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Detection of a circadian enhancer in the mDbp promoter using prokaryotic transposon vector-based strategy.

Kiyohara YB, Nishii K, Ukai-Tadenuma M, Ueda HR, Uchiyama Y, Yagita K - Nucleic Acids Res. (2008)

Bottom Line: This enhancer is classified as a CANNTG type non-canonical E-box.These findings strongly suggest that CANNTG-type non-canonical E-boxes may contribute, at least in part, to the regulation of robust circadian gene expression.Furthermore, these data may help explain the wider effects of the CLOCK/BMAL1 complex in control of clock output genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita, Osaka 565-0871, Japan.

ABSTRACT
In mammals, the expression of 5-10% of genes occurs with circadian fluctuation in various organs and tissues. This cyclic transcription is thought to be directly or indirectly regulated through circadian transcriptional/translational feedback loops consisting of a set of clock genes. Among the clock genes in mammals, expression of the Dbp mRNA robustly oscillates both in vivo and in culture cells. Here, we present circadian enhancer detection strategy using prokaryotic transposon system. The mDbp promoter drives reporter gene expression in robust circadian cycles in rat-1 fibroblasts. To identify the circadian enhancer generating this robust rhythm, we developed a prokaryotic transposon-based enhancer detecting vector for in vitro transposition. Using this system, we identified a strong circadian enhancer region containing the CATGTG sequence in the 5' flanking region of the mDbp gene; this enhancer region is critical for the ability of the mDbp promoter to drive robust oscillation in living cells. This enhancer is classified as a CANNTG type non-canonical E-box. These findings strongly suggest that CANNTG-type non-canonical E-boxes may contribute, at least in part, to the regulation of robust circadian gene expression. Furthermore, these data may help explain the wider effects of the CLOCK/BMAL1 complex in control of clock output genes.

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BMAL1/CLOCK binds the endogenous non-canonical E-box region of 5′ flank end of the Dbp gene in NIH3T3 cells. Chromatin immunoprecipitation (ChIP) was performed to analyze the binding of BMAL1/CLOCK with the endogenous non-canonical E-box identified in this study. (A) The position of identified non-canonical E-box is indicated as underline (NC E-box). Arrows below the DNA sequences indicate the primers [P(DBP)-F and P(DBP)-R] to detect co-immunoprecipitated target Dbp promoter region. (B) ChIP is performed using flag-tagged BMAL1/CLOCK expressed NIH3T3 cells. Quantitative PCR (Q-PCR) using P(DBP)-F and P(DBP)-R primers revealed that amplified signals from the precipitants with anti-Flag antibody are significantly stronger than the signals amplified from the precipitants using anti-V5 antibody as a control (left two columns). As a negative control, Q-PCR using specific primers for beta-actin promoter (Act5′) shows that amplified signals from precipitant samples using anti-flag or anti-V5 antibodies both are low and no significant difference between them (right two columns). Error bars indicate standard deviation (SD).
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Figure 7: BMAL1/CLOCK binds the endogenous non-canonical E-box region of 5′ flank end of the Dbp gene in NIH3T3 cells. Chromatin immunoprecipitation (ChIP) was performed to analyze the binding of BMAL1/CLOCK with the endogenous non-canonical E-box identified in this study. (A) The position of identified non-canonical E-box is indicated as underline (NC E-box). Arrows below the DNA sequences indicate the primers [P(DBP)-F and P(DBP)-R] to detect co-immunoprecipitated target Dbp promoter region. (B) ChIP is performed using flag-tagged BMAL1/CLOCK expressed NIH3T3 cells. Quantitative PCR (Q-PCR) using P(DBP)-F and P(DBP)-R primers revealed that amplified signals from the precipitants with anti-Flag antibody are significantly stronger than the signals amplified from the precipitants using anti-V5 antibody as a control (left two columns). As a negative control, Q-PCR using specific primers for beta-actin promoter (Act5′) shows that amplified signals from precipitant samples using anti-flag or anti-V5 antibodies both are low and no significant difference between them (right two columns). Error bars indicate standard deviation (SD).

Mentions: To confirm the identified non-canonical E-box actually functioned in living cells, we performed chromatin immunoprecipitation (ChIP) using flag-tagged BMAL1 and CLOCK expressed NIH3T3 cells. Co-precipitated genomic DNA fragments by anti-flag immunoprecipitation were analyzed by quantitative PCR (Q-PCR) method to detect the identified non-canonical E-box region. Position of primer sequences for Q-PCR and the non-canonical E-box of Dbp promoter region are presented in Figure 7A. Q-PCR analysis revealed that the significant recruitment of flag-tagged BMAL1 and CLOCK around the non-canonical E-box region of Dbp promoter [P(DBP)] was observed, comparing with the beta-actin promoter region (Act5′) as a negative control (Figure 7B). ChIP using anti-V5 antibody for flag-BMAL1 and flag-CLOCK expressed NIH3T3 showed no significant difference between P(DBP) targeted and Act5′-targeted Q-PCR (Figure 7B). These results confirmed that BMAL1/CLOCK actually bound the 5′ flank region of Dbp gene around the identified non-canonical E-box.Figure 7.


Detection of a circadian enhancer in the mDbp promoter using prokaryotic transposon vector-based strategy.

Kiyohara YB, Nishii K, Ukai-Tadenuma M, Ueda HR, Uchiyama Y, Yagita K - Nucleic Acids Res. (2008)

BMAL1/CLOCK binds the endogenous non-canonical E-box region of 5′ flank end of the Dbp gene in NIH3T3 cells. Chromatin immunoprecipitation (ChIP) was performed to analyze the binding of BMAL1/CLOCK with the endogenous non-canonical E-box identified in this study. (A) The position of identified non-canonical E-box is indicated as underline (NC E-box). Arrows below the DNA sequences indicate the primers [P(DBP)-F and P(DBP)-R] to detect co-immunoprecipitated target Dbp promoter region. (B) ChIP is performed using flag-tagged BMAL1/CLOCK expressed NIH3T3 cells. Quantitative PCR (Q-PCR) using P(DBP)-F and P(DBP)-R primers revealed that amplified signals from the precipitants with anti-Flag antibody are significantly stronger than the signals amplified from the precipitants using anti-V5 antibody as a control (left two columns). As a negative control, Q-PCR using specific primers for beta-actin promoter (Act5′) shows that amplified signals from precipitant samples using anti-flag or anti-V5 antibodies both are low and no significant difference between them (right two columns). Error bars indicate standard deviation (SD).
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Figure 7: BMAL1/CLOCK binds the endogenous non-canonical E-box region of 5′ flank end of the Dbp gene in NIH3T3 cells. Chromatin immunoprecipitation (ChIP) was performed to analyze the binding of BMAL1/CLOCK with the endogenous non-canonical E-box identified in this study. (A) The position of identified non-canonical E-box is indicated as underline (NC E-box). Arrows below the DNA sequences indicate the primers [P(DBP)-F and P(DBP)-R] to detect co-immunoprecipitated target Dbp promoter region. (B) ChIP is performed using flag-tagged BMAL1/CLOCK expressed NIH3T3 cells. Quantitative PCR (Q-PCR) using P(DBP)-F and P(DBP)-R primers revealed that amplified signals from the precipitants with anti-Flag antibody are significantly stronger than the signals amplified from the precipitants using anti-V5 antibody as a control (left two columns). As a negative control, Q-PCR using specific primers for beta-actin promoter (Act5′) shows that amplified signals from precipitant samples using anti-flag or anti-V5 antibodies both are low and no significant difference between them (right two columns). Error bars indicate standard deviation (SD).
Mentions: To confirm the identified non-canonical E-box actually functioned in living cells, we performed chromatin immunoprecipitation (ChIP) using flag-tagged BMAL1 and CLOCK expressed NIH3T3 cells. Co-precipitated genomic DNA fragments by anti-flag immunoprecipitation were analyzed by quantitative PCR (Q-PCR) method to detect the identified non-canonical E-box region. Position of primer sequences for Q-PCR and the non-canonical E-box of Dbp promoter region are presented in Figure 7A. Q-PCR analysis revealed that the significant recruitment of flag-tagged BMAL1 and CLOCK around the non-canonical E-box region of Dbp promoter [P(DBP)] was observed, comparing with the beta-actin promoter region (Act5′) as a negative control (Figure 7B). ChIP using anti-V5 antibody for flag-BMAL1 and flag-CLOCK expressed NIH3T3 showed no significant difference between P(DBP) targeted and Act5′-targeted Q-PCR (Figure 7B). These results confirmed that BMAL1/CLOCK actually bound the 5′ flank region of Dbp gene around the identified non-canonical E-box.Figure 7.

Bottom Line: This enhancer is classified as a CANNTG type non-canonical E-box.These findings strongly suggest that CANNTG-type non-canonical E-boxes may contribute, at least in part, to the regulation of robust circadian gene expression.Furthermore, these data may help explain the wider effects of the CLOCK/BMAL1 complex in control of clock output genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita, Osaka 565-0871, Japan.

ABSTRACT
In mammals, the expression of 5-10% of genes occurs with circadian fluctuation in various organs and tissues. This cyclic transcription is thought to be directly or indirectly regulated through circadian transcriptional/translational feedback loops consisting of a set of clock genes. Among the clock genes in mammals, expression of the Dbp mRNA robustly oscillates both in vivo and in culture cells. Here, we present circadian enhancer detection strategy using prokaryotic transposon system. The mDbp promoter drives reporter gene expression in robust circadian cycles in rat-1 fibroblasts. To identify the circadian enhancer generating this robust rhythm, we developed a prokaryotic transposon-based enhancer detecting vector for in vitro transposition. Using this system, we identified a strong circadian enhancer region containing the CATGTG sequence in the 5' flanking region of the mDbp gene; this enhancer region is critical for the ability of the mDbp promoter to drive robust oscillation in living cells. This enhancer is classified as a CANNTG type non-canonical E-box. These findings strongly suggest that CANNTG-type non-canonical E-boxes may contribute, at least in part, to the regulation of robust circadian gene expression. Furthermore, these data may help explain the wider effects of the CLOCK/BMAL1 complex in control of clock output genes.

Show MeSH
Related in: MedlinePlus