The methyl-CpG binding transcriptional repressor MeCP2 stably associates with nucleosomal DNA.

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.

Architectural specificity in chromatin structure at the TATA box in vivo: nucleosome displacement upon beta-phaseolin gene activation.

Extensive studies of the beta-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 histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.

Blood pressure reactivity in urban youth during angry and normal talking.

Distinctions between male and female adult patterns of cardiovascular disease are well established. Although risk for cardiovascular disease begins early in life, little research has been focused on the beginning of gender difference patterns. This pilot study examined blood pressure reactivity, gender, and biologic maturity in adolescents talking about angry life circumstances and the usual progression of their day. Systolic and diastolic blood pressure increased during talking, and the addition of biologic maturity to the analysis distinguished males from females. Males had higher systolic blood pressures than did females throughout the protocol, and, generally, mature subjects had higher systolic and diastolic blood pressures than did less mature subjects. These pilot data confirm the effects of talking on blood pressure. The data suggest that biologic maturity enhances understanding of gender differences in adolescent blood pressure reactivity and continued study of the gender-blood pressure reactivity distinctions in adolescents may further understanding of adult patterns of gender differences in cardiovascular disease.

Specificity of antiparallel DNA triple helix formation.

We have used DNase I footprinting to examine the formation of antiparallel DNA triple helices on DNA fragments containing the homopurine target sites (GGA)2GGX(GGA)2GG.(CCT)2CCZ(CCT)2CC (where X.Z is each base pair in turn), with the GA- and GT-rich oligonucleotides, (GGA)2GGN(GGA)2GG and (GGT)2GGN(GGT)2GG (N = each base in turn). These were designed to form G.GC and A.AT or T.AT triplets with a central N.XZ mismatch, which should bind in an antiparallel orientation. We find that almost all combinations generate DNase I footprints at low micromolar concentrations. At each target site, the relative binding of the GA- and GT-containing oligonucleotides was not the same, suggesting that these two triplexes adopt different conformations. For a central GC base pair, the most stable complex is observed with a third strand generating a G.GC triplet as expected. A.GC is also stable, especially in the GT oligonucleotides. For a central AT base pair, all four bases form stable complexes though T.AT is favored for the GA-rich thirds strands and A.AT for the GT-rich strands. For a central CG base pair, the stable complexes are seen with third strands generating T.CG triplets, though A.CG and C.CG are stable with GT- and GA-containing oligonucleotides, respectively. C.TA is the best triplet at a central TA base pair. The third strands with central guanines avoided the formation of G.YR triplets on the fragments containing central pyrimidines, producing DNase I footprints which had slipped relative to the target site. These oligonucleotides bound at a different location, generating complexes containing 11 contiguous stable triplets at the 3'-end of the third strand. The results suggest rules for designing the best third strand oligonucleotides for targeting sequences in which homopurine tracts are interrupted by pyrimidines.

Footprinting studies on ligands which stabilize DNA triplexes: effects on stringency within a parallel triple helix.

We have examined the effect of four triplex-binding ligands on the interaction of the oligodeoxynucleotides T8NT8 (N = A, G, C, T) with DNA fragments containing the sequences A8XA8.T8YT8 (X = G, C, T; Y = C, G, A) by DNase I footprinting. The ligands form a series of quinoline derivatives with an alkylamine chain in the 4-position and different aryl substituents in the 2-position. By themselves these compounds do not alter DNase I digestion of the DNA duplexes at concentrations up to 100 microM. At a concentration of 10 microM they potentiate triplex formation, lowering the concentration of oligonucleotide required to produce a clear footprint by as much as 100-fold. As well as stabilizing triplexes which consist of well-characterized DNA triplets, they also promote the formation of complexes which contain central triplet mismatches. This reduction in the stringency of triple helix formation may be used to broaden the range of triplex target sequences and enable recognition at sites which contain short regions for which there are no good triplet matches.

Extension of DNA triple helix formation to a neighbouring (AT)n site.

We have used DNase I footprinting to examine the formation of intermolecular triple helices at a fragment containing the target sequence A11(AT)6.(AT)6T11, using oligonucleotides designed to form parallel T.AT and G.TA triplets. We find that, although (TG)6 does not form a complex with (AT)6.(AT)6, T11(TG)6 forms a stable structure producing a clear footprint which includes the (AT)6 portion of the target site. This complex is not formed in the presence of magnesium, but can be stabilised by either manganese or a triplex-binding ligand.

Triple helix formation at A8XA8.T8YT8.

We have examined the formation of DNA triple helices between the oligonucleotides T8XT8 (X = A,C,G,T) and DNA fragments containing the target sequences A8XA8.T8YT8 (X = T,C,G; Y = A,G,C), by DNase I footprinting. We find that A8GA8.T8CT8 yields a footprint with T8CT8 and shows a weaker interaction with T17 and T8GT8. A8CA8.T8GT8 yields a footprint with T17, and shows weaker interaction with T8CT8. A8TA8.T8AT8 yields a footprint with T8GT8 and shows weaker interaction with T17. Each of the successful complexes is characterised 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 from triplexes with third strands of the type A8XA8.