Additional file 1: of Deficiency and haploinsufficiency of histone macroH2A1.1 in mice recapitulate hematopoietic defects of human myelodysplastic syndrome

Supplemental Material and Methods. Figure S1. Conditional KO of macroH2A1.1 in mice. A. Upper panel. Targeting construct containing the sequence encoding mouse macroH2A1 (H2AFY), a loxP-flanked neomycin (neo) cassette 3′ of exon 6b (included in macroH2A1.2), and a rox-flanked cassette 3’of exon 6a (included in macroH2A1.1). Lower panel. Target construct upon Cre-mediated excision. B. Southern Blot strategy to screen for positive clones: genomic NheI-digested DNA from individual ES-cell-derived clones with a 3′ probe was used to identify homologous recombinants. A 12.3-kb DNA fragment corresponds to the wild-type macroH2A1.1 locus; integration of the loxP-flanked neomycin cassette 3′ of exon 6b introduced an additional NheI site, thus increasing the size of the NheI DNA fragment to 16.2-kb in the targeted allele. Figure S2. Peripheral blood counts and biochemical parameters in macroH2A1.1 Fl/- and 1 KO mice. Blood samples were collected into heparinized containers from wild type (WT), macroH2A1.1 Fl/Fl, macroH2A1.1 Fl/- and KO mice via tail vein. Data are expressed as mean ± SEM. * p < 0.05; **p < 0.01; ***p < 0.001 as compared to macroH2A1.1 Fl/Fl. N=12-15 per each group. Figure S3. Effect of macroH2A1.1 knock/down (KD) on ribosomal protein gene expression in HL-60 and THP-1 cells. A, B. RNA was extracted from HL-60 (A) or THP-1 (B) cells stably overexpressing a vector carrying scrambled shRNA (CTL) or a vector carrying shRNA for macroH2A1.1 (KD), and processed for qPCR using specific primers against macroH2A1.1 or macroH2A1.2 transcripts. C, D. RNA was extracted from HL-60 (C) or THP-1 (D) cells stably overexpressing a lentiviral vector carrying scrambled shRNA (CTL) or a vector carrying shRNA for macroH2A1 (KD), and processed for qPCR using specific primers against Rpl19, Rpl29, Rpl38, Rps15a and Rps21 transcripts. Data are presented as means relative to CTL cells, +/- SD, n = 4. *** P < 0.001 relative to CTL. (DOCX 680 kb)