Strome and users of the Hobert lab for reagents, I

Strome and users of the Hobert lab for reagents, I. generally accepted paradigm that transcription factors can exert their activities only in specific cellular contexts (1,2). Classic examples for such context dependency are the cell-type restricted ability of MyoD to induce muscle mass cell features (3), the region-restricted ability of ectopically expressed Pax-6/eyeless to induce ectopic eyes (4) or, as a more recent example, the restricted ability of a cocktail of transcription factors to directly reprogram the identity of pancreatic cell types (5). Our understanding of the mechanistic basis of the context dependency of transcription factor activity is limited. Overcoming such context dependency would have major implications for a variety of different applications. For example, the generation of specific cell type through transcription factor-mediated reprogramming strategies may allow the establishment ofin vitrodisease models for basic research or the provision of source material for cellular alternative therapies. We sought to establish a system in which we can study the mechanistic basis of the context-dependency of transcription factor activity. To this end, we employed a genetic approach in the nematodeC.elegans, using the Zn finger transcription factor CHE-1, which is required to induce the identity Rabbit Polyclonal to OPRK1 of a specific class of gustatory neurons, called ASE neurons (6,7). CHE-1 exerts this activity through binding directly to acis-regulatory motif (termed the “ASE motif”) present in many ASE-specific terminal differentiation genes, such as chemoreceptors, signaling proteins, neurotransmitter transporters, receptors as well as others (6). Like Delamanid (OPC-67683) CHE-1, several other invertebrate Delamanid (OPC-67683) and vertebrate transcription factors are known to also co-regulate many terminal features of differentiated neurons in such a manner and have been termed “terminal selectors” (8). To test whether CHE-1 is not only required but also sufficient to induce ASE fate, we ectopically expressed CHE-1 throughout the entire animal in either larval or adult stages, using an inducible heat-shock promoter. Such misexpression results in broad ectopic expression of an artificial reporter of CHE-1 transcription factor activity, which is composed of a multimerized ASE motif (“8 ASE motif reporter”)(6)(Fig.1). This indicates that, in theory, CHE-1 can exert its biochemical activity of DNA binding and transcriptional activation without spatial or temporal constraints. In contrast, postembryonic ectopic expression ofche-1during Delamanid (OPC-67683) larval or in adult stages is able to induce markers for terminal ASE fate (thegcy-5chemoreceptor and theceh-36homeobox gene; seeTable S1for list of markers) only in a small number of head sensory neurons but nowhere else in the animal (Fig.1; Table S2). This result illustrates the context-dependency of the ASE-fate inducing activity of CHE-1. == Physique 1. Context-dependency of CHE-1 induction of target genes. == Ectopic expression of CHE-1 can induce an artificial reporter of CHE-1 activity (a multimerized binding site, called the “8 ASE motif”) throughout the animal (upper two panels; observed in 50/50 heat-shocked animals), but can induce ASE cell fate markers, such as thegcy-5chemoreceptor (exclusively expressed in ASER) or theceh-36homeobox gene (expressed in ASEL/R and AWCL/R) only in 13 types of other sensory neurons (blue arrows), consistent with previous reports (6,7). Heat-shock induction of CHE-1 was carried out at late larval stages.Table S1contains details on transgenic reporters andTable S2shows data quantification. To test the hypothesis that this mechanistic basis for such context-dependency lies in inhibitory, perhaps chromatin-based mechanisms that may prevent CHE-1 from reprogramming the identity of other cells, we screened through an RNAi library that targets all genes in theC.elegansgenome with predicted functions in chromatin regulation, based on the presence of characteristic protein domains (9)(Table S3). We found that RNAi-mediated knock down oflin-53, theC.elegansortholog of the phylogenetically conserved, WD40 domain-containing Retinoblastoma (Rb)-binding protein RbAp46/48 (10), permits ectopically expressed CHE-1 to induce the ASE neuronal fate markersgcy-5andceh-36in a large number of normally non-neuronal cells in the midbody region of larval and adult animals (Fig.2). Up to 52% of animals (n=227) show this effect (Table S4) and this effect can be observed using distinct, non-overlapping dsRNA clones that targetlin-53and six out of six tested transgenicche-1lines (observe Supplementary Methods). == Physique.