Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine

DNA cytosine-5 methylation is a well-studied epigenetic pathway implicated in gene expression control and disease pathogenesis. Different technologies have been developed to examine the distribution of 5-methylcytosine (5mC) in specific sequences of the genome. Recently, substantial amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5mC by TET1, have been detected in certain mammalian tissues. Here, we have examined the ability of several commonly used DNA methylation profiling methods to distinguish between 5mC and 5hmC. We show that techniques based on sodium bisulfite treatment of DNA are incapable of distinguishing between the two modified bases. In contrast, techniques based on immunoprecipitation with anti-5mC antibody (methylated DNA immunoprecipitation, MeDIP) or those based on proteins that bind to methylated CpG sequences (e.g. methylated-CpG island recovery assay, MIRA) do not detect 5hmC and are specific for 5mC unless both modified bases occur in the same DNA fragment. We also report that several methyl-CpG binding proteins including MBD1, MBD2 and MBD4 do not bind to sequences containing 5hmC. Selective mapping of 5hmC will require the development of unique tools for the detection of this modified base

Genomic mapping of 5-hydroxymethylcytosine in the human brain

Methylation at the 5-position of cytosine is a well-studied epigenetic pathway. In addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) also referred to as the sixth DNA base have been detected in certain tissues, most notably the brain. However, the genomic distribution of this cytosine modification is unknown. Here, we have used an immunoprecipitation technique (5hmC-IP) to examine the occurrence of 5hmC in DNA from human brain frontal lobe tissue. The distribution of 5hmC was compared to that of 5mC. We show that 5hmC is more selectively targeted to genes than is 5mC. 5hmC is particularly enriched at promoters and in intragenic regions (gene bodies) but is largely absent from non-gene regions. 5hmC peaks at transcription start sites did not correlate with gene expression levels for promoters with intermediate or high CpG content. However, the presence of 5hmC in gene bodies was more positively correlated with gene expression levels than was the presence of 5mC. Promoters of testis-specific genes showed strong 5mC peaks in brain DNA but were almost completely devoid of 5hmC. Our data provide an overview of the genomic distribution of 5hmC in human brain and will set the stage for further functional characterization of this novel DNA modification

DNA demethylation pathways: Additional players and regulators.

DNA demethylation can occur passively by "dilution" of methylation marks by DNA replication, or actively and independently of DNA replication. Direct conversion of 5-methylcytosine (5mC) to cytosine (C), as originally proposed, does not occur. Instead, active DNA methylation involves oxidation of the methylated base by ten-eleven translocations (TETs), or deamination of the methylated or a nearby base by activation induced deaminase (AID). The modified nucleotide, possibly together with surrounding nucleotides, is then replaced by the BER pathway. Recent data clarify the roles and the regulation of well-known enzymes in this process. They identify base excision repair (BER) glycosylases that may cooperate with or replace thymine DNA glycosylase (TDG) in the base excision step, and suggest possible involvement of DNA damage repair pathways other than BER in active DNA demethylation. Here, we review these new developments

TET family dioxygenases and DNA demethylation in stem cells and cancers

The methylation of cytosine and subsequent oxidation constitutes a fundamental epigenetic modification in mammalian genomes, and its abnormalities are intimately coupled to various pathogenic processes including cancer development. Enzymes of the Ten-eleven translocation (TET) family catalyze the stepwise oxidation of 5-methylcytosine in DNA to 5-hydroxymethylcytosine and further oxidation products. These oxidized 5-methylcytosine derivatives represent intermediates in the reversal of cytosine methylation, and also serve as stable epigenetic modifications that exert distinctive regulatory roles. It is becoming increasingly obvious that TET proteins and their catalytic products are key regulators of embryonic development, stem cell functions and lineage specification. Over the past several years, the function of TET proteins as a barrier between normal and malignant states has been extensively investigated. Dysregulation of TET protein expression or function is commonly observed in a wide range of cancers. Notably, TET loss-of-function is causally related to the onset and progression of hematologic malignancy in vivo. In this review, we focus on recent advances in the mechanistic understanding of DNA methylation-demethylation dynamics, and their potential regulatory functions in cellular differentiation and oncogenic transformation

Påvirker mors sykefravær fosterets helse? En studie av sykefravær blant gravide og barnets fødselstilstand

I perioden fra 1993 til 2005 økte sykefravær blant gravide kvinner i Norge fra rundt 18 prosent til rundt 30 prosent (Markussen, 2012). Gravides sykefravær kan skyldes at mor har behov for mindre belastning, men kan også begrunnes med hensyn til det ufødte barnets helse. Denne studien forsøker å utrede hvorvidt gravides sykefravær påvirker fosterets helse. Eksisterende litteratur på området indikerer at barnets fødselstilstand har stor betydning til helse senere i livet. På samme måte tror mange forskere og leger at sunn livstil, bedre ernæring og balansert fysisk aktivitet virker positivt på svangerskapsutfallet. I denne studien forsøker jeg å finne ut av om barnets helse, målt ved vekt, lengde, hodeomkrets ved fødselen og fødetidspunkt relativt til termin, påvirkes av hvorvidt en gravid kvinne er i arbeid eller er sykmeldt. Siden det er all grunn til å tro at gravides sykefravær har sammenheng med deres helsetilstand, og at mors helse samvarierer med barnets helse, kan jeg ikke kun sammenligne fødselsutfall hos gravide med og uten sykefravær i svangerskapet. For å studere hvorvidt det kan være en årsakssammenheng mellom sykefravær og fødselsutfall har jeg i stedet benyttet en strategi der fastlegers "strenghet" som sykmelder benyttes som instrumentvariabel for gravides sykefravær. Jeg har benyttet en rekke administrative registre som dekker den norske befolkningen, innsamlet og organisert av Statistisk sentralbyrå og bygget opp av Frischsenteret som datakilde. Gjennom en totrinns minste kvadraters metode (2SLS) ble sykefraværsratens effekt estimert til å ha gunstige konsekvenser for fosteret; økt vekt, økt lengde, økt hodeomkrets og redusert sannsynlighet for premature fødsel

Evolutionary aspects of physiological function and molecular diversity of the oxytocin/vasopressin signaling system

Neuropeptides and regulatory peptide hormones control important developmental, physiological, and behavioral processes in animals. One of the best studied peptidergic systems is oxytocin (OXT) and arginine vasopressin (AVP) signaling. In humans and other mammalian species, OXT and AVP mediate a range of peripheral and central physiological functions that are important for osmoregulation, reproduction, complex social behaviors, memory, and learning. The origin of the OXT/AVP system is thought to date back more than 600 million years. All vertebrate OXT- and AVP-like peptides presumably have evolved from the ancestral nonapeptide vasotocin by gene duplication. These nonapeptides and their receptors are found in mammals, birds, reptiles, amphibians, and fish. OXT- and AVP-like peptides have also been identified in several invertebrate species, including mollusks, annelids, nematodes, and arthropods. Members of this peptide family share high sequence similarity and are functionally related across the entire animal kingdom. Whereas the physiology of OXT and AVP signaling components has been studied in detail in vertebrates and mammals, there is little known about the diversity and function in invertebrates. It is evident that not all invertebrates express OXT- or AVP-like neuropeptides and their receptors. Hence it is of great interest to identify novel invertebrate OXT-like signaling systems and to elucidate their physiological function. The discovery and characterization of these novel peptide hormones will provide opportunities for pharmacology and drug design, and importantly this information may be useful for comparative studies to identify common features of OXT and AVP physiology throughout the animal kingdom that may yield translational insights into evolutionary aspects of human behavior