HS2 enhancer function is blocked by a transcriptional terminator inserted between the enhancer and the promoter.
The HS2 enhancer in the beta-globin locus control region regulates transcription of the globin genes 10-50 kb away. How the HS2 enhancer acts over this distance is not clearly understood. Earlier studies show that in erythroid cells the HS2 enhancer initiates synthesis of intergenic RNAs from sites within and downstream of the enhancer, and the enhancer-initiated RNAs are transcribed through the intervening DNA into the cis-linked promoter and gene. To investigate the functional significance of the enhancer-initiated transcription, here we inserted the lac operator sequence in the intervening DNA between the HS2 enhancer and the epsilon-globin promoter in reporter plasmids and integrated the plasmids into erythroid K562 cells expressing the lac repressor protein. We found that the interposed lac operator/repressor complex blocked the elongation of enhancer-initiated transcription through the intervening DNA and drastically reduced HS2 enhancer function as measured by the level of mRNA synthesized from the epsilon-globin promoter. The results indicate that the tracking and transcription mechanism of the HS2 enhancer-assembled transcriptional machinery from the enhancer through the intervening DNA into the cis-linked promoter can mediate enhancer-promoter interaction over a long distance.
Department of Biochemistry and Molecular Biology, School of Medicine, Medical College of Georgia, Augusta, Georgia 30912, USA.
Jianhua
J
Ling
Lincoyan
L
Ainol
Ling
L
Zhang
Xiuping
X
Yu
Wenhu
W
Pi
Dorothy
D
Tuan
D015152:Blotting, Northern; D015153:Blotting, Western; D002469:Cell Separation; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004742:Enhancer Elements, Genetic; D005434:Flow Cytometry; D005914:Globins / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D019901:Locus Control Region; D008957:Models, Genetic; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D009841:Oligonucleotides; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D012313:RNA; D005914:Globins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase