BLOOD: 2. 1. 2014

BLOOD: Mortalin and DJ-1 coordinately regulate hematopoietic stem cell function through the control of oxidative stress

"Hematopoietic stem cells (HSCs) maintain stemness through various mechanisms that protect against stressful conditions. Heat shock proteins (HSPs) preserve cell homeostasis during stress responses through protein quality control, suggesting that HSPs may safeguard HSCs against numerous traumas. Here, we show that mortalin, a mitochondrial HSP, plays an essential role in maintaining HSC properties by regulating oxidative stress. Mortalin is primarily localized in hematopoietic stem and progenitor cell (HSPC) compartments. In this study, the inhibition of mortalin function caused abnormal reactive oxygen species (ROS) elevation in HSCs and reduced HSC numbers. Knockdown (KD) of mortalin in HSPCs impaired their ability to repopulate and form colonies. Moreover, mortalin-KD HSCs could not maintain quiescence and showed severe downregulation of cyclin-dependent kinase inhibitor– and antioxidant-related genes. Conversely, HSCs that overexpressed mortalin maintained a high reconstitution capacity and low ROS levels. Furthermore, DJ-1, one of the genes responsible for Parkinson’s disease, directly bound to mortalin and acted as a negative ROS regulator. Using DJ-1–deficient mice, we demonstrated that mortalin and DJ-1 coordinately maintain normal ROS levels and HSC numbers. Collectively, these results indicate that the mortalin/DJ-1 complex guards against mitochondrial oxidative stress and is indispensable for the maintenance of HSCs."

http://bloodjournal.hematologylibrary.org/content/123/1/41.abstract

SCIENCE: Genetic Screens in Human Cells Using the CRISPR-Cas9 SYSTEM

"The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells."

http://www.sciencemag.org/content/343/6166/80.abstract