Professor Skirmantas Kriaucionis

Research Area: Cell and Molecular Biology
Technology Exchange: Bioinformatics, ES cell / homologous recombination, Immunohistochemistry, Mass spectrometry, Protein interaction and Transcript profiling
Scientific Themes: Genetics & Genomics and Cancer Biology
Keywords: Epigenetics, DNA modifications, Neuronal Cell Biology, Chromatin, Cancer, Purkinje neurons and Nucleotide metabolism
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Our research program aims to elucidate the molecular function of DNA modifications in normal cells and cancer. We employ biochemistry and in vivo approaches to investigate roles of DNA modifications in transcription, heritability, mutability and nuclear organisation. Together with advancing knowledge of the basic biology of DNA, we delineate defects in cancer and aim to target them for the therapy.

Until recently, methylation of cytosine (5-methylcytosine, 5mC) was the only recognized DNA modification present in mammalian DNA. We and others have demonstrated the existence of 5-hydroxymethylcytosine (5hmC), which is a product of TET family of oxygenases acting on methylated cytosine. In cycling cells, 5hmC is a demethylation intermediate, but it is found as a long-lived base in post-mitotic cells, where its function is less well understood.

An example of our recent work is an investigation of the hypothesis that stable 5hmC may have an impact on the mutability of DNA, since 5mC is known to relate to increased C-to-T transitions. By employing computational analysis relating 5hmC localisation in normal human tissues with mutational frequencies in cancer, we demonstrated that 5hmCs have lower mutation frequency than 5mCs.

While 5hmC in the DNA carry a benefit of reduce mutability, its presence in the nucleotide pool (used to synthesize new strands of DNA) is mutagenic. 5hmC and other biologically modified bases can enter nucleotide pool via recycling of epigenetically modified DNA. Our research on the metabolism of epigenetic nucleotides demonstrated that cytidine monophosphate kinase 1 (CMPK1) restricts production of 5hmdCTP and prevents incorporation of the modified base directly into DNA. However, cytidine deaminase (CDA) deaminates 5hmdC resulting 5hmdU, which is phosphorylated and incorporated into DNA, causing DNA damage. We are currently exploring whether this phenomenon can be exploited for personalised cancer therapy, targeting tumours overexpressing CDA.

Name Department Institution Country
Professor Benedikt M Kessler Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Dr Benjamin Schuster-Böckler Oxford Ludwig Institute Oxford University, Old Road Campus Research Building United Kingdom
Professor Ian Tomlinson Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Professor Xin Lu Oxford Ludwig Institute Oxford University, Old Road Campus Research Building United Kingdom
Prof Paresh Vyas MRCP FRCP FRCPath Nuffield Division of Clinical Laboratory Sciences Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Kriaucionis S. 2017. DNA Extras SCIENTIST, 31 (9), pp. 48-53.

Kriaucionis S. 2017. The role of DNA base modifications: Researchers are just beginning to scratch the surface of how several newly recognized epigenetic changes function in the genome Scientist, 31 (9),

Brown DA, Di Cerbo V, Feldmann A, Ahn J, Ito S, Blackledge NP, Nakayama M, McClellan M, Dimitrova E, Turberfield AH et al. 2017. The SET1 Complex Selects Actively Transcribed Target Genes via Multivalent Interaction with CpG Island Chromatin. Cell Rep, 20 (10), pp. 2313-2327. | Show Abstract | Read more

Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome.

Bardella C, Al-Dalahmah O, Krell D, Brazauskas P, Al-Qahtani K, Tomkova M, Adam J, Serres S, Lockstone H, Freeman-Mills L et al. 2016. Expression of Idh1R132H in the Murine Subventricular Zone Stem Cell Niche Recapitulates Features of Early Gliomagenesis. Cancer Cell, 30 (4), pp. 578-594. | Show Abstract | Read more

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.

Tomkova M, McClellan M, Kriaucionis S, Schuster-Boeckler B. 2016. 5-hydroxymethylcytosine marks regions with reduced mutation frequency in human DNA. Elife, 5 (MAY2016), | Show Abstract | Read more

CpG dinucleotides are the main mutational hot-spot in most cancers. The characteristic elevated C>T mutation rate in CpG sites has been related to 5-methylcytosine (5mC), an epigenetically modified base which resides in CpGs and plays a role in transcription silencing. In brain nearly a third of 5mCs have recently been found to exist in the form of 5-hydroxymethylcytosine (5hmC), yet the effect of 5hmC on mutational processes is still poorly understood. Here we show that 5hmC is associated with an up to 53% decrease in the frequency of C>T mutations in a CpG context compared to 5mC. Tissue specific 5hmC patterns in brain, kidney and blood correlate with lower regional CpG>T mutation frequency in cancers originating in the respective tissues. Together our data reveal global and opposing effects of the two most common cytosine modifications on the frequency of cancer causing somatic mutations in different cell types.

Graham B, Marcais A, Dharmalingam G, Carroll T, Kanellopoulou C, Graumann J, Nesterova TB, Bermange A, Brazauskas P, Xella B et al. 2016. MicroRNAs of the miR-290-295 Family Maintain Bivalency in Mouse Embryonic Stem Cells. Stem Cell Reports, 6 (5), pp. 635-642. | Show Abstract | Read more

Numerous developmentally regulated genes in mouse embryonic stem cells (ESCs) are marked by both active (H3K4me3)- and polycomb group (PcG)-mediated repressive (H3K27me3) histone modifications. This bivalent state is thought to be important for transcriptional poising, but the mechanisms that regulate bivalent genes and the bivalent state remain incompletely understood. Examining the contribution of microRNAs (miRNAs) to the regulation of bivalent genes, we found that the miRNA biogenesis enzyme DICER was required for the binding of the PRC2 core components EZH2 and SUZ12, and for the presence of the PRC2-mediated histone modification H3K27me3 at many bivalent genes. Genes that lost bivalency were preferentially upregulated at the mRNA and protein levels. Finally, reconstituting Dicer-deficient ESCs with ESC miRNAs restored bivalent gene repression and PRC2 binding at formerly bivalent genes. Therefore, miRNAs regulate bivalent genes and the bivalent state itself.

Zauri M, Berridge G, Thézénas M-L, Pugh KM, Goldin R, Kessler BM, Kriaucionis S. 2015. CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer. Nature, 524 (7563), pp. 114-118. | Show Abstract | Read more

Cells require nucleotides to support DNA replication and repair damaged DNA. In addition to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Because epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2'deoxycytidine (5mdC) has been investigated before, it remains unknown how cells deal with the recently identified oxidized forms of 5mdC: 5-hydroxymethyl-2'deoxycytidine (5hmdC), 5-formy-2'deoxycytidine (5fdC) and 5-carboxyl-2'deoxycytidine (5cadC). Here we show that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus, cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiology. Notably, by screening cancer cell lines for growth defects after exposure to 5hmdC, we unexpectedly identify a subset of cell lines in which 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches, we show that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage, and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metabolism of oxidized epigenetic bases, and suggest a new therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment with other cytidine analogues.

Brazauskas P, Kriaucionis S. 2014. DNA modifications: Another stable base in DNA. Nat Chem, 6 (12), pp. 1031-1033. | Read more

Duman EA, Kriaucionis S, Dunn JJ, Hatchwell E. 2015. A simple modification to the luminometric methylation assay to control for the effects of DNA fragmentation. Biotechniques, 58 (5), pp. 262-264. | Show Abstract | Read more

Variations in DNA methylation have been implicated in a number of disorders. Changes in global DNA methylation levels have long been associated with various types of cancer. One of the recently described methods for determining global DNA methylation levels is the LUminometric Methylation Assay (LUMA), which utilizes methylation sensitive and insensitive restriction endonucleases and pyrosequencing technology for quantification. Here we provide evidence suggesting that the global methylation level reported by LUMA is affected by the integrity of the DNA being analyzed. The less intact the DNA, the lower the global methylation levels reported by LUMA. In order to overcome this problem, we propose the use of undigested DNA alongside digested samples. Finally, we demonstrate that this results in a more accurate assessment of global DNA methylation levels.

Kriaucionis S, Tahiliani M. 2014. Expanding the epigenetic landscape: novel modifications of cytosine in genomic DNA. Cold Spring Harb Perspect Biol, 6 (10), pp. a018630. | Show Abstract | Read more

Methylation of the base cytosine in DNA is critical for silencing endogenous retroviruses, regulating gene expression, and establishing cellular identity, and has long been regarded as an indelible epigenetic mark. The recent discovery that the ten eleven translocation (TET) proteins can oxidize 5-methylcytosine (5mC) resulting in the formation of 5-hydroxymethylcytosine (5hmC) and other oxidized cytosine variants in the genome has triggered a paradigm shift in our understanding of how dynamic changes in DNA methylation regulate transcription and cellular differentiation, thus influencing normal development and disease.

Lercher L, McDonough MA, El-Sagheer AH, Thalhammer A, Kriaucionis S, Brown T, Schofield CJ. 2014. Structural insights into how 5-hydroxymethylation influences transcription factor binding. Chem Commun (Camb), 50 (15), pp. 1794-1796. | Show Abstract | Read more

Transcription factor binding and high resolution crystallographic studies (1.3 Å) of Dickerson-Drew duplexes with cytosine, methylcytosine and hydroxymethylcytosine bases provide evidence that C-5 cytosine modifications could regulate transcription by context dependent effects on DNA transcription factor interactions.

Piccolo FM, Bagci H, Brown KE, Landeira D, Soza-Ried J, Feytout A, Mooijman D, Hajkova P, Leitch HG, Tada T et al. 2013. Errata to Different roles for tet1 and tet2 proteins in reprogramming-mediated erasure of imprints induced by egc fusion [Molecular Cell, 49 (2013) 1023-1033] Molecular Cell, 49 (6), pp. 1176. | Read more

Piccolo FM, Bagci H, Brown KE, Landeira D, Soza-Ried J, Feytout A, Mooijman D, Hajkova P, Leitch HG, Tada T et al. 2013. Different roles for Tet1 and Tet2 proteins in reprogramming-mediated erasure of imprints induced by EGC fusion. Mol Cell, 49 (6), pp. 1023-1033. | Show Abstract | Read more

Genomic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGC) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells.

Rommel PC, Bosque D, Gitlin AD, Croft GF, Heintz N, Casellas R, Nussenzweig MC, Kriaucionis S, Robbiani DF. 2013. Fate mapping for activation-induced cytidine deaminase (AID) marks non-lymphoid cells during mouse development. PLoS One, 8 (7), pp. e69208. | Show Abstract | Read more

The Aicda gene encodes Activation-Induced cytidine Deaminase (AID), an enzyme essential for remodeling antibody genes in mature B lymphocytes. AID is also responsible for DNA damage at oncogenes, leading to their mutation and cancer-associated chromosome translocation in lymphoma. We used fate mapping and AID(GFP) reporter mice to determine if AID expression in the mouse extends beyond lymphocytes. We discovered that AID(cre) tags a small fraction of non-lymphoid cells starting at 10.5 days post conception (dpc), and that AID(GFP+) cells are detectable at dpc 11.5 and 12.5. Embryonic cells are tagged by AID(cre) in the submandibular region, where conditional deletion of the tumor suppressor PTEN causes squamous papillomas. AID(cre) also tags non-lymphoid cells in the embryonic central nervous system. Finally, in the adult mouse brain, AID(cre) marks a small fraction of diverse neurons and distinct neuronal populations, including pyramidal cells in cortical layer IV.

Cited:

58

Scopus

Piccolo FM, Bagci H, Brown KE, Landeira D, Soza-Ried J, Feytout A, Mooijman D, Hajkova P, Leitch HG, Tada T et al. 2013. Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion Molecular Cell, 49 (6), pp. 1023-1033. | Show Abstract | Read more

Genomic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGC) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells. © 2013 Elsevier Inc.

Piccolo FM, Bagci H, Brown KE, Landeira D, Soza-Ried J, Feytout A, Mooijman D, Hajkova P, Leitch HG, Tada T et al. 2013. Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion Molecular Cell, 49 (6), pp. 1176-1176. | Read more

Kriaucionis S. 2013. Covering the bases: loss of 5-hydroxymethylcytosine in melanoma Pigment Cell & Melanoma Research, 26 (2), pp. 159-161. | Read more

Mellén M, Ayata P, Dewell S, Kriaucionis S, Heintz N. 2012. MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system. Cell, 151 (7), pp. 1417-1430. | Show Abstract | Read more

The high level of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes suggests that mechanisms interpreting 5hmC in the CNS may differ from those present in embryonic stem cells. Here, we present quantitative, genome-wide analysis of 5hmC, 5-methylcytosine (5mC), and gene expression in differentiated CNS cell types in vivo. We report that 5hmC is enriched in active genes and that, surprisingly, strong depletion of 5mC is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG-binding protein 2 (MeCP2) as the major 5hmC-binding protein in the brain and demonstrate that MeCP2 binds 5hmC- and 5mC-containing DNA with similar high affinities. The Rett-syndrome-causing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell-specific epigenetic mechanism for regulation of chromatin structure and gene expression.

Blackledge NP, Long HK, Zhou JC, Kriaucionis S, Patient R, Klose RJ. 2012. Bio-CAP: a versatile and highly sensitive technique to purify and characterise regions of non-methylated DNA. Nucleic Acids Res, 40 (4), pp. e32. | Show Abstract | Read more

Across vertebrate genomes methylation of cytosine residues within the context of CpG dinucleotides is a pervasive epigenetic mark that can impact gene expression and has been implicated in various developmental and disease-associated processes. Several biochemical approaches exist to profile DNA methylation, but recently an alternative approach based on profiling non-methylated CpGs was developed. This technique, called CxxC affinity purification (CAP), uses a ZF-CxxC (CxxC) domain to specifically capture DNA containing clusters of non-methylated CpGs. Here we describe a new CAP approach, called biotinylated CAP (Bio-CAP), which eliminates the requirement for specialized equipment while dramatically improving and simplifying the CxxC-based DNA affinity purification. Importantly, this approach isolates non-methylated DNA in a manner that is directly proportional to the density of non-methylated CpGs, and discriminates non-methylated CpGs from both methylated and hydroxymethylated CpGs. Unlike conventional CAP, Bio-CAP can be applied to nanogram quantities of genomic DNA and in a magnetic format is amenable to efficient parallel processing of samples. Furthermore, Bio-CAP can be applied to genome-wide profiling of non-methylated DNA with relatively small amounts of input material. Therefore, Bio-CAP is a simple and streamlined approach for characterizing regions of the non-methylated DNA, whether at specific target regions or genome wide.

Kriaucionis S. 2012. Covering the bases: Loss of 5-hydroxymethylcytosine in melanoma Pigment Cell and Melanoma Research,

Kriaucionis S, Heintz N. 2009. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science, 324 (5929), pp. 929-930. | Show Abstract | Read more

Despite the importance of epigenetic regulation in neurological disorders, little is known about neuronal chromatin. Cerebellar Purkinje neurons have large and euchromatic nuclei, whereas granule cell nuclei are small and have a more typical heterochromatin distribution. While comparing the abundance of 5-methylcytosine in Purkinje and granule cell nuclei, we detected the presence of an unusual DNA nucleotide. Using thin-layer chromatography, high-pressure liquid chromatography, and mass spectrometry, we identified the nucleotide as 5-hydroxymethyl-2'-deoxycytidine (hmdC). hmdC constitutes 0.6% of total nucleotides in Purkinje cells, 0.2% in granule cells, and is not present in cancer cell lines. hmdC is a constituent of nuclear DNA that is highly abundant in the brain, suggesting a role in epigenetic control of neuronal function.

Nan X, Hou J, Maclean A, Nasir J, Lafuente MJ, Shu X, Kriaucionis S, Bird A. 2007. Interaction between chromatin proteins MECP2 and ATRX is disrupted by mutations that cause inherited mental retardation. Proc Natl Acad Sci U S A, 104 (8), pp. 2709-2714. | Show Abstract | Read more

Mutations in the human methyl-CpG-binding protein gene MECP2 cause the neurological disorder Rett syndrome and some cases of X-linked mental retardation (XLMR). We report that MeCP2 interacts with ATRX, a SWI2/SNF2 DNA helicase/ATPase that is mutated in ATRX syndrome (alpha-thalassemia/mental retardation, X-linked). MeCP2 can recruit the helicase domain of ATRX to heterochromatic foci in living mouse cells in a DNA methylation-dependent manner. Also, ATRX localization is disrupted in neurons of Mecp2-null mice. Point mutations within the methylated DNA-binding domain of MeCP2 that cause Rett syndrome or X-linked mental retardation inhibit its interaction with ATRX in vitro and its localization in vivo without affecting methyl-CpG binding. We propose that disruption of the MeCP2-ATRX interaction leads to pathological changes that contribute to mental retardation.

Bird A, Berger J, Kriaucionis S, Klose R, Guy J. 2007. Reading the DNA methylation signal CALCIFIED TISSUE INTERNATIONAL, 80 pp. S18-S18.

Kriaucionis S, Paterson A, Curtis J, Guy J, Macleod N, Bird A. 2006. Gene expression analysis exposes mitochondrial abnormalities in a mouse model of Rett syndrome. Mol Cell Biol, 26 (13), pp. 5033-5042. | Show Abstract | Read more

Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the X-linked MECP2 gene, which encodes a methyl-CpG binding transcriptional repressor. Using the Mecp2-null mouse (an animal model for RTT) and differential display, we found that mice with neurological symptoms overexpress the nuclear gene for ubiquinol-cytochrome c reductase core protein 1 (Uqcrc1). Chromatin immunoprecipitation demonstrated that MeCP2 interacts with the Uqcrc1 promoter. Uqcrc1 encodes a subunit of mitochondrial respiratory complex III, and isolated mitochondria from the Mecp2-null brain showed elevated respiration rates associated with respiratory complex III and an overall reduction in coupling. A causal link between Uqcrc1 gene overexpression and enhanced complex III activity was established in neuroblastoma cells. Our findings raise the possibility that mitochondrial dysfunction contributes to pathology of the Mecp2-null mouse and may contribute to the long-known resemblance between Rett syndrome and certain mitochondrial disorders.

Nuber UA, Kriaucionis S, Roloff TC, Guy J, Selfridge J, Steinhoff C, Schulz R, Lipkowitz B, Ropers HH, Holmes MC, Bird A. 2005. Up-regulation of glucocorticoid-regulated genes in a mouse model of Rett syndrome. Hum Mol Genet, 14 (15), pp. 2247-2256. | Show Abstract | Read more

Rett syndrome (RTT) is a severe form of mental retardation, which is caused by spontaneous mutations in the X-linked gene MECP2. How the loss of MeCP2 function leads to RTT is currently unknown. Mice lacking the Mecp2 gene initially show normal postnatal development but later acquire neurological phenotypes, including heightened anxiety, that resemble RTT. The MECP2 gene encodes a methyl-CpG-binding protein that can act as a transcriptional repressor. Using cDNA microarrays, we found that Mecp2-null animals differentially express several genes that are induced during the stress response by glucocorticoids. Increased levels of mRNAs for serum glucocorticoid-inducible kinase 1 (Sgk) and FK506-binding protein 51 (Fkbp5) were observed before and after onset of neurological symptoms, but plasma glucocorticoid was not significantly elevated in Mecp2-null mice. MeCP2 is bound to the Fkbp5 and Sgk genes in brain and may function as a modulator of glucocorticoid-inducible gene expression. Given the known deleterious effect of glucocorticoid exposure on brain development, our data raise the possibility that disruption of MeCP2-dependent regulation of stress-responsive genes contributes to the symptoms of RTT.

Kriaucionis S, Bird A. 2004. The major form of MeCP2 has a novel N-terminus generated by alternative splicing. Nucleic Acids Res, 32 (5), pp. 1818-1823. | Show Abstract | Read more

MeCP2 is a methyl-CpG binding protein that can repress transcription of nearby genes. In humans, mutations in the MECP2 gene are the major cause of Rett syndrome. By searching expressed sequence tag (EST) databases we have found a novel MeCP2 splice isoform (MeCP2alpha) which encodes a distinct N-terminus. We demonstrate that the MeCP2alpha mRNA splice variant is more abundant than the previously annotated MeCP2 mRNA (MeCP2beta) in mouse tissues and human brain. Furthermore, MeCP2beta mRNA has an upstream open reading frame that inhibits its translation. As a result of these differences, >90% of MeCP2 in mouse brain is MeCP2alpha. Both protein isoforms are nuclear and colocalize with densely methylated heterochromatic foci in mouse cells. The presence of a previously unknown MeCP2 isoform has implications for the genetic screening of Rett syndrome patients and for studies of the functional significance of MeCP2.

Cited:

89

Scopus

Kriaucionis S, Bird A. 2003. DNA methylation and Rett syndrome HUMAN MOLECULAR GENETICS, 12 (REV. ISS. 2), pp. R221-R227. | Show Abstract | Read more

Methylation of cytosine in human DNA has been studied for over 60 years, but has only recently been confirmed as an important player in human disease. Rett syndrome is a neurological disorder caused by mutations in the MeCP2 protein, which has been shown to bind methylated DNA and repress transcription. This review will focus on experiments addressing the basic properties of MeCP2 and on mouse models of Rett syndrome that are starting to yield insights into this condition.

Kriaucionis S, Bird A. 2003. DNA methylation and Rett syndrome. Hum Mol Genet, 12 Spec No 2 (suppl 2), pp. R221-R227. | Show Abstract | Read more

Methylation of cytosine in human DNA has been studied for over 60 years, but has only recently been confirmed as an important player in human disease. Rett syndrome is a neurological disorder caused by mutations in the MeCP2 protein, which has been shown to bind methylated DNA and repress transcription. This review will focus on experiments addressing the basic properties of MeCP2 and on mouse models of Rett syndrome that are starting to yield insights into this condition.

Tomkova M, McClellan M, Kriaucionis S, Schuster-Böckler B. 2017. DNA Replication and associated repair pathways are involved in the mutagenesis of methylated cytosine. DNA Repair (Amst), 62 pp. 1-7. | Show Abstract | Read more

Transitions of cytosine to thymine in CpG dinucleotides are the most frequent type of mutations observed in cancer. This increased mutability is commonly explained by the presence of 5-methylcytosine (5mC) and its spontaneous hydrolytic deamination into thymine. Here, we describe observations that question whether spontaneous deamination alone causes the elevated mutagenicity of 5mC. Tumours with somatic mutations in DNA mismatch-repair genes or in the proofreading domain of DNA polymerase ε (Pol ε) exhibit more 5mC to T transitions than would be expected, given the kinetics of hydrolytic deamination. This enrichment is asymmetrical around replication origins with a preference for the leading strand template, in particular in methylated cytosines flanked by guanines (GCG). Notably, GCG to GTG mutations also exhibit strand asymmetry in mismatch-repair and Pol ε wild-type tumours. Together, these findings suggest that mis-incorporation of A opposite 5mC during replication of the leading strand might be a contributing factor in the mutagenesis of methylated cytosine.

Zauri M, Berridge G, Thézénas M-L, Pugh KM, Goldin R, Kessler BM, Kriaucionis S. 2015. CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer. Nature, 524 (7563), pp. 114-118. | Show Abstract | Read more

Cells require nucleotides to support DNA replication and repair damaged DNA. In addition to de novo synthesis, cells recycle nucleotides from the DNA of dying cells or from cellular material ingested through the diet. Salvaged nucleosides come with the complication that they can contain epigenetic modifications. Because epigenetic inheritance of DNA methylation mainly relies on copying of the modification pattern from parental strands, random incorporation of pre-modified bases during replication could have profound implications for epigenome fidelity and yield adverse cellular phenotypes. Although the salvage mechanism of 5-methyl-2'deoxycytidine (5mdC) has been investigated before, it remains unknown how cells deal with the recently identified oxidized forms of 5mdC: 5-hydroxymethyl-2'deoxycytidine (5hmdC), 5-formy-2'deoxycytidine (5fdC) and 5-carboxyl-2'deoxycytidine (5cadC). Here we show that enzymes of the nucleotide salvage pathway display substrate selectivity, effectively protecting newly synthesized DNA from the incorporation of epigenetically modified forms of cytosine. Thus, cell lines and animals can tolerate high doses of these modified cytidines without any deleterious effects on physiology. Notably, by screening cancer cell lines for growth defects after exposure to 5hmdC, we unexpectedly identify a subset of cell lines in which 5hmdC or 5fdC administration leads to cell lethality. Using genomic approaches, we show that the susceptible cell lines overexpress cytidine deaminase (CDA). CDA converts 5hmdC and 5fdC into variants of uridine that are incorporated into DNA, resulting in accumulation of DNA damage, and ultimately, cell death. Our observations extend current knowledge of the nucleotide salvage pathway by revealing the metabolism of oxidized epigenetic bases, and suggest a new therapeutic option for cancers, such as pancreatic cancer, that have CDA overexpression and are resistant to treatment with other cytidine analogues.

Lercher L, McDonough MA, El-Sagheer AH, Thalhammer A, Kriaucionis S, Brown T, Schofield CJ. 2014. Structural insights into how 5-hydroxymethylation influences transcription factor binding. Chem Commun (Camb), 50 (15), pp. 1794-1796. | Show Abstract | Read more

Transcription factor binding and high resolution crystallographic studies (1.3 Å) of Dickerson-Drew duplexes with cytosine, methylcytosine and hydroxymethylcytosine bases provide evidence that C-5 cytosine modifications could regulate transcription by context dependent effects on DNA transcription factor interactions.

Piccolo FM, Bagci H, Brown KE, Landeira D, Soza-Ried J, Feytout A, Mooijman D, Hajkova P, Leitch HG, Tada T et al. 2013. Different roles for Tet1 and Tet2 proteins in reprogramming-mediated erasure of imprints induced by EGC fusion. Mol Cell, 49 (6), pp. 1023-1033. | Show Abstract | Read more

Genomic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGC) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells.

Mellén M, Ayata P, Dewell S, Kriaucionis S, Heintz N. 2012. MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system. Cell, 151 (7), pp. 1417-1430. | Show Abstract | Read more

The high level of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes suggests that mechanisms interpreting 5hmC in the CNS may differ from those present in embryonic stem cells. Here, we present quantitative, genome-wide analysis of 5hmC, 5-methylcytosine (5mC), and gene expression in differentiated CNS cell types in vivo. We report that 5hmC is enriched in active genes and that, surprisingly, strong depletion of 5mC is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG-binding protein 2 (MeCP2) as the major 5hmC-binding protein in the brain and demonstrate that MeCP2 binds 5hmC- and 5mC-containing DNA with similar high affinities. The Rett-syndrome-causing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell-specific epigenetic mechanism for regulation of chromatin structure and gene expression.

Blackledge NP, Long HK, Zhou JC, Kriaucionis S, Patient R, Klose RJ. 2012. Bio-CAP: a versatile and highly sensitive technique to purify and characterise regions of non-methylated DNA. Nucleic Acids Res, 40 (4), pp. e32. | Show Abstract | Read more

Across vertebrate genomes methylation of cytosine residues within the context of CpG dinucleotides is a pervasive epigenetic mark that can impact gene expression and has been implicated in various developmental and disease-associated processes. Several biochemical approaches exist to profile DNA methylation, but recently an alternative approach based on profiling non-methylated CpGs was developed. This technique, called CxxC affinity purification (CAP), uses a ZF-CxxC (CxxC) domain to specifically capture DNA containing clusters of non-methylated CpGs. Here we describe a new CAP approach, called biotinylated CAP (Bio-CAP), which eliminates the requirement for specialized equipment while dramatically improving and simplifying the CxxC-based DNA affinity purification. Importantly, this approach isolates non-methylated DNA in a manner that is directly proportional to the density of non-methylated CpGs, and discriminates non-methylated CpGs from both methylated and hydroxymethylated CpGs. Unlike conventional CAP, Bio-CAP can be applied to nanogram quantities of genomic DNA and in a magnetic format is amenable to efficient parallel processing of samples. Furthermore, Bio-CAP can be applied to genome-wide profiling of non-methylated DNA with relatively small amounts of input material. Therefore, Bio-CAP is a simple and streamlined approach for characterizing regions of the non-methylated DNA, whether at specific target regions or genome wide.

Kriaucionis S, Heintz N. 2009. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science, 324 (5929), pp. 929-930. | Show Abstract | Read more

Despite the importance of epigenetic regulation in neurological disorders, little is known about neuronal chromatin. Cerebellar Purkinje neurons have large and euchromatic nuclei, whereas granule cell nuclei are small and have a more typical heterochromatin distribution. While comparing the abundance of 5-methylcytosine in Purkinje and granule cell nuclei, we detected the presence of an unusual DNA nucleotide. Using thin-layer chromatography, high-pressure liquid chromatography, and mass spectrometry, we identified the nucleotide as 5-hydroxymethyl-2'-deoxycytidine (hmdC). hmdC constitutes 0.6% of total nucleotides in Purkinje cells, 0.2% in granule cells, and is not present in cancer cell lines. hmdC is a constituent of nuclear DNA that is highly abundant in the brain, suggesting a role in epigenetic control of neuronal function.

Nan X, Hou J, Maclean A, Nasir J, Lafuente MJ, Shu X, Kriaucionis S, Bird A. 2007. Interaction between chromatin proteins MECP2 and ATRX is disrupted by mutations that cause inherited mental retardation. Proc Natl Acad Sci U S A, 104 (8), pp. 2709-2714. | Show Abstract | Read more

Mutations in the human methyl-CpG-binding protein gene MECP2 cause the neurological disorder Rett syndrome and some cases of X-linked mental retardation (XLMR). We report that MeCP2 interacts with ATRX, a SWI2/SNF2 DNA helicase/ATPase that is mutated in ATRX syndrome (alpha-thalassemia/mental retardation, X-linked). MeCP2 can recruit the helicase domain of ATRX to heterochromatic foci in living mouse cells in a DNA methylation-dependent manner. Also, ATRX localization is disrupted in neurons of Mecp2-null mice. Point mutations within the methylated DNA-binding domain of MeCP2 that cause Rett syndrome or X-linked mental retardation inhibit its interaction with ATRX in vitro and its localization in vivo without affecting methyl-CpG binding. We propose that disruption of the MeCP2-ATRX interaction leads to pathological changes that contribute to mental retardation.

Kriaucionis S, Paterson A, Curtis J, Guy J, Macleod N, Bird A. 2006. Gene expression analysis exposes mitochondrial abnormalities in a mouse model of Rett syndrome. Mol Cell Biol, 26 (13), pp. 5033-5042. | Show Abstract | Read more

Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the X-linked MECP2 gene, which encodes a methyl-CpG binding transcriptional repressor. Using the Mecp2-null mouse (an animal model for RTT) and differential display, we found that mice with neurological symptoms overexpress the nuclear gene for ubiquinol-cytochrome c reductase core protein 1 (Uqcrc1). Chromatin immunoprecipitation demonstrated that MeCP2 interacts with the Uqcrc1 promoter. Uqcrc1 encodes a subunit of mitochondrial respiratory complex III, and isolated mitochondria from the Mecp2-null brain showed elevated respiration rates associated with respiratory complex III and an overall reduction in coupling. A causal link between Uqcrc1 gene overexpression and enhanced complex III activity was established in neuroblastoma cells. Our findings raise the possibility that mitochondrial dysfunction contributes to pathology of the Mecp2-null mouse and may contribute to the long-known resemblance between Rett syndrome and certain mitochondrial disorders.

Nuber UA, Kriaucionis S, Roloff TC, Guy J, Selfridge J, Steinhoff C, Schulz R, Lipkowitz B, Ropers HH, Holmes MC, Bird A. 2005. Up-regulation of glucocorticoid-regulated genes in a mouse model of Rett syndrome. Hum Mol Genet, 14 (15), pp. 2247-2256. | Show Abstract | Read more

Rett syndrome (RTT) is a severe form of mental retardation, which is caused by spontaneous mutations in the X-linked gene MECP2. How the loss of MeCP2 function leads to RTT is currently unknown. Mice lacking the Mecp2 gene initially show normal postnatal development but later acquire neurological phenotypes, including heightened anxiety, that resemble RTT. The MECP2 gene encodes a methyl-CpG-binding protein that can act as a transcriptional repressor. Using cDNA microarrays, we found that Mecp2-null animals differentially express several genes that are induced during the stress response by glucocorticoids. Increased levels of mRNAs for serum glucocorticoid-inducible kinase 1 (Sgk) and FK506-binding protein 51 (Fkbp5) were observed before and after onset of neurological symptoms, but plasma glucocorticoid was not significantly elevated in Mecp2-null mice. MeCP2 is bound to the Fkbp5 and Sgk genes in brain and may function as a modulator of glucocorticoid-inducible gene expression. Given the known deleterious effect of glucocorticoid exposure on brain development, our data raise the possibility that disruption of MeCP2-dependent regulation of stress-responsive genes contributes to the symptoms of RTT.

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Kriaucionis S, Bird A. 2003. DNA methylation and Rett syndrome HUMAN MOLECULAR GENETICS, 12 (REV. ISS. 2), pp. R221-R227. | Show Abstract | Read more

Methylation of cytosine in human DNA has been studied for over 60 years, but has only recently been confirmed as an important player in human disease. Rett syndrome is a neurological disorder caused by mutations in the MeCP2 protein, which has been shown to bind methylated DNA and repress transcription. This review will focus on experiments addressing the basic properties of MeCP2 and on mouse models of Rett syndrome that are starting to yield insights into this condition.

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