The CONJUDOR pipeline for multiplexed knockdown of gene pairs identifies RBBP-5 as a germ cell reprogramming barrier in C. elegans.

Multiple gene activities control complex biological processes such as cell fate specification during development and cellular reprogramming. Investigating the manifold gene functions in biological systems requires also simultaneous depletion of two or more gene activities. RNA interference-mediated knockdown (RNAi) is commonly used in Caenorhabditis elegans to assess essential genes, which otherwise lead to lethality or developmental arrest upon full knockout. RNAi application is straightforward by feeding worms with RNAi plasmid-containing bacteria. However, the general approach of mixing bacterial RNAi clones to deplete two genes simultaneously often yields poor results. To address this issue, we developed a bacterial conjugation-mediated double RNAi technique 'CONJUDOR'. It allows combining RNAi bacteria for robust double RNAi with high-throughput. To demonstrate the power of CONJUDOR for large scale double RNAi screens we conjugated RNAi against the histone chaperone gene lin-53 with more than 700 other chromatin factor genes. Thereby, we identified the Set1/MLL methyltransferase complex member RBBP-5 as a novel germ cell reprogramming barrier. Our findings demonstrate that CONJUDOR increases efficiency and versatility of RNAi screens to examine interconnected biological processes in C. elegans with high-throughput.

MRG-1/MRG15 Is a Barrier for Germ Cell to Neuron Reprogramming in Caenorhabditis elegans.

Chromatin regulators play important roles in the safeguarding of cell identities by opposing the induction of ectopic cell fates and, thereby, preventing forced conversion of cell identities by reprogramming approaches. Our knowledge of chromatin regulators acting as reprogramming barriers in living organisms needs improvement as most studies use tissue culture. We used Caenorhabditis elegans as an in vivo gene discovery model and automated solid-phase RNA interference screening, by which we identified 10 chromatin-regulating factors that protect cells against ectopic fate induction. Specifically, the chromodomain protein MRG-1 safeguards germ cells against conversion into neurons. MRG-1 is the ortholog of mammalian MRG15 (MORF-related gene on chromosome 15) and is required during germline development in C. elegans However, MRG-1's function as a barrier for germ cell reprogramming has not been revealed previously. Here, we further provide protein-protein and genome interactions of MRG-1 to characterize its molecular functions. Conserved chromatin regulators may have similar functions in higher organisms, and therefore, understanding cell fate protection in C. elegans may also help to facilitate reprogramming of human cells.

The calcineurin dependent transcription factor TacA is involved in development and the stress response of Dictyostelium discoideum.

Calcineurin is an important signalling protein in a plethora of Ca(2+)-regulated cellular processes. In contrast to what is known about the function of calcineurin in various organisms, information on calcineurin substrates is still limited. Here we describe the identification and characterisation of the transcription factor activated by calcineurin (TacA) in the model organism Dictyostelium discoideum. TacA is a putative zinc-finger transcription factor orthologue of yeast Crz1. In resting unstimulated cells the protein is located in the cytosol and translocates to the nucleus in a calcineurin-dependent manner after Ca(2+)-stimulation. Nuclear export of TacA is partially dependent on GskA, the Dictyostelium orthologue of mammalian GSK3. The expression of tacA is developmentally regulated with its kinetics roughly paralleling calcineurin regulation. Silencing of tacA via RNAi leads to developmental defects and dysregulation of developmentally regulated and Ca(2+)-regulated marker genes. Additionally, TacA is involved in the stress response of D. discoideum during development in a separate pathway to the well-known stress response in Dictyostelium via STATc. Finally we provide evidence that TacA is not only an orthologue of yeast Crz1 but also functionally related to mammalian NFAT.