Genomics and Proteomics
Enigmatic MELK: The controversy surrounding its complex role in cancerThe Ser/Thr protein kinase MELK (maternal embryonic leucine zipper kinase) has been considered an attractive therapeutic target for managing cancer since 2005. Studies using expression analysis have indicated that MELK expression is higher in numerous cancer cells and tissues than in their normal, nonneoplastic counterparts. Further, RNAi-mediated MELK depletion impairs proliferation of multiple cancers, including triple-negative breast cancer (TNBC), and these growth defects can be rescued with exogenous WT MELK, but not kinase-dead MELK complementation.
Microhomology-based CRISPR tagging tools for protein tracking, purification, and depletionWork in yeast models has benefitted tremendously from the insertion of epitope or fluorescence tags at the native gene locus to study protein function and behavior under physiological conditions. In contrast, work in mammalian cells largely relies on overexpression of tagged proteins because high-quality antibodies are only available for a fraction of the mammalian proteome. CRISPR/Cas9-mediated genome editing has recently emerged as a powerful genome-modifying tool that can also be exploited to insert various tags and fluorophores at gene loci to study the physiological behavior of proteins in most organisms, including mammals.
Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-G374R mutation in achondroplasia in miceCRISPR/Cas9 is a powerful technology widely used for genome editing, with the potential to be used for correcting a wide variety of deleterious disease-causing mutations. However, the technique tends to generate more indels (insertions and deletions) than precise modifications at the target sites, which might not resolve the mutation and could instead exacerbate the initial genetic disruption. We sought to develop an improved protocol for CRISPR/Cas9 that would correct mutations without unintended consequences.
A kidney-specific genetic control module in mice governs endocrine regulation of the cytochrome P450 gene Cyp27b1 essential for vitamin D3 activationThe vitamin D endocrine system regulates mineral homeostasis through its activities in the intestine, kidney, and bone. Terminal activation of vitamin D3 to its hormonal form, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), occurs in the kidney via the cytochrome P450 enzyme CYP27B1. Despite its importance in vitamin D metabolism, the molecular mechanisms underlying the regulation of the gene for this enzyme, Cyp27b1, are unknown. Here, we identified a kidney-specific control module governed by a renal cell-specific chromatin structure located distal to Cyp27b1 that mediates unique basal and parathyroid hormone (PTH)-, fibroblast growth factor 23 (FGF23)-, and 1,25(OH)2D3-mediated regulation of Cyp27b1 expression.
Selective Distal Enhancer Control of the Mmp13 Gene Identified through Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Genomic DeletionsBackground: Mmp13 is vital to bone homeostasis and controlled by a plethora of stimuli.Results: Mmp13 is modulated by distinct distal enhancers for basal (−30 kb) and vitamin D regulation (−10 kb).Conclusion: Enhancer deletions lead to altered transcription factor occupancy and expression for Mmp13.Significance: Specific CRISPR deletions reveal the repressive secondary effect of VDR, coordinated multi-enhancer gene control, and distal basal regulation of Mmp13.