translation.Recently a conditional allele of pash-1 gene has been isolated in C. elegans. At restrictive temperatures, miRNA biosynthesis is inhibited; worms paralyze and die at young ages. A point mutation in PASH-1 protein results in temperature sensitive phenotypes. Cystein at 515th position located between two RNA-binding domains (RBD) is substituted by tyrosine (C515Y). How aforementioned phenotypes are generated by C515Y mutation at molecular level has not been elucidated yet. We characterize PASH-1 and PASH-1 C515Y mutant by using 3-D algorithm programs, molecular, biochemical and immunocytochemistry techniques. Our project will mainly compromise those characteristics: 1) cellular physiology and localization, 2) microprocessor complex formation and protein stabilities, 3) self-dimerization, 4) iron binding capacity, 5) protein modifications, 6) changes in 3-D protein structure. 3-dimentional structure of PASH-1 is resolved with simulation modelling over DGCR8 by using computational algorithms. Thus, the effect of C515Y mutation on PASH-1 3-D structure will be elucidated.

Gulseren-Cakmakci, Nihal et al. (2015) International Worm Meeting "Defects in miRNA biosynthesis leads to decline in proteostasis."

Ozdemir, Isa, Sar, Funda, Gulseren-Cakmakci, Nihal

[International Worm Meeting, 2015]

microRNAs (miRNAs) are ~22 nucleotides long, non-coding RNAs that down-regulate gene expression by inhibiting translation and/or by increasing mRNA degradation. Studies with knockouts of miRNA biogenesis genes showed that miRNAs are essential for both embryonic and larval development in C. elegans. However, post-developmental roles of miRNAs have not been fully explored. Recently, a temperature sensitive allele of miRNA biogenesis gene pash-1 has been isolated, in which miRNA production can be inactivated by temperature up-shift. pash-1 gene encoding PASH-1 protein that is essential for miRNA biogenesis. PASH-1/DGCR8 guides cleavage of primary miRNA, generating precursor miRNA Using this allele, it has been shown that loss of miRNA production leads to a rapid aging phenotype. It is known that functional decline in stress responses, mitochondrial dysfuntion, autophagy and proteastasis contributes to aging. Strikingly, pash-1ts mutant animals display 50% paralysis at early adulthood on day 5 after temperature up-shift, while wild type animals do not show any motility defects. Also half-life of pash-1ts animals are 40% shorter than wild type animals. Furthermore, we observed higher toxic oligomer level and aggregations in pash-1ts animals compared to wild type. When we enhanced the proteastasis capacity by chemical treatment, the motility defects in pash-1ts animals were rescued by 80-100% on day 5 and their life span were increased by 20%. Expression of aggreagation-prone meta-stable proteins has a widespread effect in protein homoestasis in C. elegans. When pash-1ts animals were crossed with animals that express meta-stable proteins, the phenotypes due to aggregations were observed at earlier time point than in either pash-1ts or proteotoxic model strains. Thus far, our results showed that miRNA biosynthesis is required for functional proteastasis, and absence of miRNAs leads to protein aggregations in C. elegans. Currently, our efforts focus on understanding which step(s) of proteastasis is affected and determination of the miRNAs that contributes to protein homoestasis.

Avery L (1995) Worm Breeder's Gazette "C. elegans Internet Information Servers: Status and Statistics"

Avery L

[Worm Breeder's Gazette, 1995]

As many of you know, there is a gopher server at the CGC(elegans.cbs.umn.edu) and a World-Wide-Web (WWW) server in my lab inDallas (http://eatworms.swmed.edu/) devoted to information about Celegans. These servers are heavily used.There are several other C elegans information servers, but they are notincluded here because I don't have their usage information.The table shows that, although many connections come from net-surfers whovisit once and never again (perhaps having expired from boredom), there isa large group of repeat offenders who use the servers over and over. Many, perhaps most, come from C elegans labs. (For instance,wormworld.ucsf.edu and horvitzlab3.mit.edu are pretty obvious.)My point is, if you need to reach the C elegans community, this is a goodway to do it. There is an Announcements page on both gopher and WWW. Inthe 55-day period analyzed it was read 203 times. If you have a jobopening or a conference or anything else you'd like worm people to knowabout, you probably should be advertising there. Announcements can besubmitted by e-mail to leon@eatworms.swmed.edu, and usually appear withina few hours.

Martin JA et al. (2011) Curr Biol "Complexin has opposite effects on two modes of synaptic vesicle fusion."

Fenz KM, Hu Z, Martin JA, Dittman JS, Fernandez J

[Curr Biol, 2011]

BACKGROUND: Synaptic transmission can occur in a binary or graded fashion, depending on whether transmitter release is triggered by action potentials or by gradual changes in membrane potential. Molecular differences of these two types of fusion events and their differential regulation in a physiological context have yet to be addressed. Complexin isa conserved SNARE-binding protein that has been proposed to regulate both spontaneous and stimulus-evoked synaptic vesicle (SV) fusion. RESULTS: Here we examine complexin function at a graded synapse in C.elegans. Null complexin (cpx-1) mutants are viable, although nervous system function is significantly impaired. Loss of CPX-1 results in a 3-fold increase in the rate of tonic synaptic transmission at the neuromuscular junction, whereas stimulus-evoked SV fusion is decreased 10-fold. A truncated CPX-1 missing its C-terminal domain can rescue stimulus-evoked synaptic vesicle exocytosis but fails to suppress tonic activity, demonstrating that these two modes of exocytosis can be distinguished at the molecular level. A CPX-1 variant with impaired SNARE binding also rescues evoked, but not tonic, neurotransmitter release. Finally, tonic, but not evoked, release can be rescued in a syntaxin point mutant by removing CPX-1. Rescue of either form of exocytosis partially restores locomotory behavior, indicating that both types of synaptic transmission are relevant. CONCLUSION: These observations suggest a dual role for CPX-1: suppressing SV exocytosis, driven by low levels of endogenous neural activity, while promoting synchronous fusion of SVs driven by a depolarizing stimulus. Thus, patterns of synaptic activity regulate complexin's inhibitory and permissive roles at a graded synapse.