Abstract
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.
Highlights
The locus control region (LCR)1 of the human -globin gene domain, defined by four erythroid specific DNase I hypersensitive sites (HS1, -2, -3, and -4) and a ubiquitous HS5 site [1,2,3], regulates transcription of the far downstream embryonic ⑀-globin, fetal G␥-globin and A␥-globin, and the adult ␦-globin and -globin genes during erythroid cell differentiation
We found that the interposed lac operator/repressor complex blocked the elongation of enhancerinitiated transcription through the intervening DNA and drastically reduced HS2 enhancer function as measured by the level of mRNA synthesized from the ⑀-globin promoter
The results indicate that in the absence of Lac repressor protein (lacR) in (ϪR) nuclear extract or in nuclear extract treated with lacR antibody, the lac operator sequence (lacO) sequence could bind with weak affinity to an endogenous K562 nuclear protein
Summary
StuI-(lacO)2-SnaBI-BamHI, containing two tandem natural lacO sequences was inserted into the StuI and BglII sites at the 3Ј-end of HS2 in HS2-1.2-⑀p-CAT [15] to make the HS2-lac-1.2-⑀p-CAT plasmid. The HS2-lac-1.2-5.5-⑀p-GFP test plasmid was made by inserting the StuI-(lacO)2-BamHI oligonucleotide into the StuI and BglII sites at the 3Ј-end of HS2 in HS2-1.2-⑀p-GFP plasmid. Creation of (ϩR) K562 Clonal Cell Line Expressing a Steady Level of the lac Repressor and Generation of Cell Clones and Pools of (ϩR) K562 or (ϪR) K562 Harboring the Integrated Test or Reference CAT or GFP Plasmids—To create the (ϩR) K562 clonal cell line, the pCMVLacI plasmid (Stratagene) containing the lacR gene tagged with a nuclear localization signal and the hygromycin selectable marker gene was transfected into K562 cells. For HS2-1.2- and HS2-lac-1.2-5.5-⑀p-GFP plasmids (Fig. 6): primer pair 5: 4526 – 4550 (pEGFP-C1, Clontech), 8747– 8771(U01317); primer pair 6: 10096 –10115 (U01317), 14169 –14191 (U01317); primer pair 7: 19436 –
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