Long noncoding RNAs (lncRNAs) regulate gene expression via their RNA product or through transcriptional interference, yet a strategy to differentiate these two processes is lacking. To address this, ...we used multiple small interfering RNAs (siRNAs) to silence GNG12-AS1, a nuclear lncRNA transcribed in an antisense orientation to the tumour-suppressor DIRAS3. Here we show that while most siRNAs silence GNG12-AS1 post-transcriptionally, siRNA complementary to exon 1 of GNG12-AS1 suppresses its transcription by recruiting Argonaute 2 and inhibiting RNA polymerase II binding. Transcriptional, but not post-transcriptional, silencing of GNG12-AS1 causes concomitant upregulation of DIRAS3, indicating a function in transcriptional interference. This change in DIRAS3 expression is sufficient to impair cell cycle progression. In addition, the reduction in GNG12-AS1 transcripts alters MET signalling and cell migration, but these are independent of DIRAS3. Thus, differential siRNA targeting of a lncRNA allows dissection of the functions related to the process and products of its transcription.
Cytosine hydroxymethylation (5hmC) in mammalian DNA is the product of oxidation of methylated cytosines (5mC) by Ten-Eleven-Translocation (TET) enzymes. While it has been shown that the TETs ...influence 5mC metabolism, pluripotency and differentiation during early embryonic development, the functional relationship between gene expression and 5hmC in adult (somatic) stem cell differentiation is still unknown. Here we report that 5hmC levels undergo highly dynamic changes during adult stem cell differentiation from intestinal progenitors to differentiated intestinal epithelium. We profiled 5hmC and gene activity in purified mouse intestinal progenitors and differentiated progeny to identify 43425 differentially hydroxymethylated regions and 5325 differentially expressed genes. These differentially marked regions showed both losses and gains of 5hmC after differentiation, despite lower global levels of 5hmC in progenitor cells. In progenitors, 5hmC did not correlate with gene transcript levels, however, upon differentiation the global increase in 5hmC content showed an overall positive correlation with gene expression level as well as prominent associations with histone modifications that typify active genes and enhancer elements. Our data support a gene regulatory role for 5hmC that is predominant over its role in controlling DNA methylation states.
Genomic imprinting is an epigenetic marking of genes in the parental germline that ensures the stable transmission of monoallelic gene expression patterns in a parent-of-origin-specific manner. ...Epigenetic marking systems are thus able to regulate gene activity independently of the underlying DNA sequence. Several imprinted gene products regulate cell proliferation and fetal growth; loss of their imprinted state, which effectively alters their dosage, might promote or suppress tumourigenic processes. Conversely, global epigenetic changes that underlie tumourigenesis might affect imprinted gene expression. Here, we review imprinted genes with regard to their roles in epigenetic predisposition to cancer, and discuss acquired epigenetic changes (DNA methylation, histone modifications and chromatin conformation) either as a result of cancer or as an early event in neoplasia. We also address recent work showing the potential role of noncoding RNA in modifying chromatin and affecting imprinted gene expression, and summarise the effects of loss of imprinting in cancer with regard to the roles that imprinted genes play in regulating growth signalling cascades. Finally, we speculate on the clinical applications of epigenetic drugs in cancer.
5-Formylcytosine (5fC) is a rare base found in mammalian DNA and thought to be involved in active DNA demethylation. Here, we show that developmental dynamics of 5fC levels in mouse DNA differ from ...those of 5-hydroxymethylcytosine (5hmC), and using stable isotope labeling in vivo, we show that 5fC can be a stable DNA modification. These results suggest that 5fC has functional roles in DNA that go beyond being a demethylation intermediate.
The transforming growth factor beta (TGF-β) signaling pathway exerts opposing effects on cancer cells, acting as either a tumor promoter or a tumor suppressor. Here, we show that these opposing ...effects are a result of the synergy between SMAD3, a downstream effector of TGF-β signaling, and the distinct epigenomes of breast-tumor-initiating cells (BTICs). These effects of TGF-β are associated with distinct gene expression programs, but genomic SMAD3 binding patterns are highly similar in the BTIC-promoting and BTIC-suppressing contexts. Our data show cell-type-specific patterns of DNA and histone modifications provide a modulatory layer by determining accessibility of genes to regulation by TGF-β/SMAD3. LBH, one such context-specific target gene, is regulated according to its DNA methylation status and is crucial for TGF-β-dependent promotion of BTICs. Overall, these results reveal that the epigenome plays a central and previously overlooked role in shaping the context-specific effects of TGF-β in cancer.
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•TGF-β has opposing effects in different breast-tumor-initiating cell (BTIC) types•Genomic SMAD3 binding patterns are similar in BTICs with opposing responses to TGF-β•BTIC type-specific epigenomes prime genes for regulation by TGF-β/SMAD3•LBH, a type-specific TGF-β target, is essential for BTIC-promoting effects of TGF-β
The TGF-β pathway uses transcriptional regulation through SMAD transcription factors to modulate cell-context-specific phenotypes. Tufegdzic Vidakovic et al. show that in breast-tumor-initiating cells (BTICs), type-specific DNA and histone modifications help determine whether the response to TGF-β is pro-oncogenic or tumor suppressive. These landscapes act both in synergy and independently of cell-type-specific SMAD3 binding to TGF-β target genes to modulate context-specific transcriptional regulation by TGF-β/SMAD3.
The imprinted insulin-like growth factor 2 (IGF2) gene is expressed predominantly from the paternal allele. Loss of imprinting (LOI) associated with hypomethylation at the promoter proximal sequence ...(DMR0) of the IGF2 gene was proposed as a predisposing constitutive risk biomarker for colorectal cancer. We used pyrosequencing to assess whether IGF2 DMR0 methylation is either present constitutively prior to cancer or whether it is acquired tissue-specifically after the onset of cancer. DNA samples from tumour tissues and matched non-tumour tissues from 22 breast and 42 colorectal cancer patients as well as peripheral blood samples obtained from colorectal cancer patients SEARCH (n=case 192, controls 96), breast cancer patients ABC (n=case 364, controls 96) and the European Prospective Investigation of Cancer EPIC-Norfolk (n=breast 228, colorectal 225, controls 895) were analysed. The EPIC samples were collected 2–5 years prior to diagnosis of breast or colorectal cancer. IGF2 DMR0 methylation levels in tumours were lower than matched non-tumour tissue. Hypomethylation of DMR0 was detected in breast (33%) and colorectal (80%) tumour tissues with a higher frequency than LOI indicating that methylation levels are a better indicator of cancer than LOI. In the EPIC population, the prevalence of IGF2 DMR0 hypomethylation was 9.5% and this correlated with increased age not cancer risk. Thus, IGF2 DMR0 hypomethylation occurs as an acquired tissue-specific somatic event rather than a constitutive innate epimutation. These results indicate that IGF2 DMR0 hypomethylation has diagnostic potential for colon cancer rather than value as a surrogate biomarker for constitutive LOI.
Abstract Background and purpose Advances in magnetic resonance imaging (MRI) and prostate sampling enable early identification of men with low to intermediate risk prostate cancer who are candidates ...for focal therapies that minimise side effects. We report dosimetry data from a pilot study evaluating the effectiveness of hemi-gland low dose rate (HG-LDR) brachytherapy as a focal therapy approach to control unilateral localised disease. Material and methods Twenty-two men underwent HG-LDR brachytherapy. Multi parametric MRI and transperineal template mapping biopsies were used to identify low volume unilateral disease. Whole gland therapy controls ( n = 120) were retrospectively obtained. All implants were performed with 4D Brachytherapy. Results Intraoperative and postimplant dosimetry complied with established brachytherapy parameters. Mean (standard deviation) postoperative D90 for the target hemi-gland was 153.8 (11.3) Gy compared to 47.5 (12.7) Gy for the contralateral hemi-gland ( P < 0.001). Mean postoperative V100% was 93.1 (3.9) and 24.6 (10.5) for the target and contralateral hemi-glands respectively ( P < 0.001). Urethra D30 was 150.4 (19.8) Gy and 174.2 (15.0) Gy for hemi-gland and whole gland implants respectively ( P < 0.001). Significantly reduced dose was also observed for rectum and neurovascular bundles. Conclusions HG-LDR focal brachytherapy is feasible with significant reduction in dose to the contralateral hemi-gland and organs at risk.
To compare survival of patients who received LDR prostate brachytherapy relative to that of peers in the general population of England, UK.
Net survival was estimated for 2472 cases treated between ...2002 and 2016 using population-based analysis guidelines. Life tables adjusted for social deprivation in England from the Office for National Statistics were used to match patients by affluence based on their postcode.
The median (range) age at time of brachytherapy was 66 (55-84) years, 84% resided in Southeast England, 51% under an index of deprivation quintile 5 (most affluent), 55% were clinical stage T1 and the remainder T2. Death from any cause occurred in 270 patients at a median (range) of 7 (1-17) years postimplant. Five and 10-year estimates (95% CI) of overall survival were 96% (95-97) and 90% (89-92), and net survival 103% (102-104) and 109% (107-110) respectively. The net survival remained above 100% in all age-at-treatment and clinical stage groups.
Net survival above 100% indicates patients survive longer than the matched general population. The study shows for the first time the net survival of patients treated with a radical therapy for localized prostate cancer in England. The impact of treatment choice on the long-term net survival advantage requires further investigation.
It is becoming clear that interconnected functional gene networks, rather than individual genes, govern stem cell self-renewal and differentiation. To identify epigenetic factors that impact on human ...epidermal stem cells we performed siRNA-based genetic screens for 332 chromatin modifiers. We developed a Bayesian mixture model to predict putative functional interactions between epigenetic modifiers that regulate differentiation. We discovered a network of genetic interactions involving EZH2, UHRF1 (both known to regulate epidermal self-renewal), ING5 (a MORF complex component), BPTF and SMARCA5 (NURF complex components). Genome-wide localization and global mRNA expression analysis revealed that these factors impact two distinct but functionally related gene sets, including integrin extracellular matrix receptors that mediate anchorage of epidermal stem cells to their niche. Using a competitive epidermal reconstitution assay we confirmed that ING5, BPTF, SMARCA5, EZH2 and UHRF1 control differentiation under physiological conditions. Thus, regulation of distinct gene expression programs through the interplay between diverse epigenetic strategies protects epidermal stem cells from differentiation.
Abstract
Topographical variations of metabolite concentrations have been reported in the duodenum, jejunum and ileum of the small intestine, and in human intestinal tumours from those regions, but ...there are no published metabolite concentrations measurements correlated with linear position in the mouse small intestine or intestinal tumours. Since DNA methylation dynamics are influenced by metabolite concentrations, they too could show linear anatomical variation. We measured metabolites by HR-MAS
1
H NMR spectroscopy and DNA cytosine modifications by LC/MS, in normal small intestines of C57BL/6J wild-type mice, and in normal and tumour samples from
Apc
Min/
+
mice. Wild-type mouse intestines showed approximately linear, negative concentration gradations from the pylorus (i.e. the junction with the stomach) of alanine, choline compounds, creatine, leucine and valine.
Apc
Min/
+
mouse tumours showed negative choline and valine gradients, but a positive glycine gradient. 5-Hydroxymethylcytosine showed a positive gradient in the tumours. The linear gradients we found along the length of the mouse small intestine and in tumours contrast with previous reports of discrete concentration changes in the duodenum, jejunum and ileum. To our knowledge, this is also the first report of a systematic measurement of global levels of DNA cytosine modification in wild-type and
Apc
Min/+
mouse small intestine.
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