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MeCP2 is the founder member of a family of methyl-CpG-binding proteins able to repress transcription from methylated DNA. To date, MeCP2 action seems to involve the delivery on modified DNA of histone deacetylase activity, followed by histone methylating activity. It has been recently demonstrated that MECP2 mutations cause Rett syndrome, a childhood neurological disorder that represents one of the most common causes of mental retardation in females. Here we show that a novel Xenopus laevis protein of 20 kDa, p20, is able to interact in vivo and in vitro with MeCP2. The p20 sequence revealed that it belongs to the family of the WAP (whey acidic protein) proteins, often functioning as a protease inhibitor. Therefore, we asked whether the p20 can influence the MeCP2 half-life. We demonstrate that, indeed, the xp20 not only can significantly increase the stability of an exogenously expressed MeCP2 in Xenopus oocytes but also can stabilize the human endogenous MeCP2. The capability of the mammalian methyl-CpG-binding protein to interact with p20 is confirmed by co-immunoprecipitation experiments performed overexpressing the WAP protein. Glutathione S-transferase pull-down assays reveal that the MeCP2 residues localized between the methyl-binding domain and the transcriptional repression domain is the primary interaction surface. Our data suggest that regulation of MeCP2 metabolism might be of relevant importance; in accordance with this, previous results have shown that some Rett syndrome mutations are characterized by a decrease in MeCP2 stability.

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.

We identify Xenopus NF-Y as a key regulator of acetylation responsiveness for the Xenopus hsp70 promoter within chromatin assembled in Xenopus oocyte nuclei. Y-box sequences are required for the assembly of DNase I-hypersensitive sites in the hsp70 promoter, and for transcriptional activation both by inhibitors of histone deacetylase and by the p300 acetyltransferase. The viral oncoprotein E1A interferes with both of these activation steps. We clone Xenopus NF-YA, NF-YB and NF-YC and establish that NF-Y is the predominant Y-box-binding protein in Xenopus oocyte nuclei. NF-Y interacts with p300 in vivo and is itself a target for acetylation by p300. Transcription from the hsp70 promoter in chromatin can be enhanced further by heat shock factor. We suggest two steps in chromatin modification at the Xenopus hsp70 promoter: first the binding of NF-Y to the Y-boxes to pre-set chromatin and second the recruitment of p300 to modulate transcriptional activity.

CpG methylation in vertebrates correlates with alterations in chromatin structure and gene silencing. Differences in DNA-methylation status are associated with imprinting phenomena and carcinogenesis. In Xenopus laevis oocytes, DNA methylation dominantly silences transcription through the assembly of a repressive nucleosomal array. Methylated DNA assembled into chromatin binds the transcriptional repressor MeCP2 which cofractionates with Sin3 and histone deacetylase. Silencing conferred by MeCP2 and methylated DNA can be relieved by inhibition of histone deacetylase, facilitating the remodelling of chromatin and transcriptional activation. These results establish a direct causal relationship between DNA methylation-dependent transcriptional silencing and the modification of chromatin.

Xenopus laevis oocytes activate transcription from the Xenopus hsp70 promoter within a chromatin template in response to heat shock. Expression of exogenous Xenopus heat shock transcription factor 1 (XHSF1) causes the activation of the wild-type hsp70 promoter within chromatin. XHSF1 activates transcription at normal growth temperatures (18 degrees C), but heat shock (34 degrees C) facilitates transcriptional activation. Titration of chromatin in vivo leads to constitutive transcription from the wild-type hsp70 promoter. The Y box elements within the hsp70 promoter facilitate transcription in the presence or absence of chromatin. The presence of the Y box elements prevents the assembly of canonical nucleosomal arrays over the promoter and facilitates transcription. In a mutant hsp70 promoter lacking Y boxes, exogenous XHSF1 activates transcription from a chromatin template much more efficiently under heat shock conditions. Activation of transcription from the mutant promoter by exogenous XHSF1 correlates with the disappearance of a canonical nucleosomal array over the promoter. Chromatin structure on a mutant hsp70 promoter lacking Y boxes can restrict XHSF1 access; however, on both mutant and wild-type promoters, chromatin assembly can also restrict the function of the basal transcriptional machinery. We suggest that chromatin assembly has a physiological role in establishing a transcriptionally repressed state on the Xenopus hsp70 promoter in vivo.

We have isolated a cDNA encoding Xenopus laevis (Xl) heat-shock factor 1 (XHSF1). XHSF1, translated from the mRNA synthesized in vitro, will bind specifically to the Xl hsp70 promoter (hsp70). Microinjection of XHSF1 mRNA into Xl oocytes leads to synthesis of XHSF1 which accumulates in the nucleus and selectively activates Xl phsp70p activity at 18 degrees C.

DNA bending has been suggested to play a role in the regulation of gene expression, initiation of DNA replication, site specific recombination and DNA packaging. In Artemia franciscana (Phillopoda anostraca) cells we have revealed that an AluI DNA family of repeats, 113-bp in length, is the major component of the constitutive heterochromatin found in the species. By analysis of cloned oligomeric (monomer to hexamer) heterochromatic fragments and electrophoretic experiments we verified that the repetitive DNA shows a stable curvature that confers a solenoidal geometry to the double helix. Using the cloned monomeric fragment, as molecular probe, we describe the detection in an A. franciscana cell extract of a protein of 82 kDa (p82) that preferentially binds to heterochromatic DNA. This protein, purified of the other DNA binding proteins present in the crude cell extract, shows a greater affinity with the tandem copies of the AluI DNA fragment than with the monomer sequence. The binding of p82 protein to heterochromatic DNA is also drastically reduced in the presence of the antibiotic distamycin A, suggesting a role of the DNA curvature in the formation of the nucleoproteic complex.