We have investigated the interactions of the methyl-CpG binding transcriptional repressor MeCP2 with nucleosomal DNA. We find that MeCP2 forms discrete complexes with nucleosomal DNA associating with methyl-CpGs exposed in the major groove via the methyl-CpG-binding domain (MBD). In addition to the MBD, the carboxyl-terminal segment of MeCP2 facilitates binding both to naked DNA and to the nucleosome core. These observations provide a molecular mechanism by which MeCP2 can gain access to chromatin in order to target corepressor complexes that further modify chromatin structure.
Lineage specificity and temporal ordering of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement are reflected in the accessibility of recombination signal sequences (RSSs) within chromatin to in vitro cleavage by the V(D)J recombinase. In this report, we investigated the basis of this regulation by testing the ability of purified RAG1 and RAG2 proteins to initiate cleavage on positioned nucleosomes containing RSS substrates. We found that nicking and double-strand DNA cleavage of RSSs positioned on the face of an unmodified nucleosome are entirely inhibited. This inhibition was independent of translational position or rotational phase and could not be overcome either by addition of the DNA-bending protein HMG-1 or by the use of hyperacetylated histones. We suggest that the nucleosome could act as the stable unit of chromatin which limits recombinase accessibility to potential RSS targets, and that actively rearranging gene segments might be packaged in a modified or disrupted nucleosome structure.
Extensive studies of the -phaseolin (phas) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues. In vivo footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the phas promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution in vitro. A comparison of DNase I and dimethyl sulfate footprints in vivo and in vitro strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histonemediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.Chromatin structure is known to regulate expression from several animal and yeast genes, and evidence that precisely positioned nucleosomes serve as general repressors of transcription has been obtained in vivo and in vitro (1-4). Precise positioning of nucleosomes on DNA is a complex process that can be governed by the primary sequence (5, 6). Whereas most positioned nucleosomes are remodeled before, or concurrent with, transcriptional activation, the nucleosome over the TATA region must be displaced to permit formation of an initiation complex, possibly through exchange with TFIID (7,8). Evidence supporting this exchange includes recent biochemical and crystallographic evidence for a histone octamer-like substructure in TFIID (9-11). Additionally, the 10-bp repeat pattern of DNase I cleavage and protection in the adenovirus major late promoter region, which is protected by TFIID (12, 13), is similar to that obtained for nucleosomes (14) and is indicative of DNA lying on the surface of a protein complex.In plants, the rapid accumulation of storage proteins during embryogenesis and seed development requires high transcriptional and translational activity. In contrast, promoters for seed storage protein genes are inactive in vegetative tissues. The spatial regulation of the promoter for -phaseolin (phas), one of eight related genes encoding the major storage protein of bean (Phaseolus vulgaris) seed, is exceptionally tight both in bean and in transgenic tobacco (15)(16)(17). This is exemplified by the contrasting results for nuclear run-on transcription and -glucuronidase (GUS) product accumulation shown in Fig. 1 for developing seeds and leaf tissues of tobacco transgenic for a Ϫ1470phas͞uidA chimeric construct (17). The absolute constraint on expression from phas in vegetative tissues was demonstrated previously in tobacco transformed with phas͞DT-A (diphtheria toxin A-chain) constructs. Although a single molecule per cell of DT-A is lethal, phenotypically normal plants were obtained (reflecting the complete absence of DT-A expression) (18). Initial zygotic developm...
Bromfenac ophthalmic solution 0.09% dosed once daily is clinically safe and effective for the treatment of ocular inflammation and the reduction of ocular pain associated with cataract surgery.
We have examined the formation of DNA triple he&es between the oligonucleotides T,XTs (X = A,C,G,T) and DNA fragments containing the target sequences A,XA,.T,YT, (X=T,C,G; Y=A,G,C), by DNase I footprinting. We find that A8GA8-T&T* yields a footprint with T&T8 and shows a weaker interaction with T,, and T,GTB. AsC&*T,GT, yields a footprint with T,,, and shows weaker interaction with T,CTs. &TAs.T,AT, yields a footprint with T,GT, and shows weaker interaction with T,,. Each of the successful complexes is character&d by enhanced DNase I cleavage at the 3' end of the purine strand of the target, as well as protection at the 5' end. We have been unable to form triplexes with third strands of the type A,%.Triple helix; Sequence recognition; Stringency; DNase I footprinting One means of achieving long-range DNA sequence recognition is by oligonucleotide-directed triple helix formation [1,2]. To date the formation of these structures has been restricted to homopurine * homopyrimidine sequences. Two major types of intermolecular triplexes have been characterized. In one the third strand contains only pyrimidines (YYR type); AT is recognised by T while GC is recognised by C+, requiring PI-I'S less than 5.5 [3-5J The other motif has a p~ne~on~~g third strand (RRY type); AT is recognised by A while GC is recognised by G [6,7]. In each case the identical strands run antiparallel to each other [3]. These two motifs cannot be easily combined within a single molecule [S]. In each case recognition is achieved by the formation of specific hydrogen bonds between the third strand bases and major groove substituents on the purines of the target site.Although triple helices show exquisite sequence specificity we are interested to examine how single base changes, in both the target sequence and the third strand o~gonucleotide, affect triplex formation. In this paper we investigate how single base changes are tolerated within triple helices containing T*AT triplets. We have prepared DNA fragments containing the target sites AsXAs -T,YT, and examined their interaction with each oligonucleotide of the type TsNT8, where N = G,C,T or A, in turn. MATERIALS AND ~E~ODS Chemicals and enzymesDeoxyoligonucleotides were synthesised on an Applied Biosystems 380 B DNA synthesizer using standard phosphotamidite chemistry and used without further purification.DNase I was purchased from Sigma and stored at -20°C at a concentration of 7,200 U/ml. Reverse transcriptase was purchased from Promega; restriction enzymes were purchased from Pharmacia or New England Biolabs. Plasmids @UC19 and SmaI-cut, alkaline phosphatase-treated pUC18) were purchased from Pharmacia. DM fkagmerttsThe ~lf~rnpl~~~~ o~gonucleoti~s A8NA8 and T&T, (N = any base) were treated with polynucleotide kinase and ATP, annealed and ligated into &a1 (CCCIGGG)-cut pUCl9 or pUCl8. The ligation mixtures were transformed into E. co& TG2. White colonies were picked from X-gal, IPTG plates containing lOO~g/ml ampicillin. The sequences of successful clones were detemined by dideoxy sequencing u...
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