Transcriptional regulation of BRD7 expression by Sp1 and c-Myc.
BACKGROUND
Bromodomain is an evolutionally conserved domain that is found in proteins strongly implicated in signal-dependent transcriptional regulation. Genetic alterations of bromodomain genes contributed to the development of many human cancers and other disorders. BRD7 is a recently identified bromodomain gene. It plays a critical role in cellular growth, cell cycle progression, and signal-dependent gene expression. Previous studies showed that BRD7 gene exhibited much higher-level of mRNA expression in normal nasopharyngeal epithelia than in nasopharyngeal carcinoma (NPC) biopsies and cell lines. However, little is known about its transcriptional regulation. In this study, we explored the transcriptional regulation of BRD7 gene.


METHODS
Potential binding sites of transcription factors within the promoter region of BRD7 gene were predicted with MatInspector Professional http://genomatix.de/cgi-bin/matinspector_prof/mat_fam.pl. Mutation construct methods and luciferase assays were performed to define the minimal promoter of BRD7 gene. RT-PCR and western blot assays were used to detect the endogenous expression of transcription factor Sp1, c-Myc and E2F6 in all cell lines used in this study. Electrophoretic mobility shift assays (EMSA) and Chromatin immunoprecipitation (ChIP) were used to detect the direct transcription factors that are responsible for the promoter activity of BRD7 gene. DNA vector-based siRNA technology and cell transfection methods were employed to establish clone pools that stably expresses SiRNA against c-Myc expression in nasopharyngeal carcinoma 5-8F cells. Real-time PCR was used to detect mRNA expression of BRD7 gene in 5-8F/Si-c-Myc cells.


RESULTS
We defined the minimal promoter of BRD7 gene in a 55-bp region (from -266 to -212bp), and identified that its promoter activity is inversely related to c-Myc expression. Sp1 binds to the Sp1/Myc-Max overlapping site of BRD7 minimal promoter, and slightly positively regulate its promoter activity. c-Myc binds to this Sp1/Myc-Max overlapping site as well, and negatively regulates the promoter activity and endogenous mRNA expression of BRD7 gene. Knock-down of c-Myc increases the promoter activity and mRNA level of BRD7 gene. The luciferase activity of the mutated promoter constructs showed that Sp1/Myc-Max overlapping site is a positive regulation element of BRD7 promoter.


CONCLUSIONS
These studies provide for the first time the evidence that c-Myc is indeed a negative regulator of BRD7 gene. These findings will help to further understand and uncover the bio-functions of BRD7 gene involved in the pathogenesis of NPC.
Liu
Huaying
H
Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Changsha, Hunan 410078, People's Republic of China. huayingcsu@yahoo.com
Zhou
Ming
M
Luo
Xiaomin
X
Zhang
Liming
L
Niu
Zhaoxia
Z
Peng
Cong
C
Ma
Jian
J
Peng
Shuping
S
Zhou
Houde
H
Xiang
Bo
B
Li
Xiayu
X
Li
Shufang
S
He
Jiajin
J
Li
Xiaoling
X
Li
Guiyuan
G
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D000092468:Bromodomain Containing Proteins
D002868:Chromosomal Proteins, Non-Histone; D016271:Proto-Oncogene Proteins c-myc; D016329:Sp1 Transcription Factor; C485694:BRD7 protein, human; D000092468:Bromodomain Containing Proteins