Journal: Haematologica

Article Title: The Sox4/Tcf7l1 axis promotes progression of BCR-ABL -positive acute lymphoblastic leukemia

doi: 10.3324/haematol.2014.104695

Figure Lengend Snippet: Identification of Sox4-regulated genes in ALL. (A) Genes differentially expressed in transformed Sox4fl/flSE-Cre and Sox4fl/+SE-Cre cells. Fetal liver (FL) or bone marrow (BM) pro-B cells from Sox4fl/fl or Sox4fl/+ mice were transformed with p190 BCR-ABL and transduced with SE-Cre. Gene expression microarray experiments were performed and results were compared between the two types of cells. Genes with a Sox4fl/flSE-Cre/Sox4fl/+SE-Cre signal intensity ratio of <1/3 and P<0.0001 were listed. (B) Analysis of Sox4 mRNA expression by real-time RT-PCR (left) and protein by western blotting (right) in transformed Sox4fl/+SE-Cre, Sox4fl/flSE-Cre, and Sox4fl/flSE-Cre;Sox4 pro-B cells. (C) Ratio (Sox4fl/flSE-Cre;Sox4 to Sox4fl/flSE-Cre) of mRNA expression levels of differentially expressed genes in both bone marrow (BM) and fetal liver (FL) derived transformed cells by real-time RT-PCR. The relative mRNA levels of specific genes were normalized to the level of Gapdh mRNA. Note that the ratios for most genes tested were substantially above 1, suggesting that expression of these genes was reversed upon ectopic Sox4 expression. (D) Scatter diagram demonstrating the correlation between Tcf7l1 and Sox4 expression. Tcf7l1 mRNA level is correlated with Sox4 mRNA level in leukemic cells from patients with ALL (n=11; r=0.666; P=0.0253). Expression of Gapdh was used for normalization of the RT-PCR results. (E) Enrichment of Tcf7l1 promoter sequence in Sox4-specific ChIP DNA by quantitative PCR in a bioChIP analysis. Biotin-conjugating enzyme, BirA ligase, and biotin acceptor peptide (BAP)-tagged Sox4 (BAP-Sox4) (BAP served as control) were introduced into the p190 BCR-ABL-transformed pro-B cells that had Sox4fl/fl deletion (Sox4fl/flSE-Cre). Chromatin was pulled down by magnetic beads conjugated with streptavidin (Dynabeads® MyOne™ Streptavidin T1; Invitrogen, Grand Island, NY, USA). The Sox4-specific and the control ChIP DNA was purified and subjected to real-time PCR for expression of the Tcf7l1 promoter sequence. (F) Mutational analysis of a potential Sox4 binding sequence in the Tcf7l1 promoter. The 460 bp fragment upstream of the transcription start site contains the potential Sox4 binding sequence in which mutations were introduced (-28 to -23bp: ‘ctttgt’ to ‘tgctag’) by PCR (Online Supplementary Methods). This fragment was used to construct mutant reporter pSIN-luc plasmid (MU). BCR-ABL-transformed BAP and BAP-Sox4 pro-B cells were transduced with retrovirus carrying wild-type sequence (WT), MU vector, or empty vector and luciferase activities were determined 2 days after transduction using empty vector as the background control. Data are representative of three independent experiments. Values are means ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: In the presence of biotin, BirA catalyzes conjugation of biotin to BAP-Sox4 which can then be specifically pulled down, together with bound DNA fragments (Sox4 specific ChIP DNA), by magnetic beads conjugated with streptavidin (Dynabeads ® MyOneTM Streptavidin T1; Invitrogen, Grand Island, NY, USA).

Techniques: Transformation Assay, Transduction, Gene Expression, Microarray, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Real-time Polymerase Chain Reaction, Control, Magnetic Beads, Purification, Binding Assay, Construct, Mutagenesis, Plasmid Preparation, Luciferase