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. 2013 Nov 18;210(12):2641–2659. doi: 10.1084/jem.20131141

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© 2013 Abdel-Wahab et al.

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Figure 8. — Identification of genes significantly dysregulated with deletion of Asxl1 alone and in concert with deletion of Tet2 and their functional impact. (A) Volcano plot of differentially expressed transcripts from RNA-Seq data of 1-yr-old control versus littermate Asxl1 KO (Mx1-cre Asxl1^fl/fl) LSK and MP (lineage⁻ Sca-1⁻ c-Kit⁺) cells (experiment included cells from two individual mice per genotype). (B) Venn diagrams of genes significantly up- and down-regulated with Asxl1 loss in LSK and MP (lineage⁻, Sca-1⁻, cKit⁺) cells from 1-yr-old Mx1-cre Asxl1^fl/fl mice and littermate Cre⁻ controls as identified in A. (C) qRT-PCR analysis of HoxA and Hox-associated transcription factor genes in LSK cells of 1-yr-old Cre⁻ Asxl1^fl/fl control versus littermate Vav-cre Asxl1^fl/fl. (D) qRT-PCR analysis of p16^INK4a in LT-HSCs (lineage⁻, Sca-1⁺, c-Kit⁺, CD150⁺, CD48⁻) and MPP cells (lineage⁻, Sca-1⁺, c-Kit⁺, CD150⁻, CD48⁺) from 6-wk- and 6-mo-old control (C) versus littermate Vav-cre Asxl1^fl/fl (KO) mice. (E) Cell cycle analysis of MPPs from 72-wk-old Vav-cre Asxl1^fl/fl or littermate Cre⁻ Asxl1^fl/fl control mice with in vivo BrdU administration. Representative FACS plot analysis showing gating on MPP cells followed by BrdU versus DAPI stain is shown on the left (parent gate is LSK cells). Relative quantification of the percentage of MPP cells in S, G2/M, and G0/1 phase is shown on the right (n = 5 mice per group). (F) Assessment of the proportion of HSPCs undergoing apoptosis was performed by Annexin V/DAPI stain of LSK cells from 72-wk-old Vav-cre Asxl1^fl/fl mice or Cre⁻ Asxl1^fl/fl littermate controls. Representative FACS plot analysis showing gating on LSK cells followed by Annexin V versus DAPI stain is shown on the left (parent gate is lineage⁻ cells). Relative quantification of the percentage of Annexin V⁺/DAPI⁻ and Annexin V⁺/DAPI⁺ LSK cells is shown on the right (n = 5 mice per group). (G) Comparison of significant differentially expressed genes in LSK cells from 6-wk-old Mx1-cre Asxl1^fl/fl, Mx1-cre Tet2^fl/fl, or Mx1-cre Asxl1^fl/fl Tet2^fl/fl relative to controls (or Mx1-cre Asxl1 WT Tet2 WT). 99 genes are uniquely down-regulated in Asxl1/Tet2 DKO mice relative to all other genotypes (left), whereas 49 genes are significantly up-regulated (right). (H) GSEA of overlapping and statistically significant gene sets enriched in the LSK cells of mice with deletion of Asxl1 alone or with combined Asxl1 and Tet2 deletion. (I) Gene sets uniquely enriched in mice with concomitant deletion of Asxl1 and Tet2 relative to all other genotypes as determined by GSEA. Error bars represent ±SD; *, P < 0.05 (Mann–Whitney U test).