The [ ( G / C ) 3 NNJn motif : A common DNA repeat that excludes nucleosomes ( electron microscopy / histones / nucleotide triplets )

Nucleosomes, the basic structural elements of chromosomes, consist of 146 bp of DNA coiled around an octamer of histone proteins, and their presence can strongly influence gene expression. Considerations of the anisotropic flexibility of nucleotide triplets containing 3 cytosines or guanines suggested that a [5'(G/C)3NN3']J motif might resist wrapping around a histone octamer. To test this, DNAs were constructed containing a 5'-CCGNN-3' pentanucleotide repeat with the Ns varied. Using in vitro nucleosome reconstitution and electron microscopy, a plasmid with 48 contiguous CCGNN repeats strongly excluded nucleosomes in the repeat region. Competitive reconstitution gel retardation experiments using DNA fragments containing 12, 24, or 48 CCGNN repeats showed that the propensity to exclude nucleosomes increased with the length of the repeat. Analysis showed that a 268-bp DNA containing a (CCGNN)," block is 4.9 ± 0.6-fold less efficient in nucleosome assembly than a similar length pUC19 fragment and -'78-fold less efficient than a similar length (CTG). sequence, based on results from previous studies. Computer searches against the GenBank database for matches with a [(G/C)3NN]48 sequence revealed numerous examples that frequently were present in the control regions of"TATA-less" genes, including the human ETS-2 and human dihydrofolate reductase genes. In both cases the (G/C)3NN repeat, present in the promoter region, co-maps with loci previously shown to be nuclease hypersensitive sites. The eukaryotic chromosome is formed by a progressive condensation of the DNA, beginning with its assembly into a string of beads or nucleosomes (1). Each nucleosome consists of 146 bp ofDNA wrapped about a histone protein octamer and their presence near genetic control elements can strongly influence gene expression (reviewed in refs. 2 and 3). Placement of a nucleosome directly over a TATA box normally maintains genetic repression, whereas assembly nearby may aid in the entry of activator proteins and thus facilitate transcription (4-7). In general, the absence of nucleosomes in promoter regions is correlated with transcriptional potency. Numerous factors can lead to the specific assembly or exclusion of nucleosomes along DNA. These factors include the primary sequence of the DNA itself, and the energetics of different DNA sequences for nucleosome formation have been examined. Repeated tracts of 4-6 adenines in phase with the helix produce sequence-directed bends, and bent DNA preferentially assembles into nucleosomes (8,9). The 5S RNA gene from several species contains a strong nucleosome positioning element (10-13) in the form of repeats of 5'(G/C)3NN(A/ T)3NN3'. Shrader and Crothers (14, 15) proposed that in this repeating motif, adenines or thymines are preferred at sites of minor-groove compression and guanines or cytosines are favored at sites of major-groove compression. Thus, in their model, by spacing these anisotropically flexible wedges and alternating them, the wrapping of DNA around the histone core would be energetically favored (14, 15). The strongest natural nucleosome positioning element yet identified was recently discovered in this laboratory. Myotonic dystrophy is one of several human genetic diseases characterized by expansions of repeating nucleotide triplets, in this case CTG triplet repeats,t located in the 3' untranslated region of the myotonic dystrophy protein kinase gene (reviewed in ref. 16). We showed that DNA containing 130 CTG repeats generates a nucleosome positioning signal 9 times stronger than 1 copy of the 5S RNA gene, and it was suggested that generation of arrays of hyperstable nucleosomes over long CTG repeats can alter the local chromatin structure and hence the expression of the myotonic dystrophy protein kinase gene (17-19). The reason why DNA containing long CTG repeats would assemble into unusually stable nucleosomes is, as yet, unclear. Sequences also exist that inhibit nucleosome formation and in vivo such sequences, were they to occur in promoter or enhancer regions, could play an extremely important role by maintaining access to the DNA. DNA-RNA hybrids (20), left-handed Z-DNA (21), and DNA containing poly(dA.dT) runs have all been shown to exclude nucleosome formation in vitro (22-25). Recently, Iyer and Struhl (25) found that poly(dA.dT) tracts in the yeast his3 promoter stimulate Gcn4activated transcription in vivo due to nucleosome exclusion and increased accessibility of Gcn4-binding sites. Consideration of the sequence motif in the 5S RNA gene (14, 15) led us to suggest that DNA containing long repeats in the form of (G/C)3NN(G/C)3NN should exclude nucleosomes for the following reason. This motif differs from the 5S RNA gene element (G/C)3NN(A/T)3NN in that each time minor groove compression is required, the DNA presents the histone octamer with a wedge that favors bending into the major groove. Thus, we would predict that DNA containing a nucleosomesized tract of, for example, repeating CCGNN pentanucleotides, which are a member of the (G/C)3NN(G/C)3NN motif family, would energetically resist nucleosome formation. The reason for particular interest in the CCG triplet is that five different fragile sites in the human genome have been found to be associated with expansions of CCG triplets (26) and fragile sites have been suggested to result from an altered, less well organized chromatin structure (27). To examine this hypothesis, we constructed three pGEM3zf(+)-based recombinant plasmids containing 12, 24, and 48 contiguous CCGNN repeats with the N positions rich in adenines or thymines. Using in vitro reconstitution and electron microscopy (EM) and a competitive reconstitution gel retardation assay, we demonstrate here that the longer Abbreviations: EM, electron microscopy; DHFR, dihydrofolate reductase. *To whom reprint requests should be addressed. tThe simple repeat sequences described here are frequently denoted by the first three letters, for example CCG or CTG. This abbreviation indicates a duplex repeat of the form CCGCGG in which both antiparallel strands are oriented 5' to 3'. 8863 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8864 Biochemistry: Wang and Griffith (CCGNN), repeat blocks strongly exclude nucleosome formation. Computer searches against the GenBank database revealed that the (G/C)3NN motif commonly occurs in the human genome with two examples co-mapping to sites known to be nucleosome free. MATERIALS AND METHODS DNA and Proteins. Two complementary oligonucleotides (5' -CCGTACCGATCCGAACCGGACCGCTCCGAGCCGTCCCGTGCCGCACCGGCCCGTTCCGAT-3' and 5'-ACGGATCGGAACGGGCCGGTGCGGCACGGGACGGCTCGGAGCGGCTCGGTTCGGATCGGT-3') were synthesized, annealed to generate a duplex with four nucleotide cohesive ends, and ligated head-to-tail. Monomers, dimers, and tetramers of (CCGNN)12 were purified by gel electrophoresis and cloned into the pGEM3zf(+) vector. The sequences of the inserts in the recombinant plasmids p(CCGNN), were verified by direct DNA sequencing. HeLa cell histone octamers were isolated by centrifugation of purified HeLa cell chromatin through sucrose gradients containing 0.6 M NaCl, followed by release of the core histones with 2 M NaCl as described (9). In Vitro Nucleosome Reconstitution and EM. Closed circular p(CCGNN)48 or pGEM3zf(+) plasmid DNA was incubated in 5 mM MgCl2 at 55°C for 30 min prior to being mixed with purified HeLa cell histone octamers in a buffer containing 2 M NaCl. The salt was slowly lowered in increments of 0.1 M to a final concentration of 0.6M by adding a solution of 20 mM Hepes, 1 mM EDTA (pH 7.5) (5 min for each step at room temperature) to form stable nucleosomes. The nucleosomeassembled DNA was fixed with 0.6% glutaraldehyde (vol/vol) for 10 min at room temperature, chromatographed over 1 ml of Sephadex G-50, then treated with EcoO109I and AatII restriction endonucleases. Only the EcoO109I end of the molecules were filled in with biotinylated dCTP using the large fragment ofDNA polymerase I. The DNA was incubated with streptavidin (150 jig/ml) and then chromatographed over 2-ml columns of BIO-GEL A-5m (Bio-Rad) to remove the excess streptavidin. The fractions containing DNA-protein complexes were mixed with a buffer containing 2 mM spermidine, applied to glow-charged carbon-coated grids, and washed with a sequential water/ethanol series. The samples were then air-dried and rotary shadowcast with tungsten (28). Samples were examined in a Philips CM12 electron microscope and micrographs were taken on 35 mm film. Measurement of the position of nucleosomes along DNA was accomplished by projecting images of molecules on the film onto a Summagraphics digitizing tablet coupled to a Macintosh computer programmed with software developed in this laboratory. Competitive Nucleosome Reconstitution and Gel Retardation Assays. The 261-bp fragment containing the (CCGNN)12 repeat was obtained by PCR amplification of a segment of p(CCGNN)12 plasmid using primers from nucleotide 5 to nucleotide 23 and from nucleotide 182 to nucleotide 205 [based on the nucleotide numbering in pGEM3zf(+)]; the 255-bp fragment containing the (CCGNN)24 repeat was obtained by PCR amplification of a segment of p(CCGNN)24 plasmid using primers from nucleotide 5 to nucleotide 23 and from nucleotide 116 to nucleotide 139 [based on the nucleotide numbering in pGEM3zf(+)]; the 268-bp fragment containing the (CCGNN)48 repeat was obtained by EcoRI and XbaI digestion of p(CCGNN)48; and the 262-bp pUC19 fragment was obtained by PCR amplification of pUC19 plasmid using primers from nucleotide 239 to nucleotide 263 and from nucleotide 477 to nucleotide 500 (based on the nucleotide numbering in pUC19). DNA fragments were purified by 5% polyacrylamide gel electrophoresis and labeled with T4 DNA kinase (New England Biolabs) in t