Systematic dissection of transcriptional regulatory networks by genome-scale and single-cell CRISPR screens.

Millions of putative transcriptional regulatory elements (TREs) have been cataloged in the human genome, yet their functional relevance in specific pathophysiological settings remains to be determined. This is critical to understand how oncogenic transcription factors (TFs) engage specific TREs to impose transcriptional programs underlying malignant phenotypes. Here, we combine cutting edge CRISPR screens and epigenomic profiling to functionally survey ≈15,000 TREs engaged by estrogen receptor (ER). We show that ER exerts its oncogenic role in breast cancer by engaging TREs enriched in GATA3, TFAP2C, and H3K27Ac signal. These TREs control critical downstream TFs, among which TFAP2C plays an essential role in ER-driven cell proliferation. Together, our work reveals novel insights into a critical oncogenic transcription program and provides a framework to map regulatory networks, enabling to dissect the function of the noncoding genome of cancer cells.

Genetic Variations of Ultraconserved Elements in the Human Genome.

Ultraconserved elements (UCEs) are among the most popular DNA markers for phylogenomic analysis. In at least three of five placental mammalian genomes (human, dog, cow, mouse, and rat), 2189 UCEs of at least 200 bp in length that are identical have been identified. Most of these regions have not yet been functionally annotated, and their associations with diseases remain largely unknown. This is an important knowledge gap in human genomics with regard to UCE roles in physiologically critical functions, and by extension, their relevance for shared susceptibilities to common complex diseases across several mammalian organisms in the event of their polymorphic variations. In the present study, we remapped the genomic locations of these UCEs to the latest human genome assembly, and examined them for documented polymorphisms in sequenced human genomes. We identified 29,983 polymorphisms within analyzed UCEs, but revealed that a vast majority exhibits very low minor allele frequencies. Notably, only 112 of the identified polymorphisms are associated with a phenotype in the Ensembl genome browser. Through literature analyses, we confirmed associations of 37 (i.e., out of the 112) polymorphisms within 23 UCEs with 25 diseases and phenotypic traits, including, muscular dystrophies, eye diseases, and cancers (e.g., familial adenomatous polyposis). Most reports of UCE polymorphism-disease associations appeared to be not cognizant that their candidate polymorphisms were actually within UCEs. The present study offers strategic directions and knowledge gaps for future computational and experimental work so as to better understand the thus far intriguing and puzzling role(s) of UCEs in mammalian genomes.

Time- and concentration-dependent expression of immune and barrier genes in the RTgutGC fish intestinal model following immune stimulation.

The fish intestine comprises an important environment-organism interface that is vital to fish growth, health and pathogen defense. Yet, knowledge about the physiology and defense mechanisms toward environmental stressors, such as bacterial or viral cues, is limited and depends largely on in vivo experiments with fish. On this background, we here explore the immune competence of a recently established in vitro intestinal barrier model based on the rainbow trout (Oncorhynchus mykiss) intestinal epithelial cell line, RTgutGC. We demonstrate that the RTgutGC cell barrier reacts to two immune stimuli, the bacterial lipopolysaccharide (LPS) from Escherichia coli and the viral Poly(I:C), by regulating the mRNA abundance of selected genes in a partly time- and concentration dependent manner. The immune stimuli activated the Myd88-and Ticam-dependent signalling cascades, which resulted in downstream activation of pro-inflammatory cytokines and interferon, comparable to the regulatory patterns known from in vivo. Stimuli exposure furthermore influenced the regulation of epithelial barrier markers and resulted in slightly impaired barrier functionality after long-term exposure to LPS. Collectively, we provide proof of the usefulness of this unique cell culture model to further gain basic understanding of the fish innate immune system and to apply it in various fields, such as fish feed development and fish health in aquaculture or the evaluation of immuno-toxicity of chemical contaminants.