Signal transducer and activator of transcription STAT5 is recruited to c-Myc super-enhancer
© Pinz et al. 2016
Received: 20 January 2016
Accepted: 7 April 2016
Published: 14 April 2016
c-Myc has been proposed as a putative target gene of signal transducer and activator of transcription 5 (STAT5). No functional STAT5 binding site has been identified so far within the c-Myc gene locus, therefore a direct transcriptional regulation by STAT5 remains uncertain. c-Myc super-enhancer, located 1.7 Mb downstream of the c-Myc gene locus, was recently reported as essential for the regulation of c-Myc gene expression by hematopoietic transcription factors and bromodomain and extra-terminal (BET) proteins and for leukemia maintenance. c-Myc super-enhancer is composed of five regulatory regions (E1–E5) which recruit transcription and chromatin-associated factors, mediating chromatin looping and interaction with the c-Myc promoter.
We now show that STAT5 strongly binds to c-Myc super-enhancer regions E3 and E4, both in normal and transformed Ba/F3 cells. We also found that the BET protein bromodomain-containing protein 2 (BRD2), a co-factor of STAT5, co-localizes with STAT5 at E3/E4 in Ba/F3 cells transformed by the constitutively active STAT5-1*6 mutant, but not in non-transformed Ba/F3 cells. BRD2 binding at E3/E4 coincides with c-Myc transcriptional activation and is lost upon treatment with deacetylase and BET inhibitors, both of which inhibit STAT5 transcriptional activity and c-Myc gene expression.
Our data suggest that constitutive STAT5 binding to c-Myc super-enhancer might contribute to BRD2 maintenance and thus allow sustained expression of c-Myc in Ba/F3 cells transformed by STAT5-1*6.
KeywordsSTAT5 c-Myc BET BRD2 Super-enhancer Chromatin
c-Myc is a master regulator of essential biological processes such as cell proliferation, survival, differentiation, metabolism, angiogenesis and pluripotency establishment and maintenance [1, 2]. c-Myc is found overexpressed in most human cancers and is a hallmark of tumor initiation and maintenance [3, 4]. Characterizing the regulatory mechanisms of c-Myc gene expression is therefore fundamental for a better understanding of its deregulation in cancer and to possibly identify novel therapies aiming at controlling its expression. c-Myc gene transcription is regulated by multiple transcription factors via responsive elements located within both its promoter and remote enhancer regions [1, 5–9]. A number of reports, including those from our lab, provided evidence that c-Myc expression is regulated by signal transducer and activator of transcription 5 (STAT5) [10–13]. STAT5 is an essential regulator of cell differentiation, proliferation and survival [11, 14, 15] and is frequently constitutively activated in cancer. STAT5 constitutive activation results in c-Myc overexpression, increased cell proliferation and reduced cell apoptosis, and is as such an important player in cancer initiation and progression [11, 16–20]. Among molecules that suppress STAT5 activity, inhibitors of tyrosine kinases, of deacetylases and of bromodomain and extra-terminal (BET) proteins represent promising therapeutic agents, either alone or in combination [21–28]. We and others showed that expression of STAT5 target genes, including c-Myc, is reduced upon treatment with deacetylase inhibitors (trichostatin A, suberoylanilide hydroxamic acid, apicidin, valproic acid, sodium butyrate) or with the BET inhibitor (+)-JQ1 [12, 29–31]. Further elucidating the process of STAT5-induced expression of c-Myc and of its inhibition by clinically relevant therapeutic agents is critical for improving cancer therapy.
Among the evidence of a direct regulation of c-Myc by STAT5, an elegant study by Lord et al. demonstrated that c-Myc expression in response to IL-2 and IL-3 is dependent on the transactivation domain of STAT5 . Accordingly, c-Myc expression is upregulated in cells expressing constitutively active STAT5, including in the BCR-ABL-transformed human leukemic cell line K562 [11–13, 32]. Constitutive activation of STAT5 by the oncogenic tyrosine kinase BCR-ABL contributes to K562 cell transformation [33, 34]. Overexpression of c-Myc in K562 cells is inhibited by the BCR-ABL tyrosine kinase inhibitor imatinib but also by the deacetylase inhibitor trichostatin A (TSA), which we showed to inhibit STAT5-mediated transcription [12, 31, 32]. In the same line, overexpression of c-Myc in Ba/F3 cells expressing the constitutively active STAT5 mutant 1*6  is repressed by TSA [12, 31]. In apparent contradiction with a direct regulation a c-Myc by STAT5, we found that STAT5 knock-down in Ba/F3 cells did not affect IL-3-induced c-Myc gene expression . Two other acknowledged direct target genes of STAT5, Bcl-x and Id-1 [10, 13, 36–38], remained equally unaffected upon STAT5 knock-down in Ba/F3 cells . Although these observations might be interpreted as an indication that these genes are not regulated by STAT5, they might also reflect an unconventional mechanism of regulation by STAT5, possibly not as sensitive to the partial (60 %) knock-down generated upon siRNA transfection . Interestingly, in contrast to most classical STAT5 target genes (e.g., Cis, Osm, Spi2.1, …) which display STAT5 responsive elements within their promoter region , functional STAT5 binding sites have been identified outside the promoter regions of Bcl-x and Id-1, notably within Bcl-x first intron [36, 38] and within Id-1 enhancer located several kb downstream of the Id-1 gene . These observations raise the possibility that regulation of c-Myc expression by STAT5 might be likewise unconventional, possibly involving distal elements. In support of this proposition, we previously attempted and failed to detect STAT5 binding along the c-Myc gene , including at GAS elements present in its promoter and known to mediate transcriptional response to other STAT family members [39–41].
Recently, several research groups described the role of a 3′ super-enhancer in the regulation of c-Myc gene expression in hematopoietic cells and its importance in c-Myc overexpression in leukemic cells [5–8, 42]. c-Myc super-enhancer is located 1.7 Mb downstream of the c-Myc coding region. It consists of five enhancer regions (E1–E5) with multiple binding sites for transcription factors. These transcription factors recruit transcriptional co-factors, in particular the BET bromodomain protein BRD4 and the SwItch/Sucrose non-fermentable (SWI/SNF) protein BRG1. These chromatin-associated factors mediate c-Myc transcription via long-range chromatin looping and selective interaction with the c-Myc promoter [5–7]. On the other hand, we recently demonstrated that STAT5 transcriptional activity is regulated by BET proteins, including BRD2 . We found that BRD2 is recruited along STAT5 at the proximal promoter and transcription start site of the conventional STAT5 target gene Cis, both upon IL-3-induced STAT5 activation and in cells expressing and transformed by constitutively active STAT5. BRD2 association with the Cis gene is lost upon treatment with the BET inhibitor (+)-JQ1 (thereafter referred to as JQ1 ), resulting in transcriptional inhibition . Interestingly, deacetylase inhibitors also repress STAT5-mediated transcription by displacing BRD2 and preventing recruitment of the transcriptional machinery [12, 31]. Our data therefore suggest that STAT5 recruits BRD2 which, in turn, supports transcriptional activation by assisting the assembly of the preinitiation complex [12, 31]. Given that STAT5-induced expression of c-Myc is also inhibited by JQ1 [30, 31, 44], and given the absence of STAT5 binding activity within c-Myc promoter and gene locus, we tested the hypothesis that STAT5 binds to the 3′ super-enhancer and recruits BRD2 proteins to regulate c-Myc gene expression.
c-Myc expression is induced by STAT5 and repressed by inhibitors of deacetylases and of BET proteins in Ba/F3-derived cells
STAT5 binds to c-Myc super-enhancer
BRD2 co-localizes with constitutively active STAT5 at c-Myc super-enhancer
To better characterize the role of STAT5 in BRD2 recruitment to STAT5 target genes, we next addressed whether STAT5A-1*6 and BRD2 physically interact. Co-immunoprecipitation experiments were carried out on nuclear lysates from formaldehyde-crosslinked Ba/F3-1*6 cells using STAT5A- and BRD2-specific antibodies and following the ChIP protocol (Additional file 1: Fig. S1A–C). Upon immunoprecipitation (IP), samples were analysed by Western blot (Additional file 1: Fig. S1B) and quantitative PCR as a control (Additional file 1: Fig. S1C). Recruitment efficiency and specificity of STAT5A-1*6 and BRD2 to the Cis gene was comparable to that observed before (Additional file 1: Fig. S1C). No co-immunoprecipitation of STAT5A-1*6 and BRD2 was detected in Western blot, following neither STAT5A nor BRD2 IP (Additional file 1: Fig. S1B). It should be noted that, while STAT5A was strongly immunoprecipitated under the ChIP experimental conditions, BRD2 was poorly pulled-down in the same conditions (Additional file 1: Fig. S1B), probably explaining the weak signals usually detected by qPCR following BRD2 ChIP (Additional file 1: Fig. S1C; Fig. 4a), but also making it difficult to evidence a co-immunoprecipitation with STAT5A. The weak immunoprecipitation efficiency observed in BRD2 ChIP is likely the consequence of the experimental conditions used (formaldehyde-mediated crosslinking and/or IP buffer composition). Indeed, conventional immunoprecipitation from nuclear lysates of non-crosslinked cells (Additional file 1: Fig. S1D) and using mild buffer conditions resulted in efficient BRD2 immunoprecipitation (Additional file 1: Fig. S1E). However, no co-immunoprecipitation of STAT5A was observed in these conditions. Altogether, these experiments suggest that STAT5A-1*6 and BRD2 do not directly interact but are rather co-recruited at the chromatin level.
Finally, we investigated BRD2 association with the c-Myc gene and its super-enhancer in IL-3-stimulated Ba/F3 cells. While BRD2 was recruited upon IL-3 stimulation at the transcription start site of the Cis gene, as previously described , no BRD2 enrichment above the IgG background was detected along the c-Myc gene and downstream enhancer in the same conditions (Fig. 4b). This observation suggests that, by contrast to STAT5A-1*6, IL-3-induced wild-type STAT5 might not be efficient in recruiting and/or stabilizing BRD2 at c-Myc enhancer.
This study identified functional STAT5 binding sites possibly regulating c-Myc transcription. We showed that STAT5 specifically binds to enhancer regions E3 and E4 of c-Myc 3′ super-enhancer. Putative STAT5 binding sites within E3/E4 are organised in clusters of consensus and non-consensus binding motifs. Similar functional STAT5 binding sites have been described at other STAT5 target genes . Binding of wild-type STAT5 to E3/E4 was induced by IL-3 in normal Ba/F3 cells while STAT5A-1*6 binding was constitutive and independent of IL-3 in transformed Ba/F3-1*6 cells. Interestingly, ChIP-Seq assays performed in human HEL leukemia cells revealed that JAK2V617F-induced constitutive active STAT5 [47, 48] is bound at c-Myc enhancer region E3 (ArrayExpress accession number E-MTAB-1096; ), providing another evidence of STAT5 constitutive binding at c-Myc super-enhancer in leukemia cells.
Constitutive binding of STAT5 at E3/E4 in Ba/F3-1*6 cells coincided with that of BRD2, suggesting that STAT5 might play a role in BRD2 maintenance at c-Myc super-enhancer in transformed cells. By contrast, BRD2 was not detected by ChIP at the c-Myc locus in IL-3-stimulated Ba/F3 cells. We cannot exclude at this point that this absence of specific signal is due to a detection problem. Alternatively, it might be the consequence of transient STAT5 binding in Ba/F3 cells, as opposed to constitutive STAT5 binding in Ba/F3-1*6 cells which might be necessary for BRD2 maintenance at c-Myc super-enhancer. We could not evidence an interaction between STAT5A-1*6 and BRD2 in co-immunoprecipitation assays. Therefore, our data suggest that constitutive binding of STAT5A-1*6 at E3/E4 might—directly or indirectly—assist and/or stabilize BRD2 association with c-Myc super-enhancer. Furthermore, it remains possible that other BET protein(s) are involved in the regulation of c-Myc by wild-type STAT5 in Ba/F3 cells. This proposition is supported by our previous observation that BRD2 was detected at the Cis but not at the Osm promoter in IL-3-stimulated Ba/F3 cells . This is also in agreement with the implication of BRD4 in long-range regulation of c-Myc transcription via its 3′ super-enhancer [5, 6]. Interestingly, it was shown that multiple transcription factors, the BET protein BRD4 and the SWI/SNF component BRG1 are recruited to c-Myc 3′ enhancer and contribute to c-Myc gene transcription [5, 6]. Notably, BRG1 was proposed to maintain transcription factor occupancy at the enhancer region and to facilitate interactions with the c-Myc promoter . Whether such a mechanism of stabilisation by BRG1 also takes place in STAT5-mediated transcription remains to be shown. Maintenance of STAT5 occupancy at c-Myc super-enhancer would be an attractive explanation for the absence of effect of STAT5 (partial) knock-down on c-Myc gene expression .
We showed that constitutive binding of STAT5 and maintenance of BRD2 at E3/E4 in transformed Ba/F3-1*6 cells correlated with transcriptional activation of c-Myc. Furthermore, BRD2 binding at E3/E4 was lost upon TSA- and JQ1-mediated inhibition of c-Myc expression, both of which inhibit STAT5-induced transcription . Our data therefore suggest that constitutive binding of STAT5A-1*6 contributes to BRD2 maintenance at c-Myc super-enhancer in transformed Ba/F3-1*6 cells, which in turn might be implicated in c-Myc overexpression. In support of a role of STAT5 in c-Myc overexpression in leukemia via c-Myc super-enhancer, E3 was recently shown to display enhancer activity in K562 leukemia cells, using a luciferase reporter assay . This finding nicely fits with our previous finding that c-Myc expression in K562 cells is dependent on BCR-ABL-induced constitutive active STAT5 . Our model is hence in line with the recently suggested leukemia maintenance function attributed to c-Myc 3′ super-enhancer, via the recruitment of BET proteins by hematopoietic transcription factors [5, 6, 8]. We propose that the transcription factor STAT5 might play a similar role in c-Myc overexpression, in leukemia exhibiting constitutive STAT5 activation. Further functional assays will be necessary to verify this proposition.
Dimethyl sulfoxide (DMSO) and trichostatin A (TSA) were purchased from SIGMA (D-2650 and T-8552 respectively). (+)-JQ1 (BPS Bioscience #27401)—hereafter abbreviated to JQ1—was purchased from BIOMOL GmbH. TSA and JQ1 were dissolved in DMSO at a final concentration of 1 mM (TSA) and 5 mM (JQ1). DMSO was used as vehicle control. Its final concentration was adjusted to 0.02 % in all conditions.
All cell lines were cultivated at 37 °C under 5 % CO2 in a humidified incubator. The interleukin-3 (IL-3)-dependent mouse pro-B cell line Ba/F3 (a kind gift from Jacqueline Marvel, IFR 128 BioSciences Gerland-Lyon Sud, France ) was grown in RPMI 1640 (PAN-Biotech P04-16500) supplemented with 10 % heat-inactivated fetal calf serum (FCS; PAN-Biotech), penicillin/streptomycin (100 U/mL penicillin, 100 μg/mL streptomycin; PAN-Biotech) and 2 ng/ml rmIL-3 (ImmunoTools). No ethical approval was required to obtain and use the Ba/F3 parental cell line. Ba/F3-derived cell lines were generated according to German GenTSV (genetic engineering safety regulations; authorization AZ.55.1-8791.7.52). The IL-3-independent Ba/F3-1*6 cell line (clone F7) stably expressing the FLAG-tagged constitutively active mouse STAT5A-1*6 mutant  has been described , and was grown in RPMI 1640 supplemented with 10 % heat-inactivated FCS, penicillin/streptomycin and 600 μg/ml G418 (SIGMA A-1720). The IL-3-dependent Ba/F3-WT cell line (clone A7) expressing FLAG-tagged wild-type (WT) mouse STAT5A was generated by electroporating Ba/F3 cells with a pcDNA3-based expression vector allowing expression of a mSTAT5A-WT-FLAG fusion protein. Stably transfected cells were selected in IL-3-containing medium in the presence of 800 μg/mL G418 (PAA). Individual clones were isolated and characterized to verify mSTAT5A-WT transgene expression and proper IL-3-dependent activation of STAT5A-WT. Ba/F3-WT clone A7 was used for this study and served as a control for potential adverse effects of FLAG-tagged protein overexpression. Ba/F3-WT cells were maintained in RPMI 1640 supplemented with 10 % heat-inactivated FCS, penicillin/streptomycin, 600 μg/ml G418 (SIGMA A-1720) and 2 ng/ml rmIL-3. For cytokine stimulation of Ba/F3 and Ba/F3-WT cells, cells were washed twice in RPMI 1640 and rested in RPMI 1640, 10 % FCS, penicillin/streptomycin for 6–12 h before addition of 5 ng/ml IL-3 for 15–120 min, as indicated. For inhibitor treatment of Ba/F3 and Ba/F3-WT cells, rested cells were pre-treated 30 min with the respective compound or with DMSO (vehicle) prior to IL-3 stimulation. Ba/F3-1*6 cells were treated with inhibitors or vehicle for 60 min.
For co-immunoprecipitation assays from non-crosslinked cells, previously described Ba/F3-tet-on-1*6 cells conditionally expressing STAT5A-1*6 in the presence of doxycycline were used . Briefly, Ba/F3-tet-on-1*6 cells were grown for 9 h in the presence of 1 μg/ml doxycycline in IL-3-free medium. Cells were harvested for nuclear fractionation and western blot analysis.
Gene expression analysis by RT-qPCR
Following inhibitor and cytokine treatments, cells were harvested, cDNA synthesized and quantitative PCR performed as previously described . Nucleotide sequence of the qPCR primers used in this study have been published [12, 13, 31]. Data were normalized to mouse S9 ribosomal mRNA and expressed as relative mRNA levels, like previously reported [12, 31]. Data are mean ± SD of the quantitative PCR performed in either duplicate or triplicate, and are representative of at least two independent experiments.
Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation (ChIP) was carried out from either whole-cell (conventional protocol) or nuclear (alternative protocol) lysates, following reported procedures [12, 31, 50]. Chromatin Immunoprecipitation from nuclear lysates yields stronger signals than from whole cell lysates . Antibodies used were as follows: STAT5A (Santa Cruz Biotechnology sc-1081), STAT5A + B (Santa Cruz Biotechnology sc-835), BRD2 (Bethyl A302-583A) and IgG from rabbit serum (SIGMA I-5006). Antibody concentrations used were as reported [12, 31]. Co-precipitated genomic DNA was measured by quantitative PCR. Mouse Cis- and Osm-specific primers have been described . Mouse c-Myc-specific forward and reverse primers were the following, respectively: STAT1, TTTATTCTAGGGTCTCTGCAGGC and GAAAACCCGGACTTCCCAG; STAT3, CCCTCCTGCCTCCTGAAGG and CAGGATCCCTCCCCTCCC; ORF, AACAACCGCAAGTGCTCCAG and GTCGTTTTCCTCCGTGTCTGAG; E1, ACGCTCAGAGTGCTTTCCAT and GGTGGTGTGGGGTGACTAATAT; E2, GTGGGAGGGACTGAAATGGAG and TGGGCAAAGCTAGAGGCAGAT; E3, GAACAGGAAGCTGGGGAAAT and TGCAAGGAGGCTTTTCCTAA; E4, CACCCCAGCCTCAATTCAGT and GCTGCGATGACTTCTAAACGG; E5, GCAACAGCAAGAACCAGTGA and TGCTTCTCCTGAACCACCTT. Results are expressed as percentage (%) of input DNA. Data are mean ± SD of the quantitative PCR performed in either duplicate or triplicate, and are representative of at least two independent experiments.
Protein analysis by immunoprecipitation and Western blot
Western blots were performed as described , using the following antibodies and respective dilutions: pSTAT5 (#9351, Cell Signaling Technology; 1:1000), STAT5A (L-20, sc-1081, Santa-Cruz Biotechnology; 1:1000), STAT5A/B (C-17, sc-835, Santa-Cruz Biotechnology; 1:1000), FLAG (M2, SIGMA F-1804; 1:500), Brd2 (Bethyl A302–583A; 1:2000), α-tubulin (DM1A, sc-32293, Santa-Cruz Biotechnology; 1:200), HDAC1 (Millipore 05-100; 1:1000), Anti-Rabbit IgG-Peroxidase (SIGMA A0545; 1:10,000), Anti-Mouse IgG-Peroxidase (SIGMA A8924; 1:10,000). HDAC1 and α-tubulin were used as nuclear and cytosolic protein markers respectively to control the quality of the nuclear fractionation, like previously reported .
Brij whole-cell protein lysis and nuclear fractionation from non-crosslinked cells were conducted as previously described . Immunoprecipitations from non-crosslinked Ba/F3-tet-on-1*6 cells was performed using 250 μg nuclear protein lysate diluted 1:10 in Brij buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.875 % Brij 97, 0.125 % NP40, 10 mM NaF, 1 mM Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride) and 1.2 μg BRD2 antibody (Bethyl A302–583A) or 1.2 μg rabbit IgG (SIGMA I-5006) as a negative control. Immunoprecipitations were conducted for 5 h and immunocomplexes were collected using protein A-Sepharose beads. Consecutive to washing in Brij buffer, beads were boiled in 60 μl Laemmli buffer. Half of the eluted bead fraction was analyzed by Western blot in parallel to input lysate (3 % or 8 μg nuclear proteins) and immunoprecipitation supernatants (same volume as input lysate).
Nuclear-enriched protein lysate preparation and immunoprecipitations (IP) from formaldehyde-crosslinked Ba/F3-1*6 cells were carried out following the alternative ChIP protocol described above [31, 50], using 7 × 106 cells for the nuclei preparation and 2.4 μg antibody (rabbit IgG, STAT5A or BRD2) per IP. Subsequent to the last washing step, ~20 % beads were further processed for genomic DNA isolation and qPCR analysis, following the ChIP protocol. The remaining beads were boiled 10 min in 50 μl Laemmli buffer and ~40 % of the eluted bead fraction (20 μl) was analysed by Western blot in parallel to input lysate (3 %) and immunoprecipitation supernatants (same volume as input lysate).
mouse pro-B IL-3-dependent cell line
IL-3-independent BaF3 cell line stably expressing constitutive active STAT5A-1*6
tyrosine kinase oncogenic fusion
bromodomain and extra-terminal protein
- BRD2 and BRD4:
bromodomain-containing protein 2 and 4 respectively, members of the BET protein family
Brahma-related gene-1 encoding an ATPase and component of the SWI/SNF complex
- GAS element:
Gamma interferon activation site (STAT-responsive) element
- IL-2 and IL-3:
interleukin-2 and -3
inhibitor of BET protein function
human leukemic cell line
suberoylanilide hydroxamic acid
signal transducer and activator of transcription 5
constitutively active STAT5A mutant
SWItch/Sucrose Non-Fermentable, chromatin remodelling complex
AR designed and performed experiments, and drafted the manuscript. SP and SU performed experiments and revised the manuscript. All authors read and approved the final manuscript.
We thank Jacqueline Marvel for providing the Ba/F3 cell line and Krystina Beer for her technical contribution while completing an internship in our group. We are grateful to Joachim Griesenbeck for critically reading the manuscript.
Availability of data and materials
All the data supporting the findings are contained within the manuscript and Additional file 1 (Fig. S1).
The authors declare that they have no competing interests.
Consent to publish
No ethics approval and consent to participate were required for this study.
This work was supported by the Deutsche Forschungsgemeinschaft [RA 2010/2-1 to A.R.], the Deutsche Krebshilfe [109750 to A.R.], and institutional research funds [Foerderlinie C to A.R.; Frauenfoerderung, Bayerisches Programm zur Realisierung der Chancengleichheit fuer Frauen in Forschung und Lehre, to S.P.]. The funding bodies had no role in the design of the study, in collection, analysis, and interpretation of data and in writing the manuscript.
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