Sequence-specific recognition of methylated DNA by human zinc-finger proteins

DNA methylation is an essential epigenetic mark. Three classes of mammalian proteins recognize methylated DNA: MBD proteins, SRA proteins and the zinc-finger proteins Kaiso, ZBTB4 and ZBTB38. The last three proteins can bind either methylated DNA or unmethylated consensus sequences; how this is achieved is largely unclear. Here, we report that the human zinc-finger proteins Kaiso, ZBTB4 and ZBTB38 can bind methylated DNA in a sequence-specific manner, and that they may use a mode of binding common to other zinc-finger proteins. This suggests that many other sequence-specific methyl binding proteins may exist

Role of intestinal mucin-2 in the effectiveness of the treatment of Helicobacter spp. infection in laboratory mice

Abnormal synthesis of the main intestinal proteo­glycan mucin-2 is typical of ulcerative colitis and Crohn’s disease in humans. Those morphological changes of the mucus layer affect the diversity of the intestinal microflora. Antibiotics may be ineffective or even dangerous to humans or animals deficient for mucin-2 because of the risk of sepsis and chronic inflammation. In this study, we investigated the potential of antibiotics (clarithromycin, amoxicillin, and metronidazole) in elimination of patho­genic infection from Muc2 knockout mice (Muc2–/–). We assayed the population sizes of pathogens (Heli­co­bacter spp.) and symbiotic (E. coli) bacteria in the intestines of animals as a criterion of antibiotic efficacy. The damaging effect of antibacterial treatment on the host body was estimated from their survival rate. Three antibiotics were ineffective in the elimination of Helicobacter spp. from mucin-2-deficient mice. Moreover, the mortality of Muc2 knockout mice during the antibacterial treatment was 60 %. The survival of wild-type mice (C57BL/6J) during the treatment was 100 %. The weight of wild-type mice showed no decrease during the treatment. The Helico­bacter spp. pathogen was fully eradicated from wild-type mice. Thus, therapy of Helicobacter spp. infection in mucin-2 deficient animals is not only poorly efficient but even deadly. The high susceptibility to antibiotics allows Muc2 knockout mice to be used as a test model to evaluate the pharmacological safety of new antibiotics

A yeast one-hybrid system to screen for methylated DNA-binding proteins

We had previously exploited a method for targeted DNA methylation in budding yeast to succeed in one-hybrid detection of methylation-dependent DNA–protein interactions. Based on this finding, we developed a yeast one-hybrid system to screen cDNA libraries for clones encoding methylated DNA-binding proteins. Concurrent use of two independent bait sequences in the same cell, or dual-bait system, effectively reduced false positive clones, which were derived from methylation-insensitive sequence-specific DNA-binding proteins. We applied the dual-bait system to screen cDNA libraries and demonstrated efficient isolation of clones for methylated DNA-binding proteins. This system would serve as a unique research tool for epigenetics

Methylated DNA recognition during the reversal of epigenetic silencing is regulated by cysteine and cerine residues in the Epstein-Barr Virus lytic switch protein

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with various malignancies, including Burkitt's lymphoma and nasopharyngeal carcinoma. Like all herpesviruses, the EBV life cycle alternates between latency and lytic replication. During latency, the viral genome is largely silenced by host-driven methylation of CpG motifs and, in the switch to the lytic cycle, this epigenetic silencing is overturned. A key event is the activation of the viral BRLF1 gene by the immediate-early protein Zta. Zta is a bZIP transcription factor that preferentially binds to specific response elements (ZREs) in the BRLF1 promoter (Rp) when these elements are methylated. Zta's ability to trigger lytic cycle activation is severely compromised when a cysteine residue in its bZIP domain is mutated to serine (C189S), but the molecular basis for this effect is unknown. Here we show that the C189S mutant is defective for activating Rp in a Burkitt's lymphoma cell line. The mutant is compromised both in vitro and in vivo for binding two methylated ZREs in Rp (ZRE2 and ZRE3), although the effect is striking only for ZRE3. Molecular modeling of Zta bound to methylated ZRE3, together with biochemical data, indicate that C189 directly contacts one of the two methyl cytosines within a specific CpG motif. The motif's second methyl cytosine (on the complementary DNA strand) is predicted to contact S186, a residue known to regulate methyl-ZRE recognition. Our results suggest that C189 regulates the enhanced interaction of Zta with methylated DNA in overturning the epigenetic control of viral latency. As C189 is conserved in many bZIP proteins, the selectivity of Zta for methylated DNA may be a paradigm for a more general phenomenon

Role of the Mucin-2 and Kaiso genes in the social behavior of mice

Inflammatory processes in the gut lead to abnormal­ities in various systems of the body, in particular, to changes in the activity of the central nervous system. Although the mechanisms of these effects are not yet known, it has been demonstrated that intestinal inflammation is associated with anxiety and depression. In this work, we used an animal model of intestinal inflammation, which might result in behavioral changes. The animals used were knock-out mice with double mutations in the Kaiso and Mucin-2 genes. The Kaiso gene encodes a transcription factor that is expressed both in the brain and in the intestine. The Mucin-2 gene encodes a protein that serves as a scaffold for the synthesis of intestinal proteoglycan. Mucin-2 is a major proteoglycan of the intestinal mucus layer and performs multiple functions, including barrier and defensive ones. We used knock-out animals with a mutation in the trans­cription factor Kaiso in tests assessing social behavior, but did not observe any difference between test subjects and wild-type animals. By contrast, double knock-out animals that additionally had a mutation in Mucin-2, a major gene for intestinal proteoglycan, displayed significant changes in social behavior: lower aggression rates and higher rates of courtship behavior toward a male intruder. These results suggest that intestinal homeostasis might have a strong impact on the nervous system of the animals. It remains unclear whether the influence of the two genes is synergistic or the knock-out of the Mucin-2 gene alone determines this behavior in mice. Further investigations will help clarify the matter

Role of the Kaiso gene in the development of inflammation in Mucin-2 defcient mice

The number of people with inflammatory bowel disease (IBD) is constantly increasing worldwide. The main factors that have effects on the etiology of the disease are genetic, environmental and immunological. However, the mechanism of disease development and effective treatment of IBD have not yet been found. Animal models help address these problems. The most popular model is considered to be transgenic models in which individual genes are knocked out. One of such models for the study of IBD are mice with a null mutation of the Muc2 gene encoding the Mucin-2 protein, which is involved in the formation of a protective mucin layer in the small and large intestine. Some of transcription factors that change the expression of intestinal genes are involved in the development of IBD and colorectal cancer. One of such transcription factors is “zinc fnger” domain-containing protein Kaiso which is able to bind to methylated DNA. In this study, we assessed the role of Kaiso in the development of intestinal inflammation using the experimental model of C57BL/6Muc2-/-Kaiso-/-. We have shown that mice with impaired intestinal barrier function that develop processes similar to human IBD also develop inflammatory responses, such as increased expression of Il1, Tnf and Il17a genes. The defciency of the Kaiso transcription factor in Mucin-2 knockout mice causes a decrease in the expression level of only the Cox2 and Tff3 genes. Perhaps a decline in the expression of the gene encoding cyclooxygenase-2 can lead to a decrease in the expression of the antibacterial factor Trefoil factor 3. However, in the experimental model of IBD, Kaiso protein did not play a signifcant role in the regulation of pro-inflammatory cytokines of tumor necrosis factor and interleukins 1 and 17

Genome-wide nucleosome map and cytosine methylation levels of an ancient human genome.

yesEpigenetic information is available from contemporary organisms, but is difficult to track back in evolutionary time. Here, we show that genome-wide epigenetic information can be gathered directly from next-generation sequence reads of DNA isolated from ancient remains. Using the genome sequence data generated from hair shafts of a 4000-yr-old Paleo- Eskimo belonging to the Saqqaq culture, we generate the first ancient nucleosome map coupled with a genome-wide survey of cytosine methylation levels. The validity of both nucleosome map and methylation levels were confirmed by the recovery of the expected signals at promoter regions, exon/intron boundaries, and CTCF sites. The top-scoring nucleosome calls revealed distinct DNA positioning biases, attesting to nucleotide-level accuracy. The ancient methylation levels exhibited high conservation over time, clustering closely with modern hair tissues. Using ancient methylation information, we estimated the age at death of the Saqqaq individual and illustrate how epigenetic information can be used to infer ancient gene expression. Similar epigenetic signatures were found in other fossil material, such as 110,000- to 130,000-yr-old bones, supporting the contention that ancient epigenomic information can be reconstructed from a deep past. Our findings lay the foundation for extracting epigenomic information from ancient samples, allowing shifts in epialleles to be tracked through evolutionary time, as well as providing an original window into modern epigenomics