Figure 5. MSI2 binds and regulates the expression of Hoxa9, c-Myc, and Ikzf2.

(A) Cumulative distribution function (CDF) showing mRNA fold enrichment of HITS-CLIP targets in the RNA sequencing data of Msi2^fl/fl and Msi2^Δ/Δ LSCs, Kolmogorov–Smirnov test. (B) Functional pathway overlap analysis from HITS-CLIP and RNA sequencing data from Msi2^fl/fl and Msi2^Δ/Δ LSCs and microarray data from Msi2^Δ/Δ LSKs. MLL-related gene sets shared among LSKs and LSCs (Supplemental Table 6). (C) RNA immunoprecipitation of MSI2 showing binding to selected MLL targets in Msi2^fl/fl (n = 4–7 independent RNA-IPs and clones), Msi2^Δ/Δ (n = 3 clones), and MSI2^O/E (n = 4 clones; MLL-AF9 leukemia cells derived from LSK transformed cells from MSI2 doxycycline transgenic mice and induced for 2 days with doxycycline) (fold change calculated over IgG for each gene SEM). Means and SEM, *P < 0.05, **P < 0.01, unpaired Student’s t test. Hebp2 was not assessed. (D) Quantitative PCR for selected MLL target genes in control and MSI2^O/E leukemic cells at 48 hours of MSI2 induction. Data are the mean of 3 independent experiments. Control, n = 3; MSI2^O/E, n = 6. Means and SEM, *P < 0.05, unpaired Student’s t test. (E) Immunoblot analysis for MLL targets and MSI2 in control and MSI2^O/E leukemic cells, from 2 independent experiments. (F) Quantitative PCR after acute deletion of Msi2 at 68 hours after 4-hydroxytestosterone (4-OHT) treatment in Msi2^fl/fl Cre-ER^– and Msi2^fl/fl Cre-ER⁺ LSCs (n = 3 Msi2^fl/fl Cre^– and n = 4 Msi2^fl/fl Cre⁺), average and SEM. (G) Immunoblot analysis for MLL targets and deletion of Msi2 at 68 hours after 4-OHT treatment in Msi2^fl/fl Cre-ER^– and Msi2^fl/fl Cre-ER⁺ leukemic cells, representative of 2 independent experiments.