Fig 2: RAR localizes to transcribed regions of the Rarβ2 gene in a P-TEFB-dependent manner.(A) Structure of the mouse Rarβ2 promoter. pRARE: proximal RARE; dRARE: distal RARE. (B) Gene expression in P19 cells. P19 cells were treated for 48 hours with DMSO, 1 µM all trans RA (atRA), 250 µM cAMP or transfected with a HA-tag COUP-TFI expression vector. Expression of Rarβ2, Top2β, Creb, Coup-TFI, Tcf19 and Rplp0 were quantified by RT-QPCR. Basal expression levels were arbitrarily set to 1 and data are expressed as the mean±SEM (n = 3). *, p<0.05; **, p<0.01; ***, p<0.005. (C) RARα and phosphorylated RNApol II loading at the Rplp0 and Pou5f1/Oct4 promoters. P19 cells were treated as in (B) and ChIP assays were performed with indicated antibodies. (D) AF9 colocalizes to the Rarβ2 promoter. P19 cells were treated for 4 hours with 1 µM atRA, and ChIP assays were carried out as described. The specific enrichment in Rarβ2 promoter sequence is expressed after normalization to background values (Myoglobin gene). Data are expressed as the mean±SEM (n = 2). *, p<0.05; **, p<0.01; ***, p<0.005. (E) DRB inhibition of the Rarβ2 gene transcription. P19 cells were treated with the indicated combination of atRA (1 µM) and varying doses of DRB (50 to 5000 nM) for 4 hours. Rarβ2 mRNA was quantified by RT-QPCR. (F) A CDK9 dominant negative mutant inhibits Rarβ2 gene expression. P19 were cotransfected with increasing amount of pCMV-lacZ (control), pCMV-HA-wtCDK9, or pCMV-HA-dnCDK9 expression vectors at the indicated ratio, then treated 24 hours with 1 µM atRA. Gene expression was quantified as above and data expressed as the mean±SEM (n = 4). *, p<0.05; **, p<0.01; ***, p<0.005. (G) RAR and phosphorylated RNApol II are detected at transcribed regions of the Rarβ2 gene. P19 cells were treated as in (D) and ChIP/reChIP assays were performed. (H) P-TEFb inhibition prevents RAR association to Rarβ2 elongated regions. P19 cells were treated for 4 hours with the indicated combination of TTNPB (1 µM) or flavopiridol (250 nM). ChIP assays (n = 2) were performed as in (D).

Fig 3: MLL-translocation cell lines are sensitive to inhibition of pan-Cullin-dependent and CKS1-dependent protein degradation. (A) Western blots for THP-1 (MLL-AF9) and KOPN-8 (MLL-ENL) cells treated with vehicle control (DMSO), 0.1 μM MLN4924 or 1 μM C1 for 16 h and 24 h. Histone H3 was used as a loading control. Western blots are representative of 3 independent experiments. (B) Cell viability was assessed for THP-1, KOPN-8, healthy PBMC and CD34+ control cells after 3 days treatment with indicated concentrations of the pan-Cullin inhibitor (MLN4924) or the SKP2-CKS1 inhibitor (C1). Results represent the mean of 3 independent experiments with standard deviation bars. (C) Proportion of apoptotic CD34+, THP-1 and KOPN-8 cells, in response to two different concentrations of C1 or MLN4924, was measured by PI and Annexin V-FITC staining using flow cytometry. Significance was tested using the Student's t-test versus DMSO treated cells (N = 3). (D) Cell cycle status was measured for KOPN-8 cells in response to DMSO vehicle control or MLN4924 (1 μM) using the Click-iT EdU incorporation kit. Graphs are representative of 3 independent experiments.

Fig 4: Modulation of Wnt signalling in MLL-translocation cell lines by CKS1 and CKS2. TCF/LEF activity was measured in (A) KOPN-8 (MLL-ENL) and (B) THP-1 (MLL-AF9) cell lines using the TOPFlash reporter plasmid (or FOPFlash plasmid control) and pRL-TK Renilla control. Cell lines were co-transfected with siRNA to knockdown expression of CKS1B, CKS2 or CKS1B + CKS2. Values are represented as fold change RLA versus Non-specific siRNA treated cells. Significance was tested using the Student's t-test (N = 3).

Fig 5: Gene expression level in response to RARα activation in wild type, AF9- and BRD4-deficient backgrounds.(A) Genes exhibiting a fold-change above 1.2 fold in TTNPB-treated P19wt cells were clusterized according to a functional gene ontology classification. Representative functional clusters from the top 10 hits are shown. The basal level in non treated P19wt was arbitrarily set to 1 and is depicted by black boxes. Numbers indicate the fold change ratio of individual genes relative to untreated wt P19 (red: upregulation; green: downregulation; black, no change). (B) Gene expression levels of known atRA-target genes. RA-target genes were selected from the literature and their expression levels were extracted from microarray data. Results are represented as in (A).