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Transcriptional regulation of an evolutionary conserved intergenic region of CDT2-INTS7.

Nakagawa H, Tategu M, Yamauchi R, Sasaki K, Sekimachi S, Yoshida K - PLoS ONE (2008)

Bottom Line: In the mammalian genome, a substantial number of gene pairs (approximately 10%) are arranged head-to-head on opposite strands within 1,000 base pairs, and separated by a bidirectional promoter(s) that generally drives the co-expression of both genes and results in functional coupling.Overexpression of E2F1 by adenoviral-mediated gene transfer resulted in an increased CDT2, but not INTS7, mRNA level.These findings add a unique example and help to understand the mechanistic insights into the regulation of gene expression through an evolutionary conserved intergenic region of the mammalian genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Meiji University School of Agriculture, Kawasaki, Kanagawa, Japan.

ABSTRACT

Background: In the mammalian genome, a substantial number of gene pairs (approximately 10%) are arranged head-to-head on opposite strands within 1,000 base pairs, and separated by a bidirectional promoter(s) that generally drives the co-expression of both genes and results in functional coupling. The significance of unique genomic configuration remains elusive.

Methodology/principal findings: Here we report on the identification of an intergenic region of non-homologous genes, CDT2, a regulator of DNA replication, and an integrator complex subunit 7 (INTS7), an interactor of the largest subunit of RNA polymerase II. The CDT2-INTS7 intergenic region is 246 and 245 base pairs long in human and mouse respectively and is evolutionary well-conserved among several mammalian species. By measuring the luciferase activity in A549 cells, the intergenic human sequence was shown to be able to drive the reporter gene expression in either direction and notably, among transcription factors E2F, E2F1 approximately E2F4, but not E2F5 and E2F6, this sequence clearly up-regulated the reporter gene expression exclusively in the direction of the CDT2 gene. In contrast, B-Myb, c-Myb, and p53 down-regulated the reporter gene expression in the transcriptional direction of the INTS7 gene. Overexpression of E2F1 by adenoviral-mediated gene transfer resulted in an increased CDT2, but not INTS7, mRNA level. Real-time polymerase transcription (RT-PCR) analyses of the expression pattern for CDT2 and INTS7 mRNA in human adult and fetal tissues and cell lines revealed that transcription of these two genes are asymmetrically regulated. Moreover, the abundance of mRNA between mouse and rat tissues was similar, but these patterns were quite different from the results obtained from human tissues.

Conclusions/significance: These findings add a unique example and help to understand the mechanistic insights into the regulation of gene expression through an evolutionary conserved intergenic region of the mammalian genome.

Show MeSH
Deletion analyses of the human CDT2 promoter to identify the E2F responsive site.(A) Structure of the human CDT2 gene and location of a series of deleted constructs. Translation start codons (represented by ATG) of CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate E2F consensus sites (threshold >85). Arrows with numbers represent the region and direction used for the luciferase (Luc) assay. (B) The levels of luciferase expression of human CDT2 deleted promoter constructs in A549 cells were tested with E2F1 coexpression, and are shown as fold induction with respect to the pcDNA3 vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3; asterisk, P<0.05 for pcDNA3 versus E2F1).
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pone-0001484-g004: Deletion analyses of the human CDT2 promoter to identify the E2F responsive site.(A) Structure of the human CDT2 gene and location of a series of deleted constructs. Translation start codons (represented by ATG) of CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate E2F consensus sites (threshold >85). Arrows with numbers represent the region and direction used for the luciferase (Luc) assay. (B) The levels of luciferase expression of human CDT2 deleted promoter constructs in A549 cells were tested with E2F1 coexpression, and are shown as fold induction with respect to the pcDNA3 vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3; asterisk, P<0.05 for pcDNA3 versus E2F1).

Mentions: To check which region of the human CDT2 promoter was responsible for E2F1-mediated CDT2 transcription, we made partial deletion constructs and checked for their promoter activity in transient transfection experiments (Fig. 4A). From the reporter assay conducted by the same protocol described above, we were able to note that pGL3-BCD (E2F A-deleted) decreased the promoter activity to a level one-half that of pGL3-ABCD, indicating that E2F B∼D could still stand as E2F regulatory elements and E2F A partially contributed to the E2F-mediated transcription (Fig. 4B). Deletion of E2F C/D (pGL3-ABSp1, pGL3-AB), E2F A/C/D (pGL3-B) or E2F D (pGL3-ABC) dramatically abolished the E2F1-induced promoter activity, suggesting the E2F D site or surrounding sequences could play a critical role in E2F1-mediated CDT2 expression (Fig. 4B). This is supported by the fact that pGL3-CD, in which E2F A/D is deleted, still exerted 5-fold promoter activity. These results suggested that E2F consensus sites adjacent to the transcriptional start site of CDT2 were most important for E2F1-mediated transcriptional regulation of CDT2.


Transcriptional regulation of an evolutionary conserved intergenic region of CDT2-INTS7.

Nakagawa H, Tategu M, Yamauchi R, Sasaki K, Sekimachi S, Yoshida K - PLoS ONE (2008)

Deletion analyses of the human CDT2 promoter to identify the E2F responsive site.(A) Structure of the human CDT2 gene and location of a series of deleted constructs. Translation start codons (represented by ATG) of CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate E2F consensus sites (threshold >85). Arrows with numbers represent the region and direction used for the luciferase (Luc) assay. (B) The levels of luciferase expression of human CDT2 deleted promoter constructs in A549 cells were tested with E2F1 coexpression, and are shown as fold induction with respect to the pcDNA3 vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3; asterisk, P<0.05 for pcDNA3 versus E2F1).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2194621&req=5

pone-0001484-g004: Deletion analyses of the human CDT2 promoter to identify the E2F responsive site.(A) Structure of the human CDT2 gene and location of a series of deleted constructs. Translation start codons (represented by ATG) of CDT2 and INTS7 genes are marked by bold arrows in white. Transcription start sites are indicated by the bent arrows. The transcription start site of CDT2 is designated as “+1”. Positive (negative) numbers are assigned to nucleotides downstream (upstream) of nucleotide +1. Arrowheads indicate E2F consensus sites (threshold >85). Arrows with numbers represent the region and direction used for the luciferase (Luc) assay. (B) The levels of luciferase expression of human CDT2 deleted promoter constructs in A549 cells were tested with E2F1 coexpression, and are shown as fold induction with respect to the pcDNA3 vector as 1. The values reported for transfection experiments are the means±standard deviation (n = 3; asterisk, P<0.05 for pcDNA3 versus E2F1).
Mentions: To check which region of the human CDT2 promoter was responsible for E2F1-mediated CDT2 transcription, we made partial deletion constructs and checked for their promoter activity in transient transfection experiments (Fig. 4A). From the reporter assay conducted by the same protocol described above, we were able to note that pGL3-BCD (E2F A-deleted) decreased the promoter activity to a level one-half that of pGL3-ABCD, indicating that E2F B∼D could still stand as E2F regulatory elements and E2F A partially contributed to the E2F-mediated transcription (Fig. 4B). Deletion of E2F C/D (pGL3-ABSp1, pGL3-AB), E2F A/C/D (pGL3-B) or E2F D (pGL3-ABC) dramatically abolished the E2F1-induced promoter activity, suggesting the E2F D site or surrounding sequences could play a critical role in E2F1-mediated CDT2 expression (Fig. 4B). This is supported by the fact that pGL3-CD, in which E2F A/D is deleted, still exerted 5-fold promoter activity. These results suggested that E2F consensus sites adjacent to the transcriptional start site of CDT2 were most important for E2F1-mediated transcriptional regulation of CDT2.

Bottom Line: In the mammalian genome, a substantial number of gene pairs (approximately 10%) are arranged head-to-head on opposite strands within 1,000 base pairs, and separated by a bidirectional promoter(s) that generally drives the co-expression of both genes and results in functional coupling.Overexpression of E2F1 by adenoviral-mediated gene transfer resulted in an increased CDT2, but not INTS7, mRNA level.These findings add a unique example and help to understand the mechanistic insights into the regulation of gene expression through an evolutionary conserved intergenic region of the mammalian genome.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Meiji University School of Agriculture, Kawasaki, Kanagawa, Japan.

ABSTRACT

Background: In the mammalian genome, a substantial number of gene pairs (approximately 10%) are arranged head-to-head on opposite strands within 1,000 base pairs, and separated by a bidirectional promoter(s) that generally drives the co-expression of both genes and results in functional coupling. The significance of unique genomic configuration remains elusive.

Methodology/principal findings: Here we report on the identification of an intergenic region of non-homologous genes, CDT2, a regulator of DNA replication, and an integrator complex subunit 7 (INTS7), an interactor of the largest subunit of RNA polymerase II. The CDT2-INTS7 intergenic region is 246 and 245 base pairs long in human and mouse respectively and is evolutionary well-conserved among several mammalian species. By measuring the luciferase activity in A549 cells, the intergenic human sequence was shown to be able to drive the reporter gene expression in either direction and notably, among transcription factors E2F, E2F1 approximately E2F4, but not E2F5 and E2F6, this sequence clearly up-regulated the reporter gene expression exclusively in the direction of the CDT2 gene. In contrast, B-Myb, c-Myb, and p53 down-regulated the reporter gene expression in the transcriptional direction of the INTS7 gene. Overexpression of E2F1 by adenoviral-mediated gene transfer resulted in an increased CDT2, but not INTS7, mRNA level. Real-time polymerase transcription (RT-PCR) analyses of the expression pattern for CDT2 and INTS7 mRNA in human adult and fetal tissues and cell lines revealed that transcription of these two genes are asymmetrically regulated. Moreover, the abundance of mRNA between mouse and rat tissues was similar, but these patterns were quite different from the results obtained from human tissues.

Conclusions/significance: These findings add a unique example and help to understand the mechanistic insights into the regulation of gene expression through an evolutionary conserved intergenic region of the mammalian genome.

Show MeSH