Mll5 is Required for Hematopoietic Stem Cell Fitness and Homeostasis

MLL5 is a novel trithorax group gene and a candidate tumor suppressor gene located within a 2.5 Mb interval of chromosome band 7q22 that is frequently deleted in human myeloid malignancy. Here we show that Mll5 is required for normal hematopoietic stem cell (HSC) homeostasis. Inactivation of the Mll5 gene in mice results in reduced cellularity of the long-term HSC compartment, which correlates with functional impairment of long-term repopulation potential under competitive conditions. Bone marrow cells from Mll5-deficient mice were defective in spleen colony-forming assays, and the mutant mice showed enhanced susceptibility to 5-Fluorouracil-induced myelosuppression. Heterozygous and homozygous Mll5 mutant mice did not spontaneously develop hematologic cancers, and loss of Mll5 did not alter the phenotype of a fatal myeloprolferative disorder induced by oncogenic Kras in vivo. Collectively, the data reveal an important role for Mll5 in HSC homeostasis, and provide a basis for further studies to explore its role in leukemogenesis. Keywords: Cell type comparision. Global gene expression analysis. Bone marrow cells were pooled from 6 pairs of eight weeks old Mll5-/- mice and sex matched wild type littermates, and then were stained with a cocktail of biotinylated anti-mouse lineage antibodies to CD3, CD4, CD5, CD8, B220, CD11b, Gr-1 and Ter119 (eBioscience). For detection and sorting, we used Streptavidin conjugated with PE-Cy7, anti-Sca-1-APC and c-Kit-APC-Alexa F750 antibodies (eBioscience). 2.5 × 105 LSK (Lin-, Sca-1+, c-kit+) cells were flow sorted. Total RNA (320 ng) was isolated by using RNeasy Mini kit (Qiagen) following the manufactures instruction. RNA amplification and array hybridization were performed in UCSF Shared Microarray Core Facilities. Briefly, total RNA quality was assessed using a Pico Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). RNA was amplified and labeled with Cy3-CTP using the Agilent low RNA input fluorescent linear amplification kits following the manufacturers protocol (Agilent). Labeled cRNA was assessed using the Nandrop ND-100 (Nanodrop Technologies, Inc., Wilmington DE), and equal amounts of Cy3 labeled target were hybridized to Agilent whole mouse genome 4x44K Ink-jet arrays (Agilent). Hybridizations were performed for 14 hours, according to the manufacturers protocol (Agilent). Arrays were scanned using the Agilent microarray scanner (Agilent) and raw signal intensities were extracted with Feature Extraction v9.1 software (Agilent,).