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.

Rett syndrome (RTT) is a severe neurodevelopmental disorder almost exclusively affecting females and characterized by a wide spectrum of clinical manifestations. Most patients affected by classic RTT and a smaller percentage of patients with the milder form 'preserved speech variant' have either point mutations or deletions/duplications in the MECP2 gene. Recently, mutations in the CDKL5 gene, coding for a putative kinase, have been found in female patients with a phenotype overlapping with that of RTT. Here, we report two patients with the early seizure variant of RTT, bearing two novel CDKL5 truncating mutations, strengthening the correlation between CDKL5 and RTT. Considering the similar phenotypes caused by mutations in MECP2 and CDKL5, it has been suggested that the two genes play a role in common pathogenic processes. We show here that CDKL5 is a nuclear protein whose expression in the nervous system overlaps with that of MeCP2, during neural maturation and synaptogenesis. Importantly, we demonstrate that MeCP2 and CDKL5 interact both in vivo and in vitro and that CDKL5 is indeed a kinase, which is able to phosphorylate itself and to mediate MeCP2 phosphorylation, suggesting that they belong to the same molecular pathway. Furthermore, this paper contributes to the clarification of the phenotype associated with CDKL5 mutations and indicates that CDKL5 should be analyzed in each patient showing a clinical course similar to RTT but characterized by a lack of an early normal period due to the presence of seizures.

RAR and AML1 transcription factors are found in leukemias as fusion proteins with PML and ETO, respectively. Association of PML-RAR and AML1-ETO with the nuclear corepressor (N-CoR)/histone deacetylase (HDAC) complex is required to block hematopoietic differentiation. We show that PML-RAR and AML1-ETO exist in vivo within high molecular weight (HMW) nuclear complexes, reflecting their oligomeric state. Oligomerization requires PML or ETO coiled-coil regions and is responsible for abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors. Fusion of RAR to a heterologous oligomerization domain recapitulated the properties of PML-RAR, indicating that oligomerization per se is sufficient to achieve transforming potential. These results show that oligomerization of a transcription factor, imposing an altered interaction with transcriptional coregulators, represents a novel mechanism of oncogenic activation.

Silencing of transcription by DNA methylation is achieved at the level of transcription initiation and involves the removal of transcriptional machinery from active templates. This transcriptional repression can occur by indirect mechanisms involving the time-dependent assembly of repressive nucleoprotein complexes, which are able to inhibit transcription more effectively than nucleosomes alone.

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome (RTT), West syndrome, and X-linked infantile spasms, sharing the common feature of mental retardation and early seizures. CDKL5 is a rather uncharacterized kinase, but its involvement in RTT seems to be explained by the fact that it works upstream of MeCP2, the main cause of Rett syndrome. To understand the role of this kinase for nervous system functions and to address if molecular mechanisms are involved in regulating its distribution and activity, we studied the ontogeny of CDKL5 expression in developing mouse brains by immunostaining and Western blotting. The expression profile of CDKL5 was compared with that of MeCP2. The two proteins share a general expression profile in the adult mouse brain, but CDKL5 levels appear to be highly modulated at the regional level. Its expression is strongly induced in early postnatal stages, and in the adult brain CDKL5 is present in mature neurons, but not in astroglia. Interestingly, the presence of CDKL5 in the cell nucleus varies at the regional level of the adult brain and is developmentally regulated. CDKL5 shuttles between the cytoplasm and the nucleus and the C-terminal tail is involved in localizing the protein to the cytoplasm in a mechanism depending on active nuclear export. Accordingly, Rett derivatives containing disease-causing truncations of the C terminus are constitutively nuclear, suggesting that they might act as gain of function mutations in this cellular compartment.

DNA methylation and chromatin modification operate along a common pathway to repress transcription; accordingly, several experiments demonstrate that the effects of DNA methylation can spread in cis and do not require promoter modification. In order to investigate the molecular details of the inhibitory effect of methylation, we microinjected into Xenopus oocytes a series of constructs containing a human CpG-rich sequence which has been differentially methylated and cloned at different positions relative to a specific promoter. The parameters influencing the diffusion of gene silencing and the importance of histone deacetylation in the spreading effect were analyzed. We demonstrate that a few methylated cytosines can inhibit a flanking promoter but a threshold of modified sites is required to organize a stable, diffusible chromatin structure. Histone deacetylation is the main cause of gene repression only when methylation does not reach levels sufficient to establish this particular structure. Moreover, contrary to the common thought, promoter modification does not lead to the greater repressive effect; the existence of a competition between transactivators and methyl-binding proteins for the establishment of an open conformation justifies the results obtained.DNA methylation is the major modification of eukaryotic genomes and is known to have a profound effect on gene expression. In mammals, this occurs predominantly at the dinucleotide CpG, and approximately 60 to 90% of the dinucleotides are modified (50). In normal cells, methylation involves mainly CpG-poor regions, while CpG-rich areas (CpG islands), located in regulatory regions of class II genes, seem to be protected from the modification (14). This lack of methylation is likely a prerequisite for active transcription; in fact, methylated CpG islands are found on the inactive X chromosome and on silenced alleles of parentally imprinted genes (41,47,48).Genetic experiments have demonstrated that proper control of DNA methylation is essential for normal mammalian development; accordingly, this epigenetic modification seems to play important roles in X chromosome inactivation, genomic imprinting, senescence, and carcinogenesis (3,4,35,36,41,44). The correlation between DNA methylation and gene silencing has been extensively documented by a large body of evidence. In particular, transfection experiments and Xenopus oocyte microinjections, performed with in vitro-methylated DNA, demonstrated that methylation inhibits gene expression (28,29,31,38,56). Conversely, modified silent genes in cultured cell lines can be activated upon treatment with 5-azacytidine, a demethylating agent (18,26).It has been proposed that this modification causes transcriptional repression by directly interfering with the binding of transcription factors to DNA. This hypothesis has been sustained by the identification of a number of transcriptional regulators that cannot bind methylated recognition elements (16). However, the existence of factors indifferent to DNA methylation status a...

Rett syndrome (RTT) is a neurodevelopmental disorder with no efficient treatment that is caused in the majority of cases by mutations in the gene methyl-CpG binding-protein 2 (MECP2). RTT becomes manifest after a period of apparently normal development and causes growth deceleration, severe psychomotor impairment and mental retardation. Effective animal models for RTT are available and show morphofunctional abnormalities of synaptic connectivity. However, the molecular consequences of MeCP2 disruption leading to neuronal and synaptic alterations are not known. Protein synthesis regulation via the mammalian target of the rapamycin (mTOR) pathway is crucial for synaptic organization, and its disruption is involved in a number of neurodevelopmental diseases. We investigated the phosphorylation of the ribosomal protein (rp) S6, whose activation is highly dependent from mTOR activity. Immunohistochemistry showed that rpS6 phosphorylation is severely affected in neurons across the cortical areas of Mecp2 mutants and that this alteration precedes the severe symptomatic phase of the disease. Moreover, we found a severe defect of the initiation of protein synthesis in the brain of presymptomatic Mecp2 mutant that was not restricted to a specific subset of transcripts. Finally, we provide evidence for a general dysfunction of the Akt/mTOR, but not extracellular-regulated kinase, signaling associated with the disease progression in mutant brains. Our results indicate that defects in the AKT/mTOR pathway are responsible for the altered translational control in Mecp2 mutant neurons and disclosed a novel putative biomarker of the pathological process. Importantly, this study provides a novel context of therapeutic interventions that can be designed to successfully restrain or ameliorate the development of RTT.

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 preset chromatin and second the recruitment of p300 to modulate transcriptional activity.