In vertebrates, densely methylated DNA is associated with inactive transcription. Actors in this process include proteins of the MBD family that can recognize methylated CpGs and repress transcription. Kaiso, a structurally unrelated protein, has also been shown to bind methylated CGCGs through its three Krüppel-like C 2 H 2 zinc fingers. The human genome contains two uncharacterized proteins, ZBTB4 and ZBTB38, that contain Kaiso-like zinc fingers. We report that ZBTB4 and ZBTB38 bind methylated DNA in vitro and in vivo. Unlike Kaiso, they can bind single methylated CpGs. When transfected in mouse cells, the proteins colocalize with foci of heavily methylated satellite DNA and become delocalized upon loss of DNA methylation. Chromatin immunoprecipitation suggests that both of these proteins specifically bind to the methylated allele of the H19/Igf2 differentially methylated region. ZBTB4 and ZBTB38 repress the transcription of methylated templates in transfection assays. The two genes have distinct tissue-specific expression patterns, but both are highly expressed in the brain. Our results reveal the existence of a family of Kaiso-like proteins that bind methylated CpGs. Like proteins of the MBD family, they are able to repress transcription in a methyl-dependent manner, yet their tissue-specific expression pattern suggests nonoverlapping functions.

Kaiso is a BTB domain protein that associates with the signaling molecule p120-catenin and binds to the methylated sequence mCGmCG or the nonmethylated sequence CTGCNA to modulate transcription. In Xenopus laevis, xKaiso deficiency leads to embryonic death accompanied by premature gene activation in blastulae and upregulation of the xWnt11 gene. Kaiso has also been proposed to play an essential role in mammalian synapsespecific transcription. We disrupted the Kaiso gene in mice to assess its role in mammalian development. Kaiso-null mice were viable and fertile, with no detectable abnormalities of development or gene expression. However, when crossed with tumor-susceptible Apc Min/؉ mice, Kaiso-null mice showed a delayed onset of intestinal tumorigenesis. Kaiso was found to be upregulated in murine intestinal tumors and is expressed in human colon cancers. Our data suggest that Kaiso plays a role in intestinal cancer and may therefore represent a potential target for therapeutic intervention.

Non-technical summary 'To be, or not to be' -thousands of neurons are facing this Shakespearean question in the brains of patients suffering from epilepsy or the consequences of a brain traumatism or stroke. The destiny of neurons in damaged brain depends on tiny equilibrium between pro-survival and pro-death signalling. Numerous studies have shown that the activity of the neuronal potassium chloride co-transporter KCC2 strongly decreases during a pathology. However, it remained unclear whether the change of the KCC2 function protects neurons or contributes to neuronal death. Here, using cultures of hippocampal neurons, we show that experimental silencing of endogenous KCC2 using an RNA interference approach or a dominant negative mutant reduces neuronal resistance to toxic insults. In contrast, the artificial gain of KCC2 function in the same neurons protects them from death. This finding highlights KCC2 as a molecule that plays a critical role in the destiny of neurons under toxic conditions and opens new avenues for the development of neuroprotective therapy.Abstract KCC2 is a neuron-specific potassium-chloride co-transporter controlling intracellular chloride homeostasis in mature and developing neurons. It is implicated in the regulation of neuronal migration, dendrites outgrowth and formation of the excitatory and inhibitory synaptic connections. The function of KCC2 is suppressed under several pathological conditions including neuronal trauma, different types of epilepsies, axotomy of motoneurons, neuronal inflammations and ischaemic insults. However, it remains unclear how down-regulation of the KCC2 contributes to neuronal survival during and after toxic stress. Here we show that in primary hippocampal neuronal cultures the suppression of the KCC2 function using two different shRNAs, dominant-negative KCC2 mutant C568A or DIOA inhibitor, increased the intracellular chloride concentration [Cl − ] i and enhanced the toxicity induced by lipofectamine-dependent oxidative stress or activation of the NMDA receptors. The rescuing of the KCC2 activity using over-expression of the active form of the KCC2, but not its non-active mutant Y1087D, effectively restored [Cl − ] i and enhanced neuronal resistance to excitotoxicity. The reparative effects of KCC2 were mimicked by over-expression of the KCC3, a homologue transporter. These data suggest an important role of KCC2-dependent potassium/chloride homeostasis under neurototoxic conditions and reveal a novel role of endogenous KCC2 as a neuroprotective molecule. Abbreviations DIV, days in vitro; shRNA, short hairpin RNA; RT, room temperature; TBSTD, tris-buffered saline, 0.1% Tween, 5% DMSO.

Using a systematic, whole-genome analysis of enhancer activity of human-specific endogenous retroviral inserts (hsERVs), we identified an element, hsERV PRODH , that acts as a tissue-specific enhancer for the PRODH gene, which is required for proper CNS functioning. PRODH is one of the candidate genes for susceptibility to schizophrenia and other neurological disorders. It codes for a proline dehydrogenase enzyme, which catalyses the first step of proline catabolism and most likely is involved in neuromediator synthesis in the CNS. We investigated the mechanisms that regulate hsERV PRODH enhancer activity. We showed that the hsERV PRODH enhancer and the internal CpG island of PRODH synergistically activate its promoter. The enhancer activity of hsERV PRODH is regulated by methylation, and in an undermethylated state it can up-regulate PRODH expression in the hippocampus. The mechanism of hsERV PRODH enhancer activity involves the binding of the transcription factor SOX2, whch is preferentially expressed in hippocampus. We propose that the interaction of hsERV PRODH and PRODH may have contributed to human CNS evolution.human-specific endogenous retrovirus | DNA methylation | central nervous system | human speciation | retroelement U nderstanding the molecular basis of phenotypic differences between humans and chimpanzees can provide important clues to human-specific behavioral peculiarities and neurological disorders. For this purpose we conducted a genome-wide analysis of human-specific endogenous retroviral (hsERV) inserts that may induce new regulatory pathways by acting as promoters and enhancers (1, 2). HsERVs of the HERV-K(HML-2) group are one of the four families of transposable elements that were able to transpose at the time of the radiation of human lineage from the lineage of its most closely related species, chimpanzee (3). At least 50% of all hsERV elements exhibit promoter activity in human tissues (4). We found only six hsERV inserts in the upstream regions of known human genes, close to transcription start sites. Three of them displayed strong enhancer activity in transient transfection experiments; of these three, only one-near the PRODH gene-matched the transcriptional activity pattern of its endogenous genomic copy. This copy of hsERV is a full-length, almost intact betaretrovirus belonging to the HERV-K(HML-2) group. PRODH encodes a mitochondrial enzyme proline, dehydrogenase (oxidase), that converts proline to D-1-pyrroline-5-carboxylate (5). PRODH regulates proline catabolism, which is vital for normal CNS functioning. Several PRODH mutations are associated with neuropsychiatric disorders, such as schizophrenia (6). Gene knockouts in mice cause severe changes in the executive functioning of the brain (7). Given the potential importance of PRODH in brain functioning and disease, we attempted to characterize its newly recognized hsERV PRODH enhancer. We showed that hsERV PRODH enhancer activity is regulated by methylation and that the hsERV PRODH enhancer and PRODH internal CpG island act syne...

Regulation of gene expression is a complex process. It includes a great number of steps from control of mRNA synthesis to posttranslational modification of proteins. Epigenetic events play essential roles in regulation of transcription. In this review, we concentrate on methylation of DNA as one of the important epigenetic marks. It is well known that DNA methylation is associated with closed chromatin state and, therefore, repressed, inactive genes. Here we describe major processes that depend on DNA methylation: imprinting, X-inactivation, and oncogenesis. Also we describe a number of known methyl-DNA-binding proteins and links between methylation of DNA and higher-order chromatin structure.

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