A Facilitated Tracking and Transcription Mechanism of the HS2 Enhancer in the β-Globin Locus Control Region.

The 100 kb human β-globin gene locus contains the embryonic ε-, the fetal Gγ- and Aγ-, and the adult δ- and β-globin genes arranged in the transcriptional order of 5′ ε-Gγ-Aγ-δ-β 3′. The β-globin Locus Control Region (LCR), defined by DNase I hypersensitive sites HS1, 2, 3, 4 and 5 located far upstream of the globin genes, is absolutely required for transcriptional activation of the globin genes in erythroid cells. The HS2 site, located respectively 11 and 55 kb upstream of the ε- and β-globin genes, possesses prominent enhancer activity and is able to activate transcription of the globin genes over the long distance. How the HS2 enhancer acts over the long distance is not fully understood. Three mechanisms have been proposed to explain long-range enhancer function: The looping model, the protein tracking/linking model and the facilitated tracking model. We have shown earlier that a transcription mechanism of the RNA polymerase II (pol II)-complex assembled by the HS2 enhancer mediates long-range HS2 enhancer function. In the present study, we used the chicken HS4 insulator to further study the mechanism of long-range HS2 enhancer function. We created the LCR(+I) and LCR(−I) lines in human erythroid K562 cells using Cre-loxP mediated in situ recombination and in zebrafish. The integrated LCR(+I) and LCR(−I) plasmids spanned the natural 11 kb human ε-globin gene locus either with or without the chicken HS4 insulator inserted between the LCR HS2 enhancer and the ε-globin promoter. Analysis of enhancer function in the integrated plasmids and the endogenous ε-globin gene locus provided evidence for a novel facilitated tracking and transcription mechanism of long-range enhancer function: The HS2 enhancer complex containing both the enhancer DNA and the associated pol II tracked and transcribed through the 10 kb intervening DNA to loop with and activate the ε-globin promoter. The interposed insulator interrupted this facilitated tracking and transcription mechanism of the enhancer complex through the intervening DNA, thereby blocking long-range enhancer function.