dextran-enhances-lentiviral-transduction-efficiency-murine-human

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

The Blood Center of Wisconsin

Recent work has shown that RNA polymerase II-mediated transcription at distal cis-regulatory elements serves as a mark of highly active enhancers. Production of noncoding RNAs at enhancers, termed eRNAs, correlates with higher expression of genes that the enhancer interacts with; hence, eRNAs provide a new tool to model gene activity in normal and disease tissues. Moreover, this unique class of noncoding RNA has diverse roles in transcriptional regulation. Transcribed enhancers can be identified by a common signature of epigenetic marks by overlaying a series of genome-wide chromatin immunoprecipitation and RNA sequencing datasets. A computational approach to filter non-enhancer elements and other classes of noncoding RNAs is essential to not cloud downstream analysis. Here we present a protocol that combines wet and dry bench methods to accurately identify transcribed enhancers genome-wide as well as an experimental procedure to validate these datasets.

Identification of Transcribed Enhancers by Genome-Wide Chromatin Immunoprecipitation Sequencing.

Super-enhancers are tissue-specific cis-regulatory elements that drive expression of genes associated with cell identity and malignancy. A cardinal feature of super-enhancers is that they are transcribed to produce enhancer-derived RNAs (eRNAs). It remains unclear whether super-enhancers robustly activate genes in situ and whether their functions are attributable to eRNAs or the DNA element. CRISPR/Cas9 was used to systematically delete three discrete super-enhancers at the Nanog locus in embryonic stem cells, revealing functional differences in Nanog transcriptional regulation. One distal super-enhancer 45 kb upstream of Nanog (-45 enhancer) regulates both nearest neighbor genes, Nanog and Dppa3. Interestingly, eRNAs produced at the -45 enhancer specifically regulate Dppa3 expression by stabilizing looping of the -45 enhancer and Dppa3. Our work illustrates that genomic editing is required to determine enhancer function and points to a method to selectively target a subset of super-enhancer-regulated genes by depleting eRNAs.

Sridhar Rao

Laboratory of Stem Cell Transcriptional Regulation,
Blood Research Institute,
Laboratory of Stem Cell Transcriptional Regulation, Blood Research Institute

The Blood Center of Wisconsin

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

Sridhar Rao

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The Blood Center of Wisconsin

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

Laboratory of Stem Cell Transcriptional Regulation,
Blood Research Institute,
Laboratory of Stem Cell Transcriptional Regulation, Blood Research Institute