The ability of transcription factors to directly reprogram the identity of cell types is usually restricted and is defined by cellular context. We show here that through ectopic expression of single C. elegans transcription factors, the identity of mitotic germ cells can be directly converted into that of specific neuron types (glutamatergic, cholinergic or GABAergic). This reprogramming event requires the removal of the histone chaperone LIN-53/RbAp48, a component of several histone remodeling and modifying complexes, and this removal can be mimicked by chemical inhibition of histone deacetylases. Our findings illustrate the ability of germ cells to be directly converted into individual, terminally differentiated neuron types and demonstrate that a specific chromatin factor provides a barrier for cellular reprogramming.

Engineering fluorescent proteins into large genomic clones, contained within BACs or fosmid vectors, is a tool to visualize and study spatiotemporal gene expression patterns in transgenic animals. Because these reporters cover large genomic regions, they most likely capture all cis-regulatory information and can therefore be expected to recapitulate all aspects of endogenous gene expression. Inserting tags at the target gene locus contained within genomic clones by homologous recombination (“recombineering”) represents the most straightforward method to generate these reporters. In this methodology paper, we describe a simple and robust pipeline for recombineering of fosmids, which we apply to generate reporter constructs in the nematode C. elegans, whose genome is almost entirely covered in an available fosmid library. We have generated a toolkit that allows for insertion of fluorescent proteins (GFP, YFP, CFP, VENUS, mCherry) and affinity tags at specific target sites within fosmid clones in a virtually seamless manner. Our new pipeline is less complex and, in our hands, works more robustly than previously described recombineering strategies to generate reporter fusions for C. elegans expression studies. Furthermore, our toolkit provides a novel recombineering cassette which inserts a SL2-spliced intercistronic region between the gene of interest and the fluorescent protein, thus creating a reporter controlled by all 5′ and 3′ cis-acting regulatory elements of the examined gene without the direct translational fusion between the two. With this configuration, the onset of expression and tissue specificity of secreted, sub-cellular compartmentalized or short-lived gene products can be easily detected. We describe other applications of fosmid recombineering as well. The simplicity, speed and robustness of the recombineering pipeline described here should prompt the routine use of this strategy for expression studies in C. elegans.

How specific cell types can be directly converted into other distinct cell types is a matter of intense investigation with wide-ranging basic and biomedical implications. We show here that the removal of the histone 3 lysine 27 (H3K27) methyltransferase complex PRC2 (“Polycomb Repressor Complex 2”) permits ectopically expressed, neuron-type-specific transcription factors (“terminal selectors”) to convert C. elegans germ cells directly into specific neuron types. Terminal selector-induced germ cell-to-neuron conversion can not only be observed upon genome-wide loss of H3K27 methylation in PRC2(−) animals, but also upon genome-wide redistribution of H3K27 methylation patterns in animals which lack the H3K36 methyltransferase MES-4. Manipulation of the H3K27 methylation status not only permits conversion of germ cells into neurons, but also permits hlh-1/MyoD-dependent conversion of germ cells into muscle cells, indicating the PRC2 protects the germline from the aberrant execution of multiple distinct somatic differentiation programs. Taken together, our findings demonstrate that the normally multi-step process of development from a germ cell via a zygote to a terminally differentiated somatic cell type can be shortcut by providing an appropriate terminal selector transcription factor and manipulating histone methylation patterns.