Increased transactivation of c-Myb-regulated promoters by p89c-Mybex9b
Two c-Myb mutans, Δ(358–452)c-Myb and Δ(389–418)c-Myb, which lack 93 and 30 amino acids, respectively, in the leucine-zipper-NRD (LZ-NRD) are more potent than wild-type p75c-Myb
in enhancing proliferation and blocking apoptosis of normal and transformed hematopoietic cells.17
p89c-Mybex9b, a naturally occurring alternatively spliced form of c-myb, which accounts for approximately 10–15% of total c-Myb levels, contains an insertion of 121 amino acids in the LZ which disrupts its structure, presumably preventing the interaction with regulatory c-Myb-interacting proteins.
A comparison () of p75c-Myb, p89c-Mybex9b and the LZ-NRD mutant Δ(358–452)c-Myb shows that the LZ is altered in p89c-Mybex9b and in the c-Myb mutant, suggesting that p89c-Mybex9b may function like the artificial LZ-NRD Δ(358–452)c-Myb.
Figure 1 Transactivation activity of p89c-Mybex9b. (a) Schematic diagram of c-Myb proteins; levels of p75c-Myb, p89c-Mybex9b and Δ(358–452)c-Myb in transfected 293T cells (b) and K562 cells (c) used for luciferase assays. Expression of β-actin (more ...)
The transactivation potential of p89c-Mybex9b, Δ(358–452)c-Myb, and p75c-Myb was compared by dual-luciferase assays using reporter plasmids consisting of a fragment of the Myb-regulated human cyclin B1 or SLUG promoter driving the luciferase gene (cyclin B1-Luc or SLUG-Luc).
Following co-transfection of reporter and effector plasmids in 293T cells, expression and transactivation of each c-Myb protein were analyzed.
Expression of the three c-Myb isoforms was essentially identical in 293T cells (). p89c-Mybex9b and Δ(358–452)c-Myb were slightly more effective than p75c-Myb in transactivation of the cyclin B1 promoter but not of the SLUG promoter (). The transactivating ability of the three c-Myb proteins was also tested in Ph1 K562 cells; because K562 cells express high levels of endogenous c-Myb, the increase in expression of p75c-Myb in transfected cells () was quantitated by densitometry (), whereas expression of p89c-Mybex9b and Δ(358–452)c-Myb was detected because of size differences and quantitated by densitometry ().
Compared with 293T cells, the effects of the c-Myb isoforms in K562 cells were markedly different: p75c-Myb induced approximately a three-fold increase in transactivation of the cyclin B1 promoter, whereas p89c-Mybex9b and Δ(358–452)c-Myb were much more potent (~fifteen- and twelve-fold increase, respectively) (, upper panel); likewise, p89c-Mybex9b and Δ(358–452)c-Myb transactivated the SLUG promoter more effectively than p75c-Myb (~five- and six- versus three-fold, respectively) (, lower panel).
p89c-Mybex9b binds to the human cyclin B1 and SLUG promoters
To assess whether p89c-Mybex9b interacted with the cyclin B1 and SLUG promoters, we performed chromatin immunoprecipitation assays in K562 cells overexpressing FLAG-tagged-p89c-Mybex9b using either an anti-FLAG antibody (which binds only the ectopically expressed protein) or an anti-c-Myb antibody (which binds the endogenous and the ectopically expressed protein). The ability of p89c-Mybex9b to bind the two promoters was assessed by real-time PCR amplification of a 5′-flanking region segment, which includes putative c-Myb-binding sites (, right). The −122 to +177 nucleotide segment of the cyclin B1 promoter and the −77 to +175 segment of the SLUG promoter were amplified above background in both the anti-c-Myb and anti-FLAG chromatin IPs, indicating that p89c-Mybex9b binds to these c-Myb-regulated promoters (, left).
Figure 2 p89c-Mybex9b binds the cyclin B1 and SLUG promoter. Chromatin immunoprecipitation assays show binding (detected by real-time PCR) of p75c-Myb and p89c-Mybex9 to a segment of the cyclin B1 promoter (a, left) and SLUG promoter (b, left). Error bars denote (more ...)
Enhanced stability of p89c-Mybex9b in BCR/ABL-positive cells
In addition to the transactivation ability, we also tested the stability of p89c-Mybex9b
because the insertion of ex9b disrupts the LZ domain and c-Myb LZ-domain mutant proteins are more stable than wild-type p75c-Myb
Thus, parental and p89c-Mybex9b
-expressing K562 cells were treated with cycloheximide for different times and cell lysates blotted with an anti-c-Myb antibody to assess half-life of endogenous p75c-Myb
and ectopic p89c-Mybex9b
, expressed at comparable levels. Expression of p75c-Myb
was more rapidly downmodulated of p89c-Mybex9b
; by densitometry analysis, the half-life of p89c-Mybex9b
min, nearly 30
min longer than that of p75c-Myb
enhances the stability of c-Myb,17
we also assessed the half-life of the two c-Myb isoforms in IM-treated cells. Inhibition of BCR/ABL tyrosine kinase activity decreased the half-life of p75c-Myb
min in IM-treated versus 60
min in untreated cells); in contrast, the half-life of p89c-Mybex9b
was unaffected by treatment with IM (). By densitometry analysis, the half-life of p89c-Mybex9b
in IM-treated cells was ~75
min longer than that of p75c-Myb
(110 versus 35
Inhibition of p89c-Mybex9b expression suppresses proliferation and colony formation of K562 cells.
We investigated the role of p89c-Mybex9b expression in transformed cells by assessing whether its specific knockdown has any effect on the proliferation and clonogenic potential of Ph1 K562 cells.
Two siRNAs targeting two different regions of human exon 9b c-Myb transcript (siRNA-1 and siRNA-2, ) suppressed very effectively p89c-Mybex9b
expression in K562 cells (, left) with no effects on levels of the p75c-Myb
isoform. As control, we also used a pool of siRNAs targeting both p75c-Myb
and the p89c-Mybex9b
transcripts; transfection with this c-Myb siRNA pool led to the disappearance of both c-Myb isoforms (, right). Real-Time PCR using primers specific for p75c-Myb
transcripts further demonstrated that silencing of p89c-Mybex9b
expression had no effect on levels of p75c-Myb
transcripts (Supplementary Figure 1
Figure 4 Effects of p89c-Mybex9b downregulation in K562 cells. (a) Schematic diagram representing the siRNAs target sequences of p89c-Mybex9b and the mutations in p89c-Mybex9b MUT. (b) Western blot shows levels of p75c-Myb and p89c-Mybex9b in K652 cells transfected (more ...)
K562 cells transfected with the c-Myb siRNA pool or with p89c-Mybex9b
-specific siRNAs proliferated less than control siRNA-transfected cells (); at 48
h, the c-Myb siRNA pool was more effective than the p89c-Mybex9b
-specific siRNAs (54% versus 30% inhibition, respectively), but at 72
h, the effects were comparable.
We also assessed the effect of p89c-Mybex9b
-specific downregulation on colony formation of K562 cells. Thus, cells were transfected with control or specific siRNAs and 24
h after the second transfection were plated in methylcellulose (500 cells per plate) and colonies were counted 6 days later. Cells treated with control siRNA formed 350±36 colonies (), whereas K562 cells transfected with the c-Myb siRNA pool formed 178±27 colonies; a significant decrease in colony formation was also noted by plating cells transfected with p89c-Mybex9b
-siRNA-2 (230±14 colonies) or p89c-Mybex9b
-siRNA-1 (230±11) ().
To assess the specificity of the biological effects induced by p89c-Mybex9b-siRNA-2, we generated K562-derivative lines ectopically expressing wild-type p89c-Mybex9b and a mutant, p89c-Mybex9bMUT, with four nucleotide substitutions in the sequence targeted by p89c-Mybex9b-siRNA-2; these mutations were designed to prevent interaction with siRNA-2 while preserving the amino-acid sequence of the protein ().
MSCV-p89c-Mybex9b and MSCV-p89c-Mybex9bMUT K562 cells were then transfected with a control or the p89c-Mybex9b-siRNA-2 and levels of p89c-Mybex9b were tested by western blotting. Levels of p89c-Mybex9b were undetectable in MSCV-p89c-Mybex9b K562 cells transfected with p89c-Mybex9b siRNA-2 (); in contrast, expression of p89c-Mybex9b was detected in siRNA-2-transfected MSCV-p89c-Mybex9bMUT K562 cells, reflecting downregulation of the endogenous protein but not of the p89c-Mybex9b protein derived from p89c-Mybex9bMUT, which is not targeted by p89c-Mybex9b-siRNA-2. As expected, MSCV-p89c-Mybex9b K562 cells transfected with p89c-Mybex9b-siRNA-2 formed fewer colonies (~50% inhibition) than the scramble siRNA-transfected counterpart (); in contrast, there were no significant differences in colony formation of scrambled- or p89c-Mybex9b-siRNA-2-transfected MSCV-p89c-Mybex9bMUT K562 cells (), further confirming that the inhibition of K562 colony formation by p89c-Mybex9b-siRNA-2 is due to specific downregulation of p89c-Mybex9b expression.
The importance of p89c-Mybex9b expression for K562 colony formation was also assessed in K562 cells expressing a DOX-inducible c-Myb shRNA, which downregulates the p75c-Myb and p89c-Mybex9b isoforms, and carrying the p75c-Myb or p89c-Mybex9b expression vector non-targetable by the DOX-inducible c-Myb shRNA (p75c-Myb_shMUT and p89c-Mybex9b_shMUT, respectively) (). As expected, colony formation of DOX-treated K562-c-Myb shRNA cells was markedly suppressed (~80% inhibition); in contrast, expression of p89c-Mybex9b_shMUT or p75c-Myb_shMUT rescued, in part, the inhibitory effect of the DOX-inducible c-Myb shRNA (), consistent with redundant and yet non-overlapping effects of the p75c-Myb and the p89c-Mybex9b-Myb isoforms.
Levels of cyclin B1 are downregulated in p89c-Mybex9b-silenced K562 cells
The reduced colony formation of c-Myb-silenced (p75c-Myb
) K562 cells may be caused by changes in the expression of c-Myb-regulated genes with a role in cell proliferation. Cyclin B1, a gene with an essential role in the G2/M transition, is one of the c-Myb targets27
whose change in expression may explain, in part, the effects of p89c-Mybex9b
Indeed, compared with control siRNA-transfected cells, cyclin B1 expression was reduced in K562 cells transfected with the c-Myb siRNA pool (~45% decrease of cyclin B1 levels); although expression of p75c-Myb was unaffected, the specific downregulation of p89c-Mybex9 led to a similar decrease (~42%) of cyclin B1 expression ().
Figure 5 Effects of p89c-Mybex9b downregulation on cyclin B1 levels and G2/M-phase cells. (a) Western blot shows levels of c-Myb isoforms and cyclin B1 in K652 cells 16h after treatment with the c-Myb siRNA pool or c-Mybex9b siRNA or control siRNA. Cyclin (more ...)
We also analyzed the fraction of G2/M-phase cells in c-Myb- or p89c-Mybex9-silenced K562 cells by evaluating DNA content of propidium iodide-stained cells and percentage of cells positive for phospho-histone-H3, which is a marker of M phase. As expected, the decrease in the cyclin B1 levels was accompanied by a reduction in the number of cells in G2/M phase (). Although not statistically significant, in three different experiments, the number of M-phase cells was lower in c-Myb- or p89c-Mybex9b-siRNA-2 than in control siRNA-transfected K562 cells ().
p89c-Mybex9b expression affects the response of K562 cells to treatment with IM
Because expression of p89c-Mybex9b appears to be important for proliferation and colony formation of K562 cells, we speculated that it may also influence the response to treatment with IM.
- or Δ(358–452)c-Myb-expressing K562 cells were treated with IM, and cell counts and DNA content analyses were performed every 24
As expected, expression of Δ(358–452)c-Myb reduced the IM sensitivity of K562 cells (); expression of p89c-Mybex9b
had a similar, although less potent, effect as IM-treated p89c-Mybex9b
-expressing K562 cells exhibited a less pronounced decrease in cell number than parental cells (). Similar effects were observed upon assessing apoptosis of IM-treated K562 cells; as expected, IM-treated parental K562 cells showed a high percentage of apoptosis, which increased from 24 to 72
h. In contrast, apoptosis of IM-treated K562 cells expressing p89c-Mybex9b
or Δ(358–452)c-Myb was approximately 50% lower than that of parental cells (). In normal growth conditions, overexpression of p89c-Mybex9b
or Δ(358–452)c-Myb has no effect on the proliferation and basal apoptosis of K562 cells (Supplementary Figure 2
Figure 6 Effects of p89c-Mybex9b levels on viability of IM-treated K562 cells. (a) Cell count and (b) apoptosis of IM-treated parental, p89c-Mybex9b or Δ(358–452)c-Myb ectopically expressing K562 cells. (c) Cell count and (d) apoptosis of IM-treated (more ...)
We also assessed the effect of c-Myb downregulation on IM-treated K562 cells. Parental cells transfected with the c-Myb siRNA pool, p89c-Mybex9b
isoform-specific or control siRNAs were counted and analyzed by flow cytometry 24, 48 and 72
h after IM treatment. Downregulation of c-Myb or p89c-Mybex9b
enhanced the proliferation inhibitory effects of IM, compared with cells transfected with the control siRNA (at 72
h, ~60% fewer cells than in control siRNA-transfected K562 cells) (). Evaluation of apoptosis by DNA content analysis showed a similar pattern: c-Myb-silenced cells exhibited increased apoptosis compared with cells transfected with control siRNAs. Apoptotic cells were slightly more numerous after transfection with c-Myb siRNAs than with the p89c-Mybex9b
-specific siRNA but the differences were not significant ().
We also assessed the relationship between levels of p89c-Mybex9b
and colony formation after IM treatment. Thus, parental or ectopically expressing p89c-Mybex9b
or Δ(358–452)c-Myb K562 cells were treated for 24
h with I
) and plated in methylcellulose (500 cells per plate, in the presence of 1
IM); colonies were counted after 6 days.
Consistent with the effects on cell proliferation, expression of the p89c-Mybex9b isoform enhanced the number of clonogenic cells after IM treatment (13.8% and 6.5% of residual clonogenic cells in p89c-Mybex9b-expressing versus parental K562 cells). Residual colonies formed by p89c-Mybex9b or Δ(358–452)c-Myb-expressing K562 cells were nearly identical ().
Colony assays were also performed after c-Myb or p89c-Mybex9b-specific siRNA tranfection and IM treatment of K562 cells. As expected, the combination of Myb downregulation and IM treatment caused a marked decrease in the clonogenic potential of K562 cells. Residual colony formation of cells transfected with the c-Myb siRNA pool or p89c-Mybex9b-siRNA was similar (1.1% and 1.9%, respectively) and lower of that from cells transfected with control siRNAs (3.1%) (). Together, these data suggest that expression of the p89c-Mybex9b isoform can modulate the effects of IM in K562 cells.
Inhibition of p89c-Mybex9b suppresses colony formation of normal and CML CD34+ progenitors
The role of p89c-Mybex9b expression was also assessed in colony-formation assays of CD34+ cells from healthy donors and CML patients.
Normal human CD34+
=3) were transfected with the c-Myb siRNA pool or a p89c-Mybex9b
-specific siRNA or the control siRNA. 24
h after transfection, levels of c-Myb proteins were tested and cells were plated for colony-formation assays. A representative western blot shows that levels of p75c-Myb
were completely downregulated in cells transfected with the c-Myb siRNA pool, whereas transfection with p89 c-Mybex9b
-siRNA reduced only the levels of the p89c-Mybex9b
isoform (). Normal CD34+
cells transfected with the c-Myb siRNA pool or the p89c-Mybex9b
-specific siRNA formed fewer colonies than control siRNA-transfected cells (~33% and 28% inhibition, respectively; range: 23–43% and 22–37%, respectively) ().
Figure 7 Colony formation of c-Myb or p89c-Mybex9b siRNA-transfected normal or CML CD34+ progenitors. (a and c) Western blots show levels of p75c-Myb and p89c-Mybex9b in a representative sample of normal (a) or CML (c) CD34+ cells after transfection (more ...)
Peripheral blood CD34+ cells from CML chronic-phase patients (n=5) were also transfected with c-Myb or control siRNAs. Like in normal CD34+ cells, expression of p75c-Myb and p89c-Mybex9b was markedly downregulated in CML CD34+ cells transfected with the c-Myb siRNA pool, whereas only p89c-Mybex9b levels decreased in cells transfected with p89c-Mybex9b-siRNA (). Transfected CML CD34+ cells were seeded in cytokine-supplemented methylcellulose and colonies were counted 9 days later. Colonies arising from cells transfected with the c-Myb siRNA pool or p89c-Mybex9b-siRNA were fewer (63% and 41% decrease, respectively; range: 50–90% and 28–58% , respectively) than those from the scramble siRNA-transfected counterpart ().
Together, these results suggest that expression of p89c-Mybex9b is more important for colony formation of CML than normal CD34+ progenitors.