Knock-down of methyl CpG-binding protein 2 (MeCP2) causes alterations in cell proliferation and nuclear lamins expression in mammalian cells.

BACKGROUND: MeCP2 (CpG-binding protein 2) is a nuclear multifunctional protein involved in several cellular processes, like large-scale chromatin reorganization and architecture, and transcriptional regulation. In recent years, a non-neuronal role for MeCP2 has emerged in cell growth and proliferation. Mutations in the MeCP2 gene have been reported to determine growth disadvantages in cultured lymphocyte cells, and its functional ablation suppresses cell growth in glial cells and proliferation in mesenchymal stem cells and prostate cancer cells. MeCP2 interacts with lamin B receptor (LBR) and with Heterochromatin Protein 1 (HP1) at the nuclear envelope (NE), suggesting that it could be part of complexes involved in attracting heterochromatin at the nuclear periphery and in mediating gene silencing. The nuclear lamins, major components of the lamina, have a role in maintaining NE integrity, in orchestrating mitosis, in DNA replication and transcription, in regulation of mitosis and apoptosis and in providing anchoring sites for chromatin domains.In this work, we inferred that MeCP2 might have a role in nuclear envelope stability, thereby affecting the proliferation pattern of highly proliferating systems. RESULTS: By performing knock-down (KD) of MeCP2 in normal murine (NIH-3 T3) and in human prostate transformed cells (PC-3 and LNCaP), we observed a strong proliferation decrease and a defect in the cell cycle progression, with accumulation of cells in S/G2M, without triggering a strong apoptotic and senescent phenotype. In these cells, KD of MeCP2 evidenced a considerable decrease of the levels of lamin A, lamin C, lamin B1 and LBR proteins. Moreover, by confocal analysis we confirmed the reduction of lamin A levels, but we also observed an alteration in the shape of the nuclear lamina and an irregular nuclear rim. CONCLUSIONS: Our results that indicate reduced levels of NE components, are consistent with a hypothesis that the deficiency of MeCP2 might cause the lack of a key "bridge" function that links the peripheral heterochromatin to the NE, thereby causing an incorrect assembly of the NE itself, together with a decreased cell proliferation and viability.

Interaction between the inner nuclear membrane lamin B receptor and the heterochromatic methyl binding protein, MeCP2.

The nuclear membrane has an important role for the dynamic regulation of the genome, besides the well-established cytoskeletal function. The nuclear lamina is emerging as an important player in the organization of the position and functional state of interphase chromosomes. Epigenetic modifications such as DNA methylation and histone modifications are required for genome reprogramming during development, tissue-specific gene expression and global gene silencing. The Methyl-CpG binding protein MeCP2 binds methyl-CpG dinucleotides in the mammalian genome and functions as a transcriptional repressor in vivo by interacting with Sin3A, thereby recruiting histone deacetylases (HDAC). MeCP2 also mediates the formation of higher-order chromatin structures contributing to determine the architectural organization of the nucleus. In this paper, we show that MeCP2 interacts in vitro and in vivo with the inner nuclear membrane protein LBR and that the unstructured aminoacidic sequence linking the MBD and TRD domains of MeCP2 is responsible for this association. The formation of an LBR-MeCP2 protein complex might help providing a molecular explanation to the distribution of part of the heterochromatin at the nuclear periphery linked to inner membrane.

Functional consequences of mutations in CDKL5, an X-linked gene involved in infantile spasms and mental retardation.

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.

CDKL5 belongs to the same molecular pathway of MeCP2 and it is responsible for the early-onset seizure variant of Rett syndrome.

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.

A novel protein, Xenopus p20, influences the stability of MeCP2 through direct interaction.

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.

Retinoic acid receptor alpha fusion to PML affects its transcriptional and chromatin-remodeling properties.

PML-RAR is an oncogenic transcription factor forming in acute promyelocytic leukemias (APL) because of a chromosomal translocation. Without its ligand, retinoic acid (RA), PML-RAR functions as a constitutive transcriptional repressor, abnormally associating with the corepressor-histone deacetylase complex and blocking hematopoietic differentiation. In the presence of pharmacological concentrations of RA, PML-RAR activates transcription and stimulates differentiation. Even though it has been suggested that chromatin alteration is important for APL onset, the PML-RAR effect on chromatin of target promoters has not been investigated. Taking advantage of the Xenopus oocyte system, we compared the wild-type transcription factor RARalpha with PML-RAR as both transcriptional regulators and chromatin structure modifiers. Without RA, we found that PML-RAR is a more potent transcriptional repressor that does not require the cofactor RXR and produces a closed chromatin configuration. Surprisingly, repression by PML-RAR occurs through a further pathway that is independent of nucleosome deposition and histone deacetylation. In the presence of RA, PML-RAR is a less efficient transcriptional activator that is unable to modify the DNA nucleoprotein structure. We propose that PML-RAR, aside from its ability to recruit aberrant quantities of histone deacetylase complexes, has acquired additional repressive mechanisms and lost important activating functions; the comprehension of these mechanisms might reveal novel targets for antileukemic intervention.

Molecular mechanisms of gene silencing mediated by DNA methylation.

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.