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Regulation of the MAD1 promoter by G-CSF.

Jiang K, Hein N, Eckert K, Lüscher-Firzlaff J, Lüscher B - Nucleic Acids Res. (2008)

Bottom Line: The MAD1 gene is expressed in distinct patterns, mainly associated with differentiation and quiescence.STAT3 does not bind directly to promoter DNA, but is recruited by C/EBPbeta.Our findings provide the base for the characterization of additional signal transduction pathways that control the expression of MAD1.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Universitätsklinikum, RWTH Aachen University, Pauwelsstrasse 30, 52057 Aachen, Germany.

ABSTRACT
MAD family proteins are transcriptional repressors that antagonize the functions of MYC oncoproteins. In particular, MAD1 has been demonstrated to interfere with MYC-induced proliferation, transformation and apoptosis. The MAD1 gene is expressed in distinct patterns, mainly associated with differentiation and quiescence. We observed that MAD1 is directly activated by G-CSF in promyelocytic cell lines. To investigate the transcriptional regulation of the human MAD1 gene, we have cloned and characterized its promoter. A region of high homology between the MAD1 orthologs of human, mouse and rat contains the core promoter, marked by open chromatin, high GC content and the lack of a TATA box. Using deletion constructs we identified two CCAAT-boxes occupied by C/EBPalpha and beta in the homology region that mediate responsiveness to G-CSF receptor signaling. The necessary signals include the activation of STAT3 and the RAS/RAF/ERK pathway. STAT3 does not bind directly to promoter DNA, but is recruited by C/EBPbeta. In summary, our studies provide a first analysis of the MAD1 promoter and suggest STAT3 functions as a C/EBPbeta cofactor in the regulation of the MAD1 gene. Our findings provide the base for the characterization of additional signal transduction pathways that control the expression of MAD1.

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The MAD1 gene is regulated by G-CSF. (A) Exponentially growing HL60 promyelocytic cells were treated with the phorbol ester TPA (1.6 × 10–8 M) or recombinant G-CSF (10 ng/ml) for the indicated times. MAD1 mRNA was quantified by qRT–PCR with β-GUS as the internal standard. Mean values and standard deviations of three independent experiments are shown. (B) Exponentially growing U937 promyelocytic cells were treated as described in (A). Cycloheximide (25 µM) was added 15 min prior to treatment with G-CSF. Total RNA was extracted, 15 µg/lane separated on a formaldehyde–agarose gel and blotted. The hybridization was performed with probes specific for human MAD1 and MYC. The two distinct MAD1 mRNA species observed are roughly 3.8 and 6.0 kb long and are most likely the result of alternative splicing in exon 6, 3′ of the open reading fame, creating an additional non-coding exon 7. For loading control, the ethidium bromide-stained gel is shown with the 28S and 18S ribosomal RNAs indicated. (C) The experiments were performed as in (A) and (B). A qRT–PCR analysis of MAD1 mRNA is displayed. The mean values and standard deviations of three independent experiments performed in duplicates are shown. (D) U937 cells were stimulated with G-CSF (10 ng/ml) for the indicated times, total cell lysates generated in RIPA buffer and the samples immunoblotted for STAT3 (lower panel) and for Tyr705 phosphorylated STAT3 (upper panel).
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Figure 1: The MAD1 gene is regulated by G-CSF. (A) Exponentially growing HL60 promyelocytic cells were treated with the phorbol ester TPA (1.6 × 10–8 M) or recombinant G-CSF (10 ng/ml) for the indicated times. MAD1 mRNA was quantified by qRT–PCR with β-GUS as the internal standard. Mean values and standard deviations of three independent experiments are shown. (B) Exponentially growing U937 promyelocytic cells were treated as described in (A). Cycloheximide (25 µM) was added 15 min prior to treatment with G-CSF. Total RNA was extracted, 15 µg/lane separated on a formaldehyde–agarose gel and blotted. The hybridization was performed with probes specific for human MAD1 and MYC. The two distinct MAD1 mRNA species observed are roughly 3.8 and 6.0 kb long and are most likely the result of alternative splicing in exon 6, 3′ of the open reading fame, creating an additional non-coding exon 7. For loading control, the ethidium bromide-stained gel is shown with the 28S and 18S ribosomal RNAs indicated. (C) The experiments were performed as in (A) and (B). A qRT–PCR analysis of MAD1 mRNA is displayed. The mean values and standard deviations of three independent experiments performed in duplicates are shown. (D) U937 cells were stimulated with G-CSF (10 ng/ml) for the indicated times, total cell lysates generated in RIPA buffer and the samples immunoblotted for STAT3 (lower panel) and for Tyr705 phosphorylated STAT3 (upper panel).

Mentions: We observed previously that the MAD1 gene is activated in the human promyelocytic cell line HL60 upon treatment with G-CSF (32). We verified this activation in a time course experiment by performing qRT–PCR analysis. G-CSF induced MAD1 expression in HL60 cells to a similar extend and with similar kinetics as TPA (Figure 1A). Similarly, G-CSF induced MAD1 in the human promyelocytic line U937 as analyzed by northern blot (Figure 1B). The stimulation was insensitive to cycloheximide and was also observed by qRT–PCR (Figure 1B and C). These findings suggested that MAD1 expression is regulated by G-CSF independent of de novo protein synthesis. An important signal transducer of the G-CSFR is STAT3 that is activated in response to G-CSF. Indeed, in both U937 and HL60 cells phosphorylation of STAT3 at Tyr705 is enhanced by G-CSF (Figure 1D and data not shown).Figure 1.


Regulation of the MAD1 promoter by G-CSF.

Jiang K, Hein N, Eckert K, Lüscher-Firzlaff J, Lüscher B - Nucleic Acids Res. (2008)

The MAD1 gene is regulated by G-CSF. (A) Exponentially growing HL60 promyelocytic cells were treated with the phorbol ester TPA (1.6 × 10–8 M) or recombinant G-CSF (10 ng/ml) for the indicated times. MAD1 mRNA was quantified by qRT–PCR with β-GUS as the internal standard. Mean values and standard deviations of three independent experiments are shown. (B) Exponentially growing U937 promyelocytic cells were treated as described in (A). Cycloheximide (25 µM) was added 15 min prior to treatment with G-CSF. Total RNA was extracted, 15 µg/lane separated on a formaldehyde–agarose gel and blotted. The hybridization was performed with probes specific for human MAD1 and MYC. The two distinct MAD1 mRNA species observed are roughly 3.8 and 6.0 kb long and are most likely the result of alternative splicing in exon 6, 3′ of the open reading fame, creating an additional non-coding exon 7. For loading control, the ethidium bromide-stained gel is shown with the 28S and 18S ribosomal RNAs indicated. (C) The experiments were performed as in (A) and (B). A qRT–PCR analysis of MAD1 mRNA is displayed. The mean values and standard deviations of three independent experiments performed in duplicates are shown. (D) U937 cells were stimulated with G-CSF (10 ng/ml) for the indicated times, total cell lysates generated in RIPA buffer and the samples immunoblotted for STAT3 (lower panel) and for Tyr705 phosphorylated STAT3 (upper panel).
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Related In: Results  -  Collection

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Figure 1: The MAD1 gene is regulated by G-CSF. (A) Exponentially growing HL60 promyelocytic cells were treated with the phorbol ester TPA (1.6 × 10–8 M) or recombinant G-CSF (10 ng/ml) for the indicated times. MAD1 mRNA was quantified by qRT–PCR with β-GUS as the internal standard. Mean values and standard deviations of three independent experiments are shown. (B) Exponentially growing U937 promyelocytic cells were treated as described in (A). Cycloheximide (25 µM) was added 15 min prior to treatment with G-CSF. Total RNA was extracted, 15 µg/lane separated on a formaldehyde–agarose gel and blotted. The hybridization was performed with probes specific for human MAD1 and MYC. The two distinct MAD1 mRNA species observed are roughly 3.8 and 6.0 kb long and are most likely the result of alternative splicing in exon 6, 3′ of the open reading fame, creating an additional non-coding exon 7. For loading control, the ethidium bromide-stained gel is shown with the 28S and 18S ribosomal RNAs indicated. (C) The experiments were performed as in (A) and (B). A qRT–PCR analysis of MAD1 mRNA is displayed. The mean values and standard deviations of three independent experiments performed in duplicates are shown. (D) U937 cells were stimulated with G-CSF (10 ng/ml) for the indicated times, total cell lysates generated in RIPA buffer and the samples immunoblotted for STAT3 (lower panel) and for Tyr705 phosphorylated STAT3 (upper panel).
Mentions: We observed previously that the MAD1 gene is activated in the human promyelocytic cell line HL60 upon treatment with G-CSF (32). We verified this activation in a time course experiment by performing qRT–PCR analysis. G-CSF induced MAD1 expression in HL60 cells to a similar extend and with similar kinetics as TPA (Figure 1A). Similarly, G-CSF induced MAD1 in the human promyelocytic line U937 as analyzed by northern blot (Figure 1B). The stimulation was insensitive to cycloheximide and was also observed by qRT–PCR (Figure 1B and C). These findings suggested that MAD1 expression is regulated by G-CSF independent of de novo protein synthesis. An important signal transducer of the G-CSFR is STAT3 that is activated in response to G-CSF. Indeed, in both U937 and HL60 cells phosphorylation of STAT3 at Tyr705 is enhanced by G-CSF (Figure 1D and data not shown).Figure 1.

Bottom Line: The MAD1 gene is expressed in distinct patterns, mainly associated with differentiation and quiescence.STAT3 does not bind directly to promoter DNA, but is recruited by C/EBPbeta.Our findings provide the base for the characterization of additional signal transduction pathways that control the expression of MAD1.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Universitätsklinikum, RWTH Aachen University, Pauwelsstrasse 30, 52057 Aachen, Germany.

ABSTRACT
MAD family proteins are transcriptional repressors that antagonize the functions of MYC oncoproteins. In particular, MAD1 has been demonstrated to interfere with MYC-induced proliferation, transformation and apoptosis. The MAD1 gene is expressed in distinct patterns, mainly associated with differentiation and quiescence. We observed that MAD1 is directly activated by G-CSF in promyelocytic cell lines. To investigate the transcriptional regulation of the human MAD1 gene, we have cloned and characterized its promoter. A region of high homology between the MAD1 orthologs of human, mouse and rat contains the core promoter, marked by open chromatin, high GC content and the lack of a TATA box. Using deletion constructs we identified two CCAAT-boxes occupied by C/EBPalpha and beta in the homology region that mediate responsiveness to G-CSF receptor signaling. The necessary signals include the activation of STAT3 and the RAS/RAF/ERK pathway. STAT3 does not bind directly to promoter DNA, but is recruited by C/EBPbeta. In summary, our studies provide a first analysis of the MAD1 promoter and suggest STAT3 functions as a C/EBPbeta cofactor in the regulation of the MAD1 gene. Our findings provide the base for the characterization of additional signal transduction pathways that control the expression of MAD1.

Show MeSH
Related in: MedlinePlus