Limits...
c-Myc modulates microRNA processing via the transcriptional regulation of Drosha.

Wang X, Zhao X, Gao P, Wu M - Sci Rep (2013)

Bottom Line: A chromatin immunoprecipitation (ChIP) experiment revealed that c-Myc binds directly to the E-box of the drosha promoter.Both in vitro and in vivo microRNA processing assays demonstrated that c-Myc promotes miRNA processing by upregulating the Drosha expression level.Overall, our study reveals a previously unrecognised function of c-Myc in miRNA processing and provides valuable insight into a new aspect of how c-Myc regulates microRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.

ABSTRACT
The c-Myc oncogenic transcription factor is known to regulate microRNA (miRNA) expression at the transcriptional level. However, little is known about the function of c-Myc in miRNA processing. Here, we report that Drosha, one of the most important components of the miRNA processing machinery, is a c-Myc target gene. c-Myc transactivates drosha mRNA expression, thus upregulating the Drosha protein level. A chromatin immunoprecipitation (ChIP) experiment revealed that c-Myc binds directly to the E-box of the drosha promoter. Both in vitro and in vivo microRNA processing assays demonstrated that c-Myc promotes miRNA processing by upregulating the Drosha expression level. Overall, our study reveals a previously unrecognised function of c-Myc in miRNA processing and provides valuable insight into a new aspect of how c-Myc regulates microRNA expression.

Show MeSH

Related in: MedlinePlus

Drosha is a direct target gene of c-Myc.(a). Schematic representation of the drosha genomic locus. The E-box (c-Myc binding site) is indicated. The deleted 6 bp of the E-box are shown (c-Myc BS mut). The exons are represented as dark boxes. The number represents the nucleotides upstream of the initiation codon (ATG) of the drosha gene. Arrows indicate the primers for the PCR reaction in the chromatin immunoprecipitation assay and luciferase constructs. (b). Chromatin immunoprecipitation was performed using A549 cells first serum-starved for 24 hours and then re-stimulated for 2 hours. The chromatin from A549 cells was fragmented and incubated with an antibody against c-Myc. An irrelevant IgG antibody was used as a negative control. (c). 293 T cells were co-transfected with Drosha-luciferase constructs (pGL3-BS) and increasing amounts of Flag-c-Myc; luciferase activity was then measured. The pGL3-vector and pGL3-BS mutant were used as controls. The error bars indicate the standard deviations. GAPDH was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3672885&req=5

f3: Drosha is a direct target gene of c-Myc.(a). Schematic representation of the drosha genomic locus. The E-box (c-Myc binding site) is indicated. The deleted 6 bp of the E-box are shown (c-Myc BS mut). The exons are represented as dark boxes. The number represents the nucleotides upstream of the initiation codon (ATG) of the drosha gene. Arrows indicate the primers for the PCR reaction in the chromatin immunoprecipitation assay and luciferase constructs. (b). Chromatin immunoprecipitation was performed using A549 cells first serum-starved for 24 hours and then re-stimulated for 2 hours. The chromatin from A549 cells was fragmented and incubated with an antibody against c-Myc. An irrelevant IgG antibody was used as a negative control. (c). 293 T cells were co-transfected with Drosha-luciferase constructs (pGL3-BS) and increasing amounts of Flag-c-Myc; luciferase activity was then measured. The pGL3-vector and pGL3-BS mutant were used as controls. The error bars indicate the standard deviations. GAPDH was used as a loading control.

Mentions: Next, we inspected the drosha promoter region for putative c-Myc binding sites. Our sequence analysis revealed the presence of a canonical E-box sequence, 5′-CACGTG-3′ (4133 bp upstream of the Drosha translational start site), which could be recognised by c-Myc (Figure 3a). To determine whether c-Myc truly binds to this region in vivo in response to serum stimulation, we performed a chromatin immunoprecipitation (ChIP) experiment. As shown in Figure 3b, c-Myc indeed bound to the E-box of the drosha promoter, and this binding was further enhanced when c-Myc was upregulated by serum stimulation.


c-Myc modulates microRNA processing via the transcriptional regulation of Drosha.

Wang X, Zhao X, Gao P, Wu M - Sci Rep (2013)

Drosha is a direct target gene of c-Myc.(a). Schematic representation of the drosha genomic locus. The E-box (c-Myc binding site) is indicated. The deleted 6 bp of the E-box are shown (c-Myc BS mut). The exons are represented as dark boxes. The number represents the nucleotides upstream of the initiation codon (ATG) of the drosha gene. Arrows indicate the primers for the PCR reaction in the chromatin immunoprecipitation assay and luciferase constructs. (b). Chromatin immunoprecipitation was performed using A549 cells first serum-starved for 24 hours and then re-stimulated for 2 hours. The chromatin from A549 cells was fragmented and incubated with an antibody against c-Myc. An irrelevant IgG antibody was used as a negative control. (c). 293 T cells were co-transfected with Drosha-luciferase constructs (pGL3-BS) and increasing amounts of Flag-c-Myc; luciferase activity was then measured. The pGL3-vector and pGL3-BS mutant were used as controls. The error bars indicate the standard deviations. GAPDH was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672885&req=5

f3: Drosha is a direct target gene of c-Myc.(a). Schematic representation of the drosha genomic locus. The E-box (c-Myc binding site) is indicated. The deleted 6 bp of the E-box are shown (c-Myc BS mut). The exons are represented as dark boxes. The number represents the nucleotides upstream of the initiation codon (ATG) of the drosha gene. Arrows indicate the primers for the PCR reaction in the chromatin immunoprecipitation assay and luciferase constructs. (b). Chromatin immunoprecipitation was performed using A549 cells first serum-starved for 24 hours and then re-stimulated for 2 hours. The chromatin from A549 cells was fragmented and incubated with an antibody against c-Myc. An irrelevant IgG antibody was used as a negative control. (c). 293 T cells were co-transfected with Drosha-luciferase constructs (pGL3-BS) and increasing amounts of Flag-c-Myc; luciferase activity was then measured. The pGL3-vector and pGL3-BS mutant were used as controls. The error bars indicate the standard deviations. GAPDH was used as a loading control.
Mentions: Next, we inspected the drosha promoter region for putative c-Myc binding sites. Our sequence analysis revealed the presence of a canonical E-box sequence, 5′-CACGTG-3′ (4133 bp upstream of the Drosha translational start site), which could be recognised by c-Myc (Figure 3a). To determine whether c-Myc truly binds to this region in vivo in response to serum stimulation, we performed a chromatin immunoprecipitation (ChIP) experiment. As shown in Figure 3b, c-Myc indeed bound to the E-box of the drosha promoter, and this binding was further enhanced when c-Myc was upregulated by serum stimulation.

Bottom Line: A chromatin immunoprecipitation (ChIP) experiment revealed that c-Myc binds directly to the E-box of the drosha promoter.Both in vitro and in vivo microRNA processing assays demonstrated that c-Myc promotes miRNA processing by upregulating the Drosha expression level.Overall, our study reveals a previously unrecognised function of c-Myc in miRNA processing and provides valuable insight into a new aspect of how c-Myc regulates microRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.

ABSTRACT
The c-Myc oncogenic transcription factor is known to regulate microRNA (miRNA) expression at the transcriptional level. However, little is known about the function of c-Myc in miRNA processing. Here, we report that Drosha, one of the most important components of the miRNA processing machinery, is a c-Myc target gene. c-Myc transactivates drosha mRNA expression, thus upregulating the Drosha protein level. A chromatin immunoprecipitation (ChIP) experiment revealed that c-Myc binds directly to the E-box of the drosha promoter. Both in vitro and in vivo microRNA processing assays demonstrated that c-Myc promotes miRNA processing by upregulating the Drosha expression level. Overall, our study reveals a previously unrecognised function of c-Myc in miRNA processing and provides valuable insight into a new aspect of how c-Myc regulates microRNA expression.

Show MeSH
Related in: MedlinePlus