About Enhancer
| Enhancer ID: | E_02_0796 |
| Species: | human |
| Position : | Chr11:45846943-45846959   |
| Biosample name: | |
| Experiment class : | Low throughput |
| Enhancer type: | Enhancer |
| Disease: | -- |
| Pubmed ID: | 28637769 |
| Enhancer experiment: | Luciferase Reporter Assay |
| Enhancer experiment description: | To identify components necessary for cell-autonomous circadian transcription of the hCry2 gene, a 3-kbp 5'-flanking region upstream from the hCry2 transcription start site was isolated and subcloned into the upstream of the luciferase gene, and the hCry2-driven luciferase activity was monitored in real time using a cell-based system (Figure 1A).To exclude systemic factors such as blood-borne factors and body temperature, it was necessary to examine cell-autonomous transcriptional regulatory mechanisms under an in vitro culture condition. The data clearly showed a robust circadian oscillation of the bioluminescence in U2OS human steosarcoma cells (Figure 1B), indicating that the cloned genomic region contained elements essential for circadian transcription.The 60-bp region is functionally divided into two; a 43-bp region (from -158 to -116) contains elements which enhance oscillation amplitude, while the 17-bp region (from -175 to -159) is indispensable for the generation of transcriptional oscillation. After narrowing down the transcriptional regulatory region required for the cell-autonomous circadian transcription of hCry2,and further narrowing down the genomic region essential for cell-autonomous circadian transcription,the results strongly suggested that the essential regulatory elements are contained in the region from -175 to -116 (60-bp length) (Figure 1D). To identify consensus sequences for transcription factor binding within the latter region, we performed an in silico search by sequence alignment, and found interspecies-conserved E-boxes within the 17-bp region (E1 and E2, Figure 1C). To investigate whether each of the E-boxes is functional for circadian transcription of the hCry2 gene, we produced constructs carrying hCry2 lacking either one of the E-boxes (Figure 2A). To confirm whether the BMAL1 and CLOCK complex actually activates hCry2 transcription via each E-box, we performed dual luciferase assays with these constructs (Figures 2B). The data showed that overexpression of BMAL1 and CLOCK activated hCry2 transcription via the region spanning from -249 to +41, and that deletion of one of the E-boxes produced severe attenuation of transcription, while deletion of both produced an almost complete loss of activation. |
About Target gene
| Target gene : | CRY2(HCRY2,PHLL2) |
| Strong evidence: | -- |
| Less strong evidence: | Luciferase Reporter Assay |
| Target gene experiment description: | To identify components necessary for cell-autonomous circadian transcription of the hCry2 gene, a 3-kbp 5'-flanking region upstream from the hCry2 transcription start site was isolated and subcloned into the upstream of the luciferase gene, and the hCry2-driven luciferase activity was monitored in real time using a cell-based system (Figure 1A).To exclude systemic factors such as blood-borne factors and body temperature, it was necessary to examine cell-autonomous transcriptional regulatory mechanisms under an in vitro culture condition. The data clearly showed a robust circadian oscillation of the bioluminescence in U2OS human steosarcoma cells (Figure 1B), indicating that the cloned genomic region contained elements essential for circadian transcription.The 60-bp region is functionally divided into two; a 43-bp region (from -158 to -116) contains elements which enhance oscillation amplitude, while the 17-bp region (from -175 to -159) is indispensable for the generation of transcriptional oscillation. After narrowing down the transcriptional regulatory region required for the cell-autonomous circadian transcription of hCry2,and further narrowing down the genomic region essential for cell-autonomous circadian transcription,the results strongly suggested that the essential regulatory elements are contained in the region from -175 to -116 (60-bp length) (Figure 1D). To identify consensus sequences for transcription factor binding within the latter region, we performed an in silico search by sequence alignment, and found interspecies-conserved E-boxes within the 17-bp region (E1 and E2, Figure 1C). To investigate whether each of the E-boxes is functional for circadian transcription of the hCry2 gene, we produced constructs carrying hCry2 lacking either one of the E-boxes (Figure 2A). To confirm whether the BMAL1 and CLOCK complex actually activates hCry2 transcription via each E-box, we performed dual luciferase assays with these constructs (Figures 2B). The data showed that overexpression of BMAL1 and CLOCK activated hCry2 transcription via the region spanning from -249 to +41, and that deletion of one of the E-boxes produced severe attenuation of transcription, while deletion of both produced an almost complete loss of activation. |
About TF
| TF name : | ARNTL(BMAL1,BMAL1c,JAP3,MOP3,PASD3,TIC,bHLHe5)CLOCK(KAT13D,bHLHe8) |
| TF experiment: | EMSA,Pull-down assay |
| TF experiment description: | We therefore performed electrophoretic mobility shift assays (EMSAs) to investigate the physical interaction of purified BMAL1 and CLOCK proteins to double-stranded DNA fragments (Figure 3A).We confirmed that the electrophoretic mobility of the probe was shifted by the physical interaction with BMAL1CLOCK. A mutation of E1 resulted in a severe attenuation of the affinity of the BMAL1 and CLOCK complex to the probe, and the position of the shifted band was slightly changed, probably because of the reduction of the mole ratio between the binding proteins and probe. A mutation of E2 led to almost complete loss of the physical interaction between them. Taken together, the present EMSA data indicate that each of the hCry2 tandem E-boxes physically interacts directly with the BMAL1 and CLOCK complex in an interdependent and not independent manner. Next, we performed hCry2-oligodeoxynucleotide (ODN) pull-down assays and confirmed that endogenous BMAL1 and CLOCK expressed in the liver bound to a fragment containing the hCry2 tandem E-boxes (Figure 3B). |
About Function
| Enhancer function : | the interdependent behavior of enhancer elements including not only E-boxboxes but also ROR response elements (ROREs) might contribute to limit cycle oscillations by increasing transcriptional nonlinearity. |
| Enhancer function experiment: | EMSA |
| Enhancer function experiment description: |
Immunoprecipitation was performed with antibody-conjugated beads, and the immunoprecipitates were eluted with an excess amount of tag-peptide.A mutation of E1 resulted in a severe attenuation of the affinity of the BMAL1 and CLOCK complex to the probe, and the position of the shifted band was slightly changed, probably because of the reduction of the mole ratio between the binding proteins and probe. A mutation of E2 led to almost complete loss of the physical interaction between them. Taken together, the present EMSA data indicate that each of the hCry2 tandem E-boxes physically interacts directly with the BMAL1 and CLOCK complex in an interdependent and not independent manner. |
About SNP
| SNP ID: | -- |
Upstream Pathway Annotation of TF
Enhancer associated network
The number on yellow line represents the distance between enhancer and
target gene