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.

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...

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome, West syndrome, and X-linked infantile spasms sharing the common features of generally intractable early seizures and mental retardation. Disease-causing mutations are distributed in both the catalytic domain and in the large COOH terminus. In this report, we examine the functional consequences of some Rett mutations of CDKL5 together with some synthetically designed derivatives useful to underline the functional domains of the protein. The mutated CDKL5 derivatives have been subjected to in vitro kinase assays and analyzed for phosphorylation of the TEY (Thr-Glu-Tyr) motif within the activation loop, their subcellular localization, and the capacity of CDKL5 to interact with itself. Whereas wild-type CDKL5 autophosphorylates and mediates the phosphorylation of the methyl-CpG-binding protein 2 (MeCP2) in vitro, Rett-mutated proteins show both impaired and increased catalytic activity suggesting that a tight regulation of CDKL5 is required for correct brain functions. Furthermore, we show that CDKL5 can self-associate and mediate the phosphorylation of its own TEY (Thr-Glu-Tyr) motif. Eventually, we show that the COOH terminus regulates CDKL5 properties; in particular, it negatively influences the catalytic activity and is required for its proper sub-nuclear localization. We propose a model in which CDKL5 phosphorylation is required for its entrance into the nucleus whereas a portion of the COOH-terminal domain is responsible for a stable residency in this cellular compartment probably through protein-protein interactions.X-linked cyclin-dependent kinase-like 5 (CDKL5, 3 previously named STK9) was originally identified in a transcriptional mapping project focused on the human chromosome Xp22.3-p21.3, spanning a region critical for several diseases. Expression studies demonstrated that CDKL5 was transcribed in several tissues, including brain (1). However, the possible link between CDKL5 and human diseases was drawn only few years later when balanced translocating events disrupting the gene were identified in two female patients affected by severe infantile spasms and mental retardation (2). Retrospectively, a previous publication had identified a large deletion involving CDKL5 in a male patient with X-linked retinoschisis and seizure (3); it has recently been hypothesized that retinoschisis is due to deletion of the XLRS1 gene, whereas epilepsy is caused by truncation of at least the last exon of CDKL5 (2). The importance of CDKL5 in early onset seizures and severe mental retardation in females has been further reinforced by five recent reports linking mutations in CDKL5 to patients with Rett syndrome but only in those affected by a variant form characterized by seizure onset before 6 months of age (4 -8). Very recently the frequency of CDKL5 mutations in patients affected by infantile spasms or early onset epilepsy of unknown cause has been investigated. The identification of several novel lik...

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