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. 2000 Jul 1;28(13):2541-50.
doi: 10.1093/nar/28.13.2541.

HMG I/Y regulates long-range enhancer-dependent transcription on DNA and chromatin by changes in DNA topology

R Bagga  1 , S Michalowski, R Sabnis, J D Griffith, B M Emerson
Affiliations

HMG I/Y regulates long-range enhancer-dependent transcription on DNA and chromatin by changes in DNA topology

R Bagga et al. Nucleic Acids Res. .

Abstract

The nature of nuclear structures that are required to confer transcriptional regulation by distal enhancers is unknown. We show that long-range enhancer-dependent beta-globin transcription is achieved in vitro upon addition of the DNA architectural protein HMG I/Y to affinity-enriched holo RNA polymerase II complexes. In this system, HMG I/Y represses promoter activity in the absence of an associated enhancer and strongly activates transcription in the presence of a distal enhancer. Importantly, nucleosome formation is neither necessary for long-range enhancer regulation in vitro nor sufficient without the addition of HMG I/Y. Thus, the modulation of DNA structure by HMG I/Y is a critical regulator of long-range enhancer function on both DNA and chromatin-assembled genes. Electron microscopic analysis reveals that HMG I/Y binds cooperatively to preferred DNA sites to generate distinct looped structures in the presence or absence of the beta-globin enhancer. The formation of DNA topologies that enable distal enhancers to strongly regulate gene expression is an intrinsic property of HMG I/Y and naked DNA.

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Figures

Figure 1
Figure 1
Transcription of β-globin genes by the holo RNA polymerase II complex. (A) Diagram of the chick β-globin gene and protein complexes on the promoter and 3′-enhancer regions. (B) In vitro transcription of the enhancer-containing (lanes 1, 3 and 5) and enhancerless (lanes 2, 4 and 6) β-globin gene plasmids using increasing amounts of affinity-enriched holo RNAP II: lanes 1 and 2, 1 µl; lanes 3 and 4, 2 µl; lanes 5 and 6, 3 µl. M indicates molecular weight markers and the lower panel shows a recovery control. Numbers below each lane indicate the relative transcription (in arbitrary units) measured by phosophorimager analysis of the gel. (C) Western blot analyses of holo RNAP II (3 µl) and HeLa whole cell extracts (10 µl ) using antibodies to various general transcription factors and activators. Not all of the general transcription factors are present in stoichiometric amounts in the holo RNA pol II preparations, as has been shown previously (21). Recombinant HMG I was detected in the concentration range 50–1000 ng protein and used as a positive control. HMG I is not a component of the holo RNA polymerase II preparation.
Figure 2
Figure 2
Long-range enhancer-dependent transcription of β-globin genes by holo RNA polymerase II is conferred by HMG I/Y. (A) rHMG I selectively represses transcription of enhancerless β-globin genes and activates transcription in the presence of the enhancer. Transcription of the enhancerless (lanes 1–4) and enhancer-containing (lanes 5–8) β-globin genes with 3 µl of holo RNAP II and increasing amounts of rHMG I: lanes 1 and 5, 0 ng; lanes 2 and 6, 15 ng; lanes 3 and 7, 30 ng; lanes 4 and 8, 60 ng. Numbers below each lane indicate the relative transcription (in arbitrary units) measured by phosophorimager analysis of the gel. (B) Mutation of the AP1/NF-E2 binding site within the β-globin gene enhancer abolishes enhancer-dependent activation by rHMG I. Transcription of β-globin genes containing the wild-type enhancer (lanes 1 and 2) or mutated enhancer (lanes 3 and 4) by 3 µl of holo RNAP II in the absence of rHMG I (lanes 1 and 3) or in the presence of 60 ng rHMG I (lanes 2 and 4). Numbers below each lane indicate the relative transcription (in arbitrary units) measured by phosophorimager analysis of the gel. (C) Enhancer-dependent transcription of chromatin-assembled β-globin genes requires HMG I/Y. Enhancer-containing (lanes 1–5) and enhancerless (lanes 6–10) β-globin gene plasmids were either mock-assembled (lanes 1 and 6) or chromatin-assembled using Drosophila S-190 extract (lanes 2–5 and 7–10). In some cases, HeLa nuclear extract (a source of holo RNA polymerase II) alone (lanes 3 and 8) or in combination with rHMG I at 30 (lanes 4 and 9) or 60 ng (lanes 5 and 10) was bound to the DNA templates prior to chromatin assembly for 10 min at 30°C. After assembly, reactions were transcribed and processed.
Figure 3
Figure 3
Interaction of holo RNA polymerase II with the β-globin promoter and enhancer regions. (A) DNase I footprinting of holo RNAP II within the β-globin promoter region using supercoiled plasmid DNA. Lane 1, control, no protein on β-globin (+enhancer); lane 2, holo RNAP II on β-globin (+enhancer); lane 3, holo RNAP II on β-globin (–enhancer). Lanes A, C, G and T contain β-globin promoter DNA sequencing ladders. Bars to the right of the autoradiogram represent holo RNAP II sites of interaction. (B) DNase I footprinting of holo RNA polymerase II within the β-globin enhancer region using supercoiled plasmid DNA. Lane 1, DNase I digestion following 15 min incubation of holo RNAP II on β-globin (+enhancer); lane 2, DNase I digestion following 2 min incubation; lane 3, no protein control. Bars to the right of the autoradiogram represent holo RNAP II sites of interaction. (C) DNase I footprinting of holo RNA polymerase II within the β-globin promoter region using supercoiled plasmid DNA in the presence of 60 ng rHMG I. Lane 1, control, no protein on β-globin (+enhancer); lanes 2–4, DNase I digestion following 15 min incubation of holo RNAP II on β-globin (+enhancer) in the absence (lane 2) or presence of rHMG I; lane 3 (+enhancer) and lane 4 (–enhancer). Bars to the right of the autoradiogram represent holo RNAP II sites of interaction.
Figure 4
Figure 4
Interaction of HMG I/Y with the β-globin enhancer. (A) DNase I footprinting of rHMG I within the β-globin enhancer region using supercoiled plasmid DNA. Lane 1, no protein on β-globin (+enhancer) plasmid; lanes 2 and 3, 30 and 60 ng rHMG I, respectively. M indicates DNA size markers. Bars to the right of the panel indicate regions protected by rHMG I. (B) Summary of transcription factors and HMG I/Y binding sites [from (A) and data not shown] within the β-globin enhancer region.
Figure 5
Figure 5
HMG I/Y forms DNA loops by cooperative binding and self-association. Electron micrographs of HMG I-mediated loops in streptavidin–biotin end-labeled β-globin DNA. (A–E) A sampling of the types of molecules seen at HMG I concentration of 15 ng for 50 ng DNA. Enhancer-containing β-globin DNA–HMG I complexes are shown in (A)–(C) and enhancerless β-globin DNA–HMG I complexes are shown in (D) and (E). (F) Demonstration of a protein-stabilized large loop in enhancer-containing β-globin DNA using the classic surface method employing a denatured film of cytochrome c protein. Arrows in (A)–(E) represent rHMG I particles bound to DNA and the short arrow in (D) represents the streptavidin–biotin tag. The size bar indicates 1 kb.
Figure 6
Figure 6
HMG I/Y particle mass. Mass analysis of rHMG I complexes formed on DNA. Complexes of HMG I were formed on linear plasmids containing the β-globin gene enhancer and promoter regions as described in Materials and Methods. The mass of these molecules was calculated based on area measurements using streptavidin (68 kDa) as the standard.
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References

    1. Blackwood E.M. and Kadonaga,J.T. (1998) Science, 281, 61–63. - PubMed
    1. Ptashne M. (1986) Nature, 322, 697–701. - PubMed
    1. Wang J.C. and Giaever,G.N. (1988) Science, 240, 300–304. - PubMed
    1. Rippe K., von Hippel,P.H. and Langowski,J. (1995) Trends Biochem. Sci., 20, 500–506. - PubMed
    1. Barton M.C. and Emerson,B.M. (1994) Genes Dev., 8, 2453–2465. - PubMed

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