QUMA: quantification tool for methylation analysis

170

168

The intestinal epithelium forms a barrier protecting the organism from microbes and other proinflammatory stimuli. The integrity of this barrier and the proper response to infection requires precise regulation of powerful immune homing signals such as tumor necrosis factor (TNF). Dysregulation of TNF leads to inflammatory bowel diseases (IBD), but the mechanism controlling the expression of this potent cytokine and the events that trigger the onset of chronic inflammation are unknown. Here, we show that loss of function of the epigenetic regulator ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1) in zebrafish leads to a reduction in tnfa promoter methylation and the induction of tnfa expression in intestinal epithelial cells (IECs). The increase in IEC tnfa levels is microbe-dependent and results in IEC shedding and apoptosis, immune cell recruitment, and barrier dysfunction, consistent with chronic inflammation. Importantly, tnfa knockdown in uhrf1 mutants restores IEC morphology, reduces cell shedding, and improves barrier function. We propose that loss of epigenetic repression and TNF induction in the intestinal epithelium can lead to IBD onset.inflammation | Uhrf1 | DNA methylation | tumor necrosis factor | zebrafish I ntestinal epithelial cells (IECs) function as a barrier to prevent luminal contents from accessing underlying tissues, and loss of barrier function is a crucial factor leading to the development of inflammatory bowel diseases (IBD) (1). IBD, including Crohn's disease and ulcerative colitis, are intestinal disorders of poorly understood origin thought to arise from genetic susceptibility, luminal microbiota, immune responses, and environmental factors (2-4). A key element in IBD onset is the up-regulation of the proinflammatory cytokine tumor necrosis factor (TNF) by various cell types including immune cells and IECs. TNF overexpression has been detected in the Paneth cells within the epithelium of human IBD patients (5), and anti-TNF treatments are used successfully to treat patients with Crohn's disease (6). Previous research in mice has demonstrated that intestinal TNF exposure leads to loss of barrier function (7), and overexpression of TNF in mouse IECs is sufficient to elicit an IBD phenotype (8). Despite its pathogenic relevance, the genetic mechanisms regulating TNF expression and IBD onset remain largely unknown.Genome-wide association studies have identified numerous susceptibility loci associated with IBD including ubiquitin-like protein containing PHD and RING finger domains 1 (UHRF1) and the DNA methyltransferases DNMT1 and DNMT3a (9, 10), which are genes involved in DNA methylation controlling epigenetic transcriptional repression. Moreover, low concordance rates have been observed in monozygotic twin studies (3), leading to the hypothesis that epigenetic regulation also contributes to IBD pathogenesis. Changes in DNA and histone modifications associated with epigenetic regulation have been detected in IBD patients (3, 4, 9, 11, 12), but direct links to the I...

Section: Methodsmentioning

confidence: 99%

Epigenetic control of intestinal barrier function and inflammation in zebrafish

Marjoram¹,

Alvers²,

Deerhake

et al. 2015

170

168

“…Recombinant plasmids were isolated (28) and sequenced with the M13 reverse primer at the University of Texas Health Science Center at San Antonio DNA sequencing facility. Raw bisulfite sequences were analyzed by using QUMA software (29) to eliminate replicate sequences with identical location and number of unconverted non-CpG-associated cytosines. Maternal and paternal alleles were distinguished by using multiple polymorphisms that are distinct between B6 and Cast sequences as described (17,23,24).…”

Section: Methodsmentioning

confidence: 99%

Primary epimutations introduced during intracytoplasmic sperm injection (ICSI) are corrected by germline-specific epigenetic reprogramming

Waal

Yamazaki

Ingale

et al. 2012

The use of assisted reproductive technologies (ART) has become increasingly common worldwide and is now responsible for 2-3% of children born in developed countries. Multiple reports have suggested that ART-conceived children are more likely to develop rare epigenetic disorders such as Beckwith-Wiedemann Syndrome or Angelman Syndrome, both of which involve dysregulation of imprinted genes. Anecdotal reports suggest that animals produced with ART that manifest apparent epigenetic defects typically do not transmit these epimutations to subsequent generations when allowed to breed naturally, but this hypothesis has not been directly studied. We analyzed allele-specific DNA methylation and expression at three imprinted genes, H19, Snrpn, and Peg3, in somatic cells from adult mice generated with the use of intracytoplasmic sperm injection (ICSI), a type of ART. Epimutations were detected in most of the ICSI-derived mice, but not in somatic cells of their offspring produced by natural mating. We examined germ cells from the ICSI mice that exhibited epimutations in their somatic cells and confirmed normal epigenetic reprogramming of the three imprinted genes analyzed. Collectively, these results confirm that ART procedures can lead to the formation of primary epimutations, but while such epimutations are likely to be maintained indefinitely in somatic cells of the ART-derived individuals, they are normally corrected in the germ line by epigenetic reprogramming and thus, not propagated to subsequent generations.gametogenesis | transgenerational inheritance E arly embryos and germ cells are unique in that they must undergo extensive epigenetic reprogramming to reestablish developmental potency during each generation. This reprogramming entails erasure of most inherited epigenetic modifications followed by the acquisition and subsequent maintenance of new epigenetic profiles (1). Genomic imprinting is an epigenetic phenomenon found in eutherian and metatherian mammals (2) that results in parent-of-origin-specific, monoallelic expression of a subset of genes (3). This functional asymmetry of imprinted genes is conferred through differential DNA methylation patterns established during gametogenesis in each parent that distinguish the maternal and paternal alleles in the ensuing offspring (4, 5). These regions are known as differentially methylated regions (DMRs), and the differential methylation profiles at these genetic elements play an important role in regulating allele-specific expression of imprinted genes (3).Because the epigenome is distinct in each cell type and is readily reversible by developmental reprogramming, it is particularly susceptible to disruption by environmental influences (6). Such epigenetic defects are known as "epimutations" and can be of two types-primary epimutations or secondary epimutations (7). Primary epimutations result from a direct disruption of an epigenetic parameter such as DNA methylation that is then propagated through DNA replication to subsequent cells. Secondary epimutations result...

“…Bisulfite-converted reads were mapped using NovoAlign alignment suite (Novocraft). Methylation status of 250 mapped reads (>98% sequence match) selected by a custom randomization algorithm was determined using QUMA (34). Cellular Analyses.…”

Section: Molecular Analysesmentioning

confidence: 99%

The histone methyltransferase SETDB1 represses endogenous and exogenous retroviruses in B lymphocytes

Collins

Kyle

Egawa

et al. 2015

Genome stability relies on epigenetic mechanisms that enforce repression of endogenous retroviruses (ERVs). Current evidence suggests that distinct chromatin-based mechanisms repress ERVs in cells of embryonic origin (histone methylation dominant) vs. more differentiated cells (DNA methylation dominant). However, the latter aspect of this model has not been tested. Remarkably, and in contrast to the prevailing model, we find that repressive histone methylation catalyzed by the enzyme SETDB1 is critical for suppression of specific ERV families and exogenous retroviruses in committed B-lineage cells from adult mice. The profile of ERV activation in SETDB1-deficient B cells is distinct from that observed in corresponding embryonic tissues, despite the loss of repressive chromatin modifications at all ERVs. We provide evidence that, on loss of SETDB1, ERVs are activated in a lineage-specific manner depending on the set of transcription factors available to target proviral regulatory elements. These findings have important implications for genome stability in somatic cells, as well as the interface between epigenetic repression and viral latency.epigenetics | histone methylation | repression | transposable elements | retroviruses R epressive chromatin is a biochemical platform for many cellular processes (1), including gene silencing during development and the suppression of potentially mutagenic repetitive elements, such as endogenous retroviruses (ERVs). The formation and maintenance of repressive chromatin rely on epigenetic modification of its DNA and nucleosome components, including hypermethylation of CpG dinucleotides by DNA methyltransferases (DNMTs). DNA methylation patterns are conserved during replication, and this modification is thought to provide the most stable form of repression (1). An important modification for initiating repressive chromatin formation is trimethylation of histone H3 at the lysine 9 position (H3K9me3) by two major histone methyltransferases (HMTs). Suppressor Of Variegation 3-9 Homolog (SUV39H)1/2 complexes maintain this modification at constitutive heterochromatin and most long interspersed nuclear elements (LINEs), which together encompass the vast majority of H3K9me3 in chromatin (2, 3). In contrast, the HMT called SET Domain, Bifurcated 1 (SETDB1 a.k.a. ESET) is targeted primarily to ERVs, which must be silenced to maintain genome and transcriptome integrity (4).Long terminal repeat (LTR)-containing ERVs represent ∼10% of human and mouse genomes (5). Although most are defunct genomic artifacts, many species contain a substantial number of active ERVs (6). In mice, ERV expression is tissue and strain specific. The intracisternal A particle (IAP) and early transposon/ Mus musculus type D (ETn/MusD) families of ERVs are expressed predominantly in early embyronic tissues. One consequence of this de-repression is the acquisition of spontaneous germ-line mutations, with IAP and ETn retrotransposition accounting for nearly 15% of these genomic alterations in mice (5). Likewise, B lymph...