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An HMG I/Y-Containing Repressor Complex and Supercolled DNA Topology Are Critical for Long-Range Enhancer-Dependent Transcription in Vitro

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Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press An HMG I/Y-containing repressor complex and supercolled DNA topology are critical for long-range enhancer-dependent transcription in vitro Rajesh Bagga and Beverly M. Emerson 1 Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037 USA The 3' enhancer of the T cell receptor s.chain (TCR~) gene directs the tissue- and stage-specific expression and V(D)Jrecombination of this gene locus. Using an in vitro system that reproduces TCRoL enhancer activity efficiently, we show that long-range promoter-enhancer regulation requires a T cell-specific repressor complex and is sensitive to DNA topology. In this system, the enhancer functions to derepress the promoter on supercoiled, but not relaxed, templates. We find that the TCRoL promoter is inactivated by a repressor complex that contains the architectural protein HMG I/Y. In the absence of this repressor complex, expression of the TCR~ gene is completely independent of the 3' enhancer and DNA topology. The interaction of the T cell-restricted protein LEF-1 with the TCR~ enhancer is required for promoter derepression. In this system, the TCR~ enhancer increases the number of active promoters rather than the rate of transcription. Thus, long-range enhancers function in a distinct manner from promoters and provide the regulatory link between repressors, DNA topology, and gene activity. [Key Words: TCR genes; transcription; enhancers; HMG I/Y; derepression; DNA topology] Received December 27, 1996; revised version accepted January 14, 1997. The widespread importance of long-range promoter- Giaever 1988; Rippe et al. 1995). Evidence supporting enhancer interactions in tissue-specific and developmen- each model has been demonstrated in different systems. tally regulated gene expression has been documented In terms of DNA topology, the critical role of DNA ar- extensively. Some well-known examples include the im- chitectural proteins, such as high mobility group I/Y munoglobulin, f~-globin, insulin, B-interferon, ~-feto- (HMG I/Y) and integration host factor (IHF) among oth- protein, ovalbumin, and T-cell receptor genes, as well as ers, in the regulation of transcription and recombination the Drosophila alcohol dehydrogenase Adh, fushi through the formation of higher-order protein-DNA tarazu, and ecdysone-regulated glue genes (for review, complexes is particularly germane (for review, see Bustin see Wasylyk 1988). These studies have yielded a wealth and Reeves 1996). For example, HMG I/Y is required to of information about enhancer-dependent transcription, generate a multicomponent complex within the inter- but they have not provided mechanistic data to explain feron-~ promoter in response to viral infection for maxi- how these control elements actually work. For example, mal synergistic activation of the gene, presumably by it is currently unknown how a gene must be packaged in changing the local DNA structure (Falvo et al. 1995). the nucleus for natural enhancers to function. In this Alterations in global DNA topology can substantially regard, the requirement for chromatin structure to gen- affect the requirements of a gene for general transcrip- erate long-range transcriptional regulation has been tion initiation factors (Parvin and Sharp 1993). Another demonstrated in vitro using reconstituted DNA tem- compelling question is whether enhancers regulate pro- plates (Laybourn and Kadonaga 1992; Barton and Emer- moters by activation or derepression. The prevailing son 1994). It also remains to be determined how promot- view is that enhancers function by increasing the local ers communicate with distal enhancers. Several models concentration of transcriptional activators in the vicin- have been proposed: DNA looping by the association of ity of the promoter through DNA looping rather than by proteins bound at distal sites, DNA tracking by protein operating in an entirely distinct manner from upstream translocation, and long-range interactions affected by promoter elements. However, evidence exists showing DNA topology (for review, see Ptashne 1986; Wang and that the primary role of enhancers injected into mouse preimplantation embryos is to relieve promoter repres- 1Corresponding author. sion (Majumder et al. 1993). Thus, open questions re- E-MAIL [email protected]; FAX (619) 535-8194. main concerning the nuclear structure required to gen- GENES & DEVELOPMENT 11:629-639 © 1997 by Cold Spring Harbor LaboratoryPress ISSN 0890°9369/97 $5.00 629 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Bagga and Emerson erate an enhancer-responsive gene including the role of hancer and DNA topology. Thus, enhancers function architectural proteins, the nature of promoter-enhancer with a specialized class of repressor proteins to regulate communication (looping, tracking, topology, or a combi- promoter activity on the basis of DNA topology and an nation thereof), and the functional consequences of this architectural factor, HMG I/Y, is a critical component of long-range interaction (activation or derepression). the repressor complex. One well-characterized system in which to examine these issues is the tissue-specific T-cell receptor o~-chain (TCRe~) gene. This gene undergoes V(D)J recombinase- Results mediated rearrangement at a discrete stage of T-cell de- velopment in a process that requires the TCR~ enhancer Distal enhancer-dependent RNA polymerase II (Strominger 1989; Capone et al. 1993). Once produc- transcription in vitro tively rearranged, the TCRc~ gene is regulated tissue spe- Previously, we have shown that in vitro transcription of cifically by the 3' ~ enhancer from distances 450 kb. The cosmids containing the chick f~-globin gene within its minimal enhancer consists of 116 bp that interacts with 40-kb chromosomal locus is dependent on the action of four proteins, cAMP response element binding/activat- an erythroid-specific enhancer residing 2 kb 3' of the ing transcription factor (CREB/ATF), lymphoid en- promoter. In this system, the DNA was incorporated hancer-binding factor 1 (LEF- 1), polyoma enhancer bind- into synthetic nuclei using Xenopus egg extracts in the ing protein (PEBP2c~), and E twenty-six transforming presence of stage-specific erythroid proteins. This re- virus protein-1 (Ets-1 ), and functions in a context-depen- sulted in the assembly of DNA into chromatin and sub- dent manner (for review, see Leiden 1993). The lym- sequent encapsulation by a nuclear membrane (Barton phoid-restricted factor LEF-1 contains an HMG-binding and Emerson 1994). Because a variety of cloned genes as domain (Waterman et al. 1991) and has been shown to naked DNA do not normally display distal enhancer- act as an architectural factor by its ability to induce a dependent expression under standard in vitro transcrip- sharp bend in the enhancer that brings the other adja- tion conditions, our results and those of others (Lay- cently bound proteins into close juxtaposition to pro- bourn and Kadonaga 1992; Schild et al. 1993) suggested duce a functional complex (Giese et al. 1992, 1995). How that a nucleosomal structure is required. We were inter- the protein-enhancer complex then regulates its cognate ested in characterizing the nature of this structure and V~ promoter at a distance is unknown. To examine the how complex it had to be to reproduce tissue-specific mechanistic processes involved in these long-range in- enhancer function at a distance. As a first step, we de- teractions, we have developed an in vitro system that vised conditions in which cloned mouse TCRe~-chain re- reproduces accurately distal enhancer-dependent tran- porter genes are transcribed in a distal enhancer-depen- scriptional regulation of cloned TCR~ genes using pro- dent manner in a soluble in vitro transcription system tein extracts from oL/~-expressing Jurkat T cells. without the need for a separate nucleosome assembly Here we show that long-range promoter-enhancer step. This system is derived from human Jurkat T cells communication does not require a separate chromatin that normally express the endogenous TCRoL/f3 genes. assembly step but is strongly dependent on DNA topol- The TCRc~ DNA template used in these studies, ogy. TCR~ genes must exist in a supercoiled topology to pTCR1700TCRen, contains the mouse V~ 11.1 promoter be transcribed efficiently in the presence of the 3' en- region from -1600, a 2.5-kb chloramphenicol acetyl- hancer. When the gene is relaxed, transcription is abol- transferase (CAT) reporter gene, and 237 bp of 3'-flank- ished by the enhancer. However, if the enhancer is de- ing sequences including the minimal TCRoL enhancer. leted, the relaxed genes are highly expressed. This indi- The 3' enhancer deletion pTCR1700 was derived from cates that a critical aspect of enhancer function is to the parental plasmid pTCR1700TCRen. These templates regulate promoter activity by sensing changes in levels have been characterized extensively by transfection of template superhelicity to insure that genes are active studies in other laboratories and V~ 11.1 promoter activ- when supercoiled and inactive when relaxed. In this sys- ity was shown to be completely enhancer dependent in tem, TCR~ enhancer-mediated gene regulation requires vivo (Winoto and Baltimore 1989; Kingsley and Winoto the action of a repressor complex that binds to topology- 1992). The 23 7-bp TCR~ enhancer binds to five proteins: dependent DNA structures. This complex is composed the ubiquitous CREB/ATF and tissue-restricted LEF-1, of T-cell factors and the architectural protein HMG I/Y, Ets-1, PEBP2~, and GATA-3, which together are essen- which is required to generate an enhancer-responsive tial for its T-cell-specific activity. Very little information DNA topology. The enhancer functions by derepression is available concerning the V~ 11.1 promoter except that of the supercoiled V~ promoter in a process that may it does not contain a TATA motif and is regulated posi- involve modulating DNA structure to regulate the bind- tively by members of the Spl family, including Sp2 and ing affinity of the HMG I/Y-repressor complex.
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