Person:

Korthauer, Keegan

The ability to quantify cellular heterogeneity is a major advantage of single-cell technologies. However, statistical methods often treat cellular heterogeneity as a nuisance. We present a novel method to characterize differences in expression in the presence of distinct expression states within and among biological conditions. We demonstrate that this framework can detect differential expression patterns under a wide range of settings. Compared to existing approaches, this method has higher power to detect subtle differences in gene expression distributions that are more complex than a mean shift, and can characterize those differences. The freely available R package scDD implements the approach. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1077-y) contains supplementary material, which is available to authorized users.

Abstract In the post‐genomic era, thousands of putative noncoding regulatory regions have been identified, such as enhancers, promoters, long noncoding RNAs (lncRNAs), and a cadre of small peptides. These ever‐growing catalogs require high‐throughput assays to test their functionality at scale. Massively parallel reporter assays have greatly enhanced the understanding of noncoding DNA elements en masse. Here, we present a massively parallel RNA assay (MPRNA) that can assay 10,000 or more RNA segments for RNA‐based functionality. We applied MPRNA to identify RNA‐based nuclear localization domains harbored in lncRNAs. We examined a pool of 11,969 oligos densely tiling 38 human lncRNAs that were fused to a cytosolic transcript. After cell fractionation and barcode sequencing, we identified 109 unique RNA regions that significantly enriched this cytosolic transcript in the nucleus including a cytosine‐rich motif. These nuclear enrichment sequences are highly conserved and over‐represented in global nuclear fractionation sequencing. Importantly, many of these regions were independently validated by single‐molecule RNA fluorescence in situ hybridization. Overall, we demonstrate the utility of MPRNA for future investigation of RNA‐based functionalities.

Cyclin-dependent kinases 4 and 6 (CDK4/6) mediate cancer cell proliferation, and CDK4/6 inhibitors effectively induce cancer cell cycle arrest. Recent reports have suggested that CDK4/6 inhibition might also exert other effects in cancer cells, impacting their immunogenicity, apoptotic responses, and differentiation. Using breast cancer cell lines, mouse models, and clinical specimens, we show that CDK4/6 inhibition induces dramatic remodelling of cancer cell chromatin, characterised by widespread enhancer activation, and that this explains many of these effects. The newly activated enhancers include classical super-enhancers that are associated with luminal differentiation and apoptotic evasion, as well as a smaller set of enhancers overlying endogenous retroviral elements that is predicted to enhance tumour cell immunogenicity. Importantly, CDK4/6 inhibition increases the level of several Activator Protein-1 (AP-1) transcription factor family members in breast cancer cells, and AP-1 factors are implicated in the activity of many of these new enhancers. Our findings offer new insights into CDK4/6 pathway biology and have implications for the future development of CDK4/6 inhibitors.