Rendeiro, A. F. | Search bioRxiv

bioRxiv2017

Functional dissection of the enhancer repertoire in human embryonic stem cells

Enhancers are genetic elements that regulate spatiotemporal gene expression. Enhancer function requires transcription factor (TF) binding and correlates with histone modifications. However, the extent to which TF binding and histone modifications can functionally define active enhancers remains unclear. Here we combine chromatin immunoprecipitation with a massively parallel reporter assay to identify functional enhancers in human embryonic stem cells (hESCs) genome-wide in a quantitative unbiased manner. While active enhancers associate with TFs, only a minority of regions marked by NANOG, OCT4, H3K27ac and H3K4me1 function as enhancers, with activity changing markedly with culture conditions. Our analysis also reveals a novel enhancer set associated with housekeeping genes. Moreover, while transposable elements associate with putative enhancers only some exhibit activity. Similarly, within super-enhancers, large tracts are non-functional, with activity restricted to small sub-domains. This catalogue of validated enhancers provides a valuable resource for further functional dissection of the regulatory genome.\n\nHighlightsO_LIA catalog of functional enhancers in hESCs including a novel housekeeping class\nC_LIO_LIActive enhancers feature specific transcription factors and transposable elements\nC_LIO_LIMajor shifts in enhancer activity occur during induction of naive pluripotency\nC_LIO_LISuper-enhancers consist of small units with enhancer function\nC_LI

genomics↗

bioRxiv2016

Pooled CRISPR screening with single-cell transcriptome read-out

CRISPR-based genetic screens have revolutionized the search for new gene functions and biological mechanisms. However, widely used pooled screens are limited to simple read-outs of cell proliferation or the production of a selectable marker protein. Arrayed screens allow for more complex molecular read-outs such as transcriptome profiling, but they provide much lower throughput. Here we demonstrate CRISPR genome editing together with single-cell RNA sequencing as a new screening paradigm that combines key advantages of pooled and arrayed screens. This approach allowed us to link guide-RNA expression to the associated transcriptome responses in thousands of single cells using a straightforward and broadly applicable screening workflow.

genomics↗