Luke CJ et al. (2014) Methods "Non-microfluidic methods for imaging live C. elegans."

O'Reilly LP, Niehaus JZ, Luke CJ, Watkins SC

[Methods, 2014]

There are many challenges to live Caenorhabditis elegans imaging including the high motility of the animals and sustaining their viability for extended periods of time. Commonly used anesthetics to immobilize the C. elegans for imaging purpose prevents feeding of the animals and can cause cellular physiologic changes. Here we present three adapted or novel methodologies to image live C. elegans over different imaging microscopy equipment to allow for visualization of animals by DIC and fluorescence without the use of microfluidic technologies. The methods present here use common microscopy consumables and equipment found in many imaging core facilities and can be easily adapted to fit on multiple microscopy systems.

Ni, J. et al. (2017) International Worm Meeting "The triggering mechanism of C. elegans germline nuclear RNAi at the native targets."

Ni, J., Gu, S., Kalinava, N.

[International Worm Meeting, 2017]

We wish to understand the molecular mechanisms that distinguish "self" and "non-self" DNA in the germline genome of C. elegans. Nuclear RNAi refers to a set of small RNA-guided chromatin-based gene-silencing pathways (e.g., heterochromatin formation and transcriptional silencing), and plays an essential role in genome surveillance in yeast, plants, and animals. We previously identified a large set of genomic loci that are targeted by germline nuclear RNAi in C. elegans [1,2]. The triggering mechanisms at these native targets appear to be highly complex and are poorly understood. We are using two different approaches to resolve this gap. (1) We are identifying aberrant features associated with germline nuclear RNAi-targeted transcripts. To this end, we are using an unbiased RNA-seq approach to characterize both polyA and non-polyA transcripts in both wild type and mutant animals. In addition, subcellular localization of the native target transcripts are being examined using single-molecule FISH and RNA-seq combined with biochemical fractionation. (2) We are using CRISPR-mediated genome editing to test whether the silencing responses at native targets are indeed triggered by the aberrant features that are identified in (1). To date, we have found that "self" and "non-self" transcripts differ in RNA-pocessing, as well as subcellular localization. By using CRISPR, we have identified cis-regulatory elements that are required for the silencing response. Reference: 1. Ni JZ, Chen E, Gu SG. BMC Genomics. 2014. PMID: 25534009 2. Ni JZ, Kalinava N, Chen E, Huang A, Trinh T, Gu SG. Epigenetics Chromatin. 2016. PMID: 26779286

Kalinava, N. et al. (2017) International Worm Meeting "H3K9me3 plays a complex role in RNAi-mediated transcriptional silencing and transgenerational epigenetic inheritance in C. elegans."

Chen, E., Kalinava, N., Ni, J., Gu, S., Peterman, K.

[International Worm Meeting, 2017]

In many organisms, nuclear siRNA can guide transcriptional silencing and/or heterochromatin formation at the homologous genomic regions. Such heterochromatin response can be maintained over several generations in C. elegans. However, the function of the heterochromatin response remains elusive. We use biochemical, genetic and whole-genome approaches to investigate the requirements of H3K9me3 heterochromatin for initiation and maintenance of transcriptional silencing in germline nuclear RNAi pathway in C. elegans. First we identified three histone methyltransferase (HMT) - MET-2, SET-25 and SET-32 that, in combination, are required for the full H3K9me3 response in the germline nuclear RNAi pathway. Our results showed that H3K9me3 is not required for the heritable RNAi. In addition, complete depletion of H3K9me3 in met-2 set-25;set-32 mutants does not result with transcriptional de-silencing of nuclear RNAi targets. Therefore, the maintenance of transcriptional silencing by nuclear RNAi pathway can occur in H3K9me3-independent manner [1]. We recently developed a CRISPR-based system to examine the establishment of transcriptional silencing by nuclear RNAi at the native targets. This work revealed additional complexity for the silencing mechanism at the native targets in that H3K9me3 and RNAi play distinct roles at the establishment and maintenance stages of transgenerational silencing. We will report these novel findings at the meeting. Reference: 1. Kalinava N, Ni JZ, Peterman K, Chen E, Gu SG. Epigenetics Chromatin. 2017. PMID: 28228846