c-MYB is a transcriptional regulator of ESPL1/Separase in BCR-ABL-positive chronic myeloid leukemia

Abstract

Background: Genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML). Recently, we have shown that clonal evolution and blast crisis correlate with altered expression and activity of Separase, a cysteine endopeptidase that is a mitotic key player in chromosomal segregation and centriole duplication. Hyperactivation of Separase in human hematopoietic cells has been linked to a feedback mechanism that posttranslationally stimulates Separase proteolytic activity after imatinib therapy-induced reduction of Separase protein levels.

Methods and results: In search for potential therapy-responsive transcriptional mechanisms we have investigated the role of the transcription factor c-MYB for Separase expression in CML cell lines (LAMA-84, K562, BV-173) and in clinical samples. Quantitative RT-PCR and Western blot immunostaining experiments revealed that c-MYB expression levels are decreased in an imatinib-dependent manner and positively correlate with Separase expression levels in cell lines and in clinical CML samples. RNA silencing of c-MYB expression in CML cell lines resulted in reduced Separase protein levels. Gelshift and ChIP assays confirmed that c-MYB binds to a putative c-MYB binding sequence located within the ESPL1 promoter.

Conclusions: Our data suggest that ESPL1/Separase is a regulatory target of c-MYB. Therefore, c-MYB, known to be required for BCR-ABL-dependent transformation of hematopoietic progenitors and leukemogenesis, may also control the Separase-dependent fidelity of mitotic chromosomal segregation and centriole duplication essential for maintenance of genomic stability.

Keywords: BCR-ABL; CML; ESPL1/Separase; Genomic stability; Imatinib; c-MYB.

Fig. 1

Analysis of c-MYB and ESPL1 transcript levels, c-MYB and Separase protein levels in LAMA-84 (a), K562 (b) and BCR-ABL-negative control cells (U937) (c) upon IM treatment. Treatment (dose, period) was performed as noted in Table 1. Level changes (Δ-values in %) are shown as calculated from comparison with the corresponding untreated cells. Upper panel. Transcript levels were analyzed by qRT-PCR, protein levels by Western blot immunostaining densitometry. Representative Western blot images are shown in the lower panel. In all qRT-PCR experiments the housekeeping gene Gus (beta-glucuronidase) served as internal standard. For Western blot immunostaining experiments Actin was used as loading control and reference parameter. Densitometric data are derived from at least triplicate experiments and are denoted in Table 1. P-values are given above the respective column. Abbreviations: ns, not significant; nd, not determined

Fig. 2

Comparative analysis of c-MYB and ESPL1 transcript levels in paired cDNA samples of CML patients (n = 5) before and under IM therapy. Percent changes (Δ-values are differences between means) are shown corresponding to expression level changes within paired samples, each pair derived from the same patient at differing time points (sample at diagnosis (before IM treatment) vs. sample after major molecular response (MMR) achievement under IM therapy). All transcript levels were normalized to Gus and represent mean values of triplicate qRT-PCR assays

Fig. 3

Separase expression after c-MYB silencing by RNAi in BV-173 and LAMA-84 cells. BV-173 (panel a) and LAMA-84 cells (panel b) were treated with negative control siRNA (nc) and c-MYB-specific siRNA (siRNA). C-MYB transcript levels are measured by qRT-PCR (left column). The house-keeping gene Gus (beta-glucuronidase) served as internal standard. C-MYB and Separase regulation on protein levels were determined by quantitative Western blot immunostaining experiments 48 h post transfection (middle and right columns, respectively). Corresponding representative Western blot images are shown in the very right panels of A and B. Actin served as loading control for Western blot immunostaining. All densitometric data are derived from at least triplicate experiments

Fig. 4

Electrophoretic mobility shift assay (EMSA) using synthetic ESPL1 promoter-derived c-MYB binding site probes and Chromatin immunoprecipitation (ChIP). a Schematic drawing depicting the ESPL1 Separase promoter and location of predicted regulatory DNA motifs (drawing not to scale) (Pati 2008). The arrow shows the predicted transcription start site (TSS). Abbreviations: TATA, TATA box; PRE, progesterone responsive element; ERE, estrogen responsive element; p53, p53 binding element; c-MYB, predicted c-MYB binding element. Numbers denote upstream distances with respect to the TSS. b A FITC-labeled double stranded DNA oligonucleotide corresponding to the putative c-MYB binding site of the ESPL1 promoter was incubated with native BV-173 nuclear extract. DNA/protein complexes were resolved on a 0.5 % TBE 1.0 % native LE/GTG agarose gel. The left panel (tonal inversion) shows FITC-related fluorescence signaling of the gel before blotting, the right panel depicts the corresponding anti-c-MYB Western blot immunostaining. The lanes represent: lane 1, DNA target (FITC-labeled oligonucleotide) without nuclear extract; lane 2, DNA target with nuclear extract; lane 3, DNA target with nuclear extract and with 100fold molar excess of analogous unlabeled oligonucleotide as binding competitor. c ChIP analysis of BV-173 cells. DNA fragments immunoprecipitated by anti-c-MYB IgG and a non-binding control IgG were amplified by qRT-PCR. Results are expressed as percentage of input (average). ChIP results are derived from at least triplicate qRT-PCR measurements