Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing.
A major challenge in genetics is to identify genetic variants driving natural phenotypic variation. However, current methods of genetic mapping havelimited resolution. To address this challenge, we developed aCRISPR-Cas9-based high-throughput genome editing approach that can introduce thousands of specific genetic variants in a single experiment. This enabled us to study the fitness consequences of 16,006 natural genetic variants in yeast. We identified 572 variants with significant fitness differences in glucose media; these are highly enriched in promoters, particularly in transcription factor binding sites, while only 19.2% affect amino acid sequences. Strikingly, nearby variants nearly always favor the same parent's alleles, suggesting that lineage-specific selection is often driven by multiple clusteredvariants. In sum, our genome editing approach reveals the genetic architecture of fitness variation at single-base resolution and could be adapted tomeasure the effects of genome-wide genetic variation in any screen for cell survival or cell-sortable markers.
View details for PubMedID 30245013
Histone H3 lysine 4 monomethylation modulates long-range chromatin interactions at enhancers.
Long-range chromatin interactions between enhancers and promoters are essential for transcription of many developmentally controlled genes in mammals and other metazoans. Currently, the exact mechanisms that connect distal enhancers to their specific target promoters remain to be fully elucidated. Here, we show that the enhancer-specific histone H3 lysine 4 monomethylation (H3K4me1) and the histone methyltransferases MLL3 and MLL4 (MLL3/4) play an active role in this process. We demonstrate that in differentiating mouse embryonic stem cells, MLL3/4-dependent deposition of H3K4me1 at enhancers correlates with increased levels of chromatin interactions, whereas loss of this histone modification leads to reduced levels of chromatin interactions and defects in gene activation during differentiation. H3K4me1 facilitates recruitment of the Cohesin complex, a known regulator of chromatin organization, to chromatin in vitro and in vivo, providing a potential mechanism for MLL3/4 to promote chromatin interactions between enhancers and promoters. Taken together, our results support a role for MLL3/4-dependent H3K4me1 in orchestrating long-range chromatin interactions at enhancers in mammalian cells.Cell Research advance online publication 9 January 2018; doi:10.1038/cr.2018.1.
View details for PubMedID 29313530
RACK-1 regulates let-7 microRNA expression and terminal cell differentiation in Caenorhabditis elegans
2014; 13 (12): 1995-2009
The let-7 microRNA (miRNA) regulates cell cycle exit and terminal differentiation in the C. elegans heterochronic gene pathway. Low expression of let-7 results in retarded vulva and hypodermal cell development in C. elegans and has been associated with several human cancers. Previously, the versatile scaffold protein receptor for activated C kinase 1 (RACK1) was proposed to facilitate recruitment of the miRNA-induced silencing complex (miRISC) to the polysome and to be required for miRNA function in C. elegans and humans. Here, we show that depletion of C. elegans RACK-1 by RNAi increases let-7 miRNA levels and suppresses the retarded terminal differentiation of lateral hypodermal seam cells in mutants carrying the hypomorphic let-7(n2853) allele or lacking the let-7 family miRNA genes mir-48 and mir-241. Depletion of RACK-1 also increases the levels of precursor let-7 miRNA. When Dicer is knocked down and pre-miRNA processing is inhibited, depletion of RACK-1 still leads to increased levels of pre-let-7, suggesting that RACK-1 affects a biogenesis mechanism upstream of Dicer. No changes in the activity of the let-7 promoter or the levels of primary let-7 miRNA are associated with depletion of RACK-1, suggesting that RACK-1 affects let-7 miRNA biogenesis at the post-transcriptional level. Interestingly, rack-1 knockdown also increases the levels of a few other precursor miRNAs. Our results reveal that RACK-1 controls the biogenesis of a subset of miRNAs, including let-7, and in this way plays a role in the heterochronic gene pathway during C. elegans development.
View details for DOI 10.4161/cc.29017
View details for Web of Science ID 000337944100023
View details for PubMedID 24776851
View details for PubMedCentralID PMC4111763