Schematic representation of molecules involved in the IL-3 signaling pathways that lead to mcl-1 gene transcription through the SIE and CRE-2 elements (see text for details).

Dominant negative mutant form of PU.1 attenuates the IL-3 inducibility of mcl-1 reporters with an intact SIE motif. Ba/F3 cells were transiently transfected with reporter genes as indicated along with a control or the HA/PU.1dl133 (DN-PU.1) expression vector. After transfection, cells were cultivated in medium with or without IL-3 for 12 h before cell lysates were prepared and analyzed for luciferase activity. The results shown are averages of three independent transfection experiments done in duplicate. For experiments with each indicated reporter, the luciferase activity of that particular reporter in cells cotransfected with a control vector and with IL-3 stimulation was assigned a value of 100% and all other results obtained with cells transfected with the same reporter were normalized to this activity. The differences between the results shown in lanes 2 and 3 and those shown in lanes 8 and 9 are statistically significant (*, P < 0.001; **, P = 0.002).

IL-3-induced phosphorylation of PU.1 occurs via a p38^MAPK-dependent pathway. (A) In vivo phosphorylation of PU.1. Ba/F3-HAPU.1 cells with (lane 3) or without (lane 2) prior treatment with SB203580 were stimulated with IL-3 in the presence of [³²P]orthophosphate as described in Materials and Methods. After labeling, the HA-PU.1 protein was immunoprecipitated from cell lysates by using the anti-HA antibody, resolved by SDS-PAGE, and subsequently revealed by autoradiography (top) or Western blotting with an anti-HA antibody (bottom). DMSO, dimethyl sulfoxide. (B) In vitro phosphorylation of PU.1 by the p38^MAPK immunocomplex. The bacterium-produced His/PU.1 protein was phosphorylated in vitro by the p38^MAPK immunocomplex as described in Materials and Methods. Lanes 1 and 2 are results of experiments with p38^MAPK immunocomplexes isolated from cells without and with IL-3 treatment, respectively. Lane 3, same as lane 2, except that the p38^MAPK complex was prepared from cells pretreated with SB203580 prior to IL-3 stimulation. Lane 4, same as lane 1, except that the p38^MAPK complex was isolated from cells pretreated with anisomycin. (C) Same conditions as described for panel B, except that bacterium-produced His/PU.1dlN133 was used as the substrate. (D) Same conditions as described for lane 2 of panel B, except that various bacterium-produced PU.1 mutant forms, as indicated, were used as the substrate.

p38^MAPK inhibitor (SB203580) attenuates IL-3 stimulation of mcl-1 mRNA and protein expression. (A) Ba/F3 cells deprived of IL-3 were pretreated with various inhibitors, as indicated, for 30 min prior to stimulation with IL-3 for 1 h. After IL-3 stimulation, total RNA was isolated from these cells and analyzed by Northern blotting with a probe specific for detection of the murine mcl-1 mRNA. The same blot was later stripped and hybridized with a glyceraldehyde-3-phosphophate dehydrogenase (G3PDH)-specific probe. At the bottom is a quantitative analysis of the mRNA levels shown at the top. The amount of mcl-1 mRNA in each group was normalized to that of G3PDH mRNA and plotted as a ratio relative to results obtained with cells deprived of IL-3 (lane 1). (B) Same conditions as in panel A, except that cells were lysed and 100 μg of protein lysates was analyzed by immunoblotting with antibodies specific to mouse Mcl-1 and α-tubulin, respectively. The asterisk points to an unknown protein that was also recognized by the mouse Mcl-1 antibody. DMSO, dimethyl sulfoxide.

Dominant negative mutant form of p38α attenuates the IL-3 inducibility of the SIE but not the CRE-2 reporter. (A) p38^MAPK is activated by IL-3. Cell lysates from Ba/F3 cells stimulated with IL-3 for various times were analyzed by Western blotting with an antibody recognizing the active form (P-p38) (top) or all forms (bottom) of p38^MAPK. (B) Ba/F3 cells transfected with the indicated reporter plasmid along with a control vector (lanes 1, 2, 8, and 9) or a vector expressing the dominant negative mutant form of p38α (lanes 3 and 10) or other proteins, as indicated (lanes 4 to 7), were stimulated with or without IL-3 for 12 h before the cell lysates were prepared and analyzed for luciferase activity. The results shown are averages of three independent experiments done in duplicate and are plotted as described in the legend to Fig. 3. The difference between the results shown in lanes 2 and 3 is statistically significant (*, P < 0.001).

PU.1 is one component of the SIE-binding complex. (A) Gel shift assay with the ³²P-labeled SIE probe using in vitro-translated HA/PU.1 (lane 2) or HA/PU.1dlN133 (lane 3). Lane 4, same as lane 2 except that the anti-PU.1 antibody was included in the assay. Lane 1 is the result of a control experiment using the reticulocyte lysates programmed with an empty expression vector. SS1, the HA/PU.1-DNA complex supershifted by the PU.1 antibody. (B) PU.1 is present in the complex pulled down by the SIE-containing oligonucleotide. The oligonucleotide pulldown assay using lysates of Ba/F3 cells with or without prior stimulation with IL-3 was carried out as described in Materials and Methods. The protein complex pulled down by this assay was analyzed by Western blotting with an antibody specific for PU.1. (C) Lane 1, same as lane 2 of panel A, except that nuclear extracts from IL-3-stimulated Ba/F3 cells were used in the gel shift assay. Lanes 2 and 3 are results of the same experiment with the inclusion of a 100-fold molar excess of unlabeled wild-type SIE- and mSIE (see Materials and Methods for the bases changed in the nucleotide sequence)-containing oligonucleotides, respectively, as a cold competitor (Comp.). SS denotes the B2 complex supershifted by the PU.1 antibody. (D) Same as lane 1 of panel C, except that the assay was carried out with nuclear extracts from Ba/F3 cells with or without IL-3 treatments.

Mutation of serine 142 to alanine attenuates IL-3-enhanced transactivation activity of PU.1. (A and B) The S142A, S148A, and SS142/148AA mutant forms all have transactivation activities similar to that of the wild-type (Wt or wt) protein on the SIE-containing reporter. Reporter gene assays were carried out with cells transfected with the p(−203/+10)mcl-luc (A) or −203/+10mS (B) reporter along with a pcDNA3 control or a PU.1 (wild-type or mutant)-expressing vector, as indicated. Both transfection assays were performed in the absence of IL-3. The luciferase activities obtained from cells transfected with the control vector were assigned a value of 100%. (C to E) Reporter gene assays carried out with cells transfected with the indicated reporter gene along with a wild-type or mutant PU.1 expression vector, as indicated. After transfection, cells were split into two groups; one remained untreated, and the other was stimulated with IL-3. Twelve hours after transfection, cells were lysed and analyzed for luciferase activity. The data shown here are normalized results to illustrate the average increase in the transactivation activity of wild-type or mutant PU.1 following IL-3 treatment. The normalization method used is described in the legend to Fig. 8B. The differences between the results shown in lanes 1 and 2 and those between the results shown in lanes 1 and 4 of panels C and D are statistically significant (*, P < 0.001; **, P < 0.01, n = 6).

IL-3 stimulates the transactivation activity of PU.1 on SIE-containing reporters via a p38α-dependent pathway. (A) Ba/F3 cells transfected with various mcl-1 reporter genes along with a control or PU.1 expression vector, as indicated, were stimulated with or without IL-3 for 12 h before the cell lysates were prepared and analyzed for luciferase activity. The data shown here are representative results of three or four independent experiments performed in duplicate. (B) Same conditions as in lanes 5 to 8 of panel A, except with the inclusion of vectors expressing DN-p38α, DN-p38β, and DN-p85 (lanes 5 to 7, respectively) during the transfection step. The data shown are means ± standard deviations of duplicates from an experiment that was repeated two to four times with similar results. Lanes 8 to 11 illustrate the average normalized results of four independent transfection experiments as described for lanes 4 to 7. For this normalization method, the percent increase in the transactivation activity of PU.1 caused by IL-3 treatment was assigned a value of 100 (i.e., the ratio of activity shown in lane 4 over that shown in lane 3). The activities shown in lanes 5 to 7 were each transformed by the same method, and the resultant quotient was normalized to that obtained from the results shown in lanes 3 and 4. The result shown in lane 9 is statistically significantly different from that shown in lane 8 (*, P = 0.002).

(A and B) p38α and PU.1 work in the same pathway to regulate the IL-3 inducibility of SIE-containing reporters. (A) Ba/F3 cells transfected with the p(−203/+10)mcl-luc reporter along with a control vector or vectors expressing DN-PU.1, DN-p38α, and DN-p85 or a combination of these expression vectors were left untreated or stimulated with IL-3 for 12 h before cell lysates were prepared and analyzed for luciferase activity. The data shown here are representative results of three to five independent experiments performed in duplicate. Luciferase activity was plotted as described in legend to Fig. 3. The differences between the results shown in lanes 3 and 6 and between those shown in lanes 4 and 7 are statistically significant. (*, P = 0.0001; **, P = 0.003). (B) Same conditions as in lanes 1 to 4 and 8 of panel A, except that the pGL-8XSIE reporter was used in the assay. (C) The p38^MAPK pathway is involved in the viability response of IL-3 in Ba/F3 cells. Ba/F3 cells cultivated in growth medium containing IL-3 were treated with various chemical inhibitors as indicated. Fifteen hours after each treatment, cells were harvested and the percentage of apoptotic cells in each group was quantified by flow cytometric analysis of cells with a sub-G₁ DNA content. The data shown are representative results of two independent experiments performed in duplicate. DMSO, dimethyl sulfoxide.

PU.1 binds to the mcl-1 gene promoter in vivo. (A) Schematic representation of the mcl-1 genomic locus spanning the promoter region. Fragments A to D (sizes are indicated in base pairs) are those predicted to be generated by a specified pair of primers as described in Materials and Methods. (B) ChIP analysis of PU.1 binding to the mcl-1 gene locus. Formaldehyde-cross-linked chromatin was immunoprecipitated with PU.1 antibody and processed for PCR amplification as described in Materials and Methods, by using primer sets that would specifically amplify fragments A to D, as indicated in panel A. As a positive control, PCR amplification was also carried out with input DNA from chromatin prior to the immunoprecipitation (I) step (lanes 2, 4, 6, 8, and 11). Serving as a negative control, a primer set that would specifically amplify the promoter region of the E4BP4 gene was included in the assay mixture (lanes 10 and 11). At the bottom is the result of a Southern blot analysis of the same gel with a probe spanning the mcl-1 promoter region between −175 and +38. All of the chromatin used in this assay was isolated from Ba/F3 cells stimulated with IL-3. (C) ChIP analysis similar to that described for lane 7 of panel B, except that chromatin was isolated from cells with (lanes 3, 5, 7, and 9) or without (even-numbered lanes) IL-3 treatments and the immunoprecipitation (IP) step was carried out with various antibodies (Ab), as indicated. At the bottom is the result of a Southern blot analysis of the same gel with the same probe as described for panel B. M stands for DNA size markers (100-bp ladders).