Figure 6
Figure 6. The human NmU promoter contains functional MREs distal to its transcription start site. (A) Schematic representation of the NmU promoter with the location of canonical MREs (black bars) and noncanonical MREs (gray bars). MRE3, MRE4, MRE9, and MRE10 are conserved between mouse and human, which are denoted with an asterisk above each MRE. The bent arrow represents the transcription start site for NmU. (B) Luciferase activity was determined using K562-MERT cells nucleofected with full-length NmU promoter, del MRE6–11, del MRE9, or del MRE10. A schematic of the promoter constructs with the locations of canonical and noncanonical MREs as well as MREs conserved between the human and mouse as described in panel A are provided to the left of the luciferase activity graph. The “X” in del MRE9 and del MRE10 indicates the deleted MRE. The luciferase activity obtained with each construct is presented as fold induction. The DNA recovered from ChIP assays with (C) c-Myb antibody or (D) B-Myb antibody was amplified with primers that flanked MREs throughout NmU's promoter. The amplified DNA with each primer set is presented as fold enrichment, which was calculated as the ratio between the enrichment obtained with either anti-c-Myb (black bars in C), anti-B-Myb (black bars in D), or nonspecific antibody (white bars in C-D) to that obtained without antibody. The results are the mean of triplicate determinations and are representative of at least 2 independent experiments. The asterisk indicates statistically significant difference in fold enrichment between the anti-c-Myb and nonspecific antibody (P < .05). (D) Inset is the fold enrichment observed when DNA recovered from B-Myb antibody and nonspecific antibody ChIP assays were amplified with primers specific for cyclin B1's promoter. The data are the mean of a triplicate determination for 2 independent experiments. (E) Relative gene expression of NmU, c-myb, and B-myb after the nucleofection with either control (white bars) or c-myb (black bars) siRNA. (F) Relative expression of NmU, c-myb, and B-myb after nucleofection with either control (white bars) or B-myb (black bars) siRNA. The data are the mean of duplicate determinations from 2 independent experiments.

The human NmU promoter contains functional MREs distal to its transcription start site. (A) Schematic representation of the NmU promoter with the location of canonical MREs (black bars) and noncanonical MREs (gray bars). MRE3, MRE4, MRE9, and MRE10 are conserved between mouse and human, which are denoted with an asterisk above each MRE. The bent arrow represents the transcription start site for NmU. (B) Luciferase activity was determined using K562-MERT cells nucleofected with full-length NmU promoter, del MRE6–11, del MRE9, or del MRE10. A schematic of the promoter constructs with the locations of canonical and noncanonical MREs as well as MREs conserved between the human and mouse as described in panel A are provided to the left of the luciferase activity graph. The “X” in del MRE9 and del MRE10 indicates the deleted MRE. The luciferase activity obtained with each construct is presented as fold induction. The DNA recovered from ChIP assays with (C) c-Myb antibody or (D) B-Myb antibody was amplified with primers that flanked MREs throughout NmU's promoter. The amplified DNA with each primer set is presented as fold enrichment, which was calculated as the ratio between the enrichment obtained with either anti-c-Myb (black bars in C), anti-B-Myb (black bars in D), or nonspecific antibody (white bars in C-D) to that obtained without antibody. The results are the mean of triplicate determinations and are representative of at least 2 independent experiments. The asterisk indicates statistically significant difference in fold enrichment between the anti-c-Myb and nonspecific antibody (P < .05). (D) Inset is the fold enrichment observed when DNA recovered from B-Myb antibody and nonspecific antibody ChIP assays were amplified with primers specific for cyclin B1's promoter. The data are the mean of a triplicate determination for 2 independent experiments. (E) Relative gene expression of NmU, c-myb, and B-myb after the nucleofection with either control (white bars) or c-myb (black bars) siRNA. (F) Relative expression of NmU, c-myb, and B-myb after nucleofection with either control (white bars) or B-myb (black bars) siRNA. The data are the mean of duplicate determinations from 2 independent experiments.

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