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The [(G/C)3Nnjn Motif: a Common DNA Repeat That Excludes Nucleosomes (Electron Microscopy/Histones/Nucleotide Triplets) YUH-HWA WANG and JACK D

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The [(G/C)3Nnjn Motif: a Common DNA Repeat That Excludes Nucleosomes (Electron Microscopy/Histones/Nucleotide Triplets) YUH-HWA WANG and JACK D Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8863-8867, August 1996 Biochemistry The [(G/C)3NNJn motif: A common DNA repeat that excludes nucleosomes (electron microscopy/histones/nucleotide triplets) YUH-HWA WANG AND JACK D. GRIFFITH* Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295 Communicated by Clyde A. Hutchinson III, University of North Carolina, Chapel Hill, NC, May 14, 1996 (received for review January 16, 1996) ABSTRACT Nucleosomes, the basic structural elements alternating them, the wrapping of DNA around the histone of chromosomes, consist of 146 bp of DNA coiled around an core would be energetically favored (14, 15). The strongest octamer of histone proteins, and their presence can strongly natural nucleosome positioning element yet identified was influence gene expression. Considerations of the anisotropic recently discovered in this laboratory. Myotonic dystrophy is flexibility of nucleotide triplets containing 3 cytosines or one of several human genetic diseases characterized by expan- guanines suggested that a [5'(G/C)3NN3']J motif might resist sions of repeating nucleotide triplets, in this case CTG triplet wrapping around a histone octamer. To test this, DNAs were repeats,t located in the 3' untranslated region of the myotonic constructed containing a 5'-CCGNN-3' pentanucleotide re- dystrophy protein kinase gene (reviewed in ref. 16). We peat with the Ns varied. Using in vitro nucleosome reconsti- showed that DNA containing 130 CTG repeats generates a tution and electron microscopy, a plasmid with 48 contiguous nucleosome positioning signal 9 times stronger than 1 copy of CCGNN repeats strongly excluded nucleosomes in the repeat the 5S RNA gene, and it was suggested that generation of region. Competitive reconstitution gel retardation experi- arrays of hyperstable nucleosomes over long CTG repeats can ments using DNA fragments containing 12, 24, or 48 CCGNN alter the local chromatin structure and hence the expression of repeats showed that the propensity to exclude nucleosomes the myotonic dystrophy protein kinase gene (17-19). The increased with the length of the repeat. Analysis showed that reason why DNA containing long CTG repeats would assem- a 268-bp DNA containing a (CCGNN)," block is 4.9 ± 0.6-fold ble into unusually stable nucleosomes is, as yet, unclear. less efficient in nucleosome assembly than a similar length Sequences also exist that inhibit nucleosome formation and pUC19 fragment and -'78-fold less efficient than a similar in vivo such sequences, were they to occur in promoter or length (CTG). sequence, based on results from previous enhancer regions, could play an extremely important role by studies. Computer searches against the GenBank database for maintaining access to the DNA. DNA-RNA hybrids (20), matches with a [(G/C)3NN]48 sequence revealed numerous left-handed Z-DNA (21), and DNA containing poly(dA.dT) examples that frequently were present in the control regions runs have all been shown to exclude nucleosome formation in of"TATA-less" genes, including the human ETS-2 and human vitro (22-25). Recently, Iyer and Struhl (25) found that dihydrofolate reductase genes. In both cases the (G/C)3NN poly(dA.dT) tracts in the yeast his3 promoter stimulate Gcn4- repeat, present in the promoter region, co-maps with loci activated transcription in vivo due to nucleosome exclusion and previously shown to be nuclease hypersensitive sites. increased accessibility of Gcn4-binding sites. Consideration of the sequence motif in the 5S RNA gene (14, 15) led us to The eukaryotic chromosome is formed by a progressive con- suggest that DNA containing long repeats in the form of densation of the DNA, beginning with its assembly into a string (G/C)3NN(G/C)3NN should exclude nucleosomes for the of beads or nucleosomes (1). Each nucleosome consists of 146 following reason. This motif differs from the 5S RNA gene bp of DNA wrapped about a histone protein octamer and their element (G/C)3NN(A/T)3NN in that each time minor groove presence near genetic control elements can strongly influence compression is required, the DNA presents the histone oc- gene expression (reviewed in refs. 2 and 3). Placement of a tamer with a wedge that favors bending into the major groove. nucleosome directly over a TATA box normally maintains we would predict that DNA containing a nucleosome- genetic repression, whereas assembly nearby may aid in the Thus, entry of activator proteins and thus facilitate transcription sized tract of, for example, repeating CCGNN pentanucleoti- (4-7). In general, the absence of nucleosomes in promoter des, which are a member of the (G/C)3NN(G/C)3NN motif regions is correlated with transcriptional potency. family, would energetically resist nucleosome formation. The Numerous factors can lead to the specific assembly or reason for particular interest in the CCG triplet is that five exclusion of nucleosomes along DNA. These factors include different fragile sites in the human genome have been found the primary sequence of the DNA itself, and the energetics of to be associated with expansions of CCG triplets (26) and different DNA sequences for nucleosome formation have been fragile sites have been suggested to result from an altered, less examined. Repeated tracts of 4-6 adenines in phase with the well organized chromatin structure (27). helix produce sequence-directed bends, and bent DNA pref- To examine this hypothesis, we constructed three erentially assembles into nucleosomes (8,9). The 5S RNA gene pGEM3zf(+)-based recombinant plasmids containing 12, 24, from several species contains a strong nucleosome positioning and 48 contiguous CCGNN repeats with the N positions rich element (10-13) in the form of repeats of 5'(G/C)3NN(A/ in adenines or thymines. Using in vitro reconstitution and T)3NN3'. Shrader and Crothers (14, 15) proposed that in this electron microscopy (EM) and a competitive reconstitution gel repeating motif, adenines or thymines are preferred at sites of retardation assay, we demonstrate here that the longer minor-groove compression and guanines or cytosines are favored at sites of major-groove compression. Thus, in their Abbreviations: EM, electron microscopy; DHFR, dihydrofolate re- model, by spacing these anisotropically flexible wedges and ductase. *To whom reprint requests should be addressed. tThe simple repeat sequences described here are frequently denoted The publication costs of this article were defrayed in part by page charge by the first three letters, for example CCG or CTG. This abbreviation payment. This article must therefore be hereby marked "advertisement" in indicates a duplex repeat of the form CCGCGG in which both accordance with 18 U.S.C. §1734 solely to indicate this fact. antiparallel strands are oriented 5' to 3'. 8863 Downloaded by guest on September 26, 2021 8864 Biochemistry: Wang and Griffith Proc. Natl. Acad. Sci. USA 93 (1996) (CCGNN), repeat blocks strongly exclude nucleosome forma- 10 ,tg of unlabeled calf thymus DNA and 2.5 ,tg of histone tion. Computer searches against the GenBank database re- octamers in a solution containing 2 M NaCl, 100 ,ug/ml BSA, vealed that the (G/C)3NN motif commonly occurs in the and 0.1% Nonidet P-40 (Sigma). Nucleosome assembly and human genome with two examples co-mapping to sites known electrophoresis was carried out as described in ref. 18. Briefly, to be nucleosome free. nucleosome assembly was accomplished by slowly lowering the salt in increments of 0.1 M to a final concentration of 0.1 M using a solution of 20 mM Hepes, 1 mM EDTA (pH 7.5) (5 min MATERIALS AND METHODS for each step at room temperature). The assembly mixtures DNA and Proteins. Two complementary oligonucleotides were then directly electrophoresed on 5% polyacrylamide gels (5' -CCGTACCGATCCGAACCGGACCGCTCCG- at 150 V for 4 hr at room temperature to separate free DNA AGCCGTCCCGTGCCGCACCGGCCCGTTCCGAT-3' from the nucleosome-assembled DNA. The amounts of DNA and 5'-ACGGATCGGAACGGGCCGGTGCGGCAC- in each band were determined by Phosphorlmager (Molecular GGGACGGCTCGGAGCGGCTCGGTTCGGATCGGT-3') Dynamics) scanning. were synthesized, annealed to generate a duplex with four Sequence Homology Search. A GenBank database search nucleotide cohesive ends, and ligated head-to-tail. Monomers, was done by using MACVECTOR (release 4.1.4) to access the dimers, and tetramers of (CCGNN)12 were purified by gel ENTREZ:SEQUENCES database (release 6.0), which was pro- electrophoresis and cloned into the pGEM3zf(+) vector. The vided by the National Center for Biotechnology Information sequences of the inserts in the recombinant plasmids p(C- (Bethesda, MD). CGNN), were verified by direct DNA sequencing. HeLa cell histone octamers were isolated by centrifugation of purified RESULTS HeLa cell chromatin through sucrose gradients containing 0.6 M NaCl, followed by release of the core histones with 2 M The (CCGNN). Repeat Generates a Strong Nucleosome NaCl as described (9). Exclusion Signal. To examine the hypothesis that long tracts In Vitro Nucleosome Reconstitution and EM. Closed circular of (G/C)3NN repeats will exclude nucleosomes three p(CCGNN)48 or pGEM3zf(+) plasmid DNA was incubated in pGEM3zf(+)-based recombinant plasmids, p(CCGNN)12, 5 mM MgCl2 at 55°C for 30 min prior to being mixed with p(CCGNN)24, and p(CCGNN)48, were constructed contain- purified HeLa cell histone octamers in a buffer containing 2 ing, respectively, 12, 24, and 48 tandem copies of the CCGNN M NaCl. The salt was slowly lowered in increments of 0.1 M repeat with the N positions varied and rich in adenines and to a final concentration of 0.6 M by adding a solution of 20 mM thymines. Two independent methods of analysis were em- Hepes, 1 mM EDTA (pH 7.5) (5 min for each step at room ployed. In the first, EM was used together with in vitro temperature) to form stable nucleosomes. The nucleosome- nucleosome reconstitution onto supertwisted p(CCGNN)48 assembled DNA was fixed with 0.6% glutaraldehyde (vol/vol) plasmid DNA.
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