Uncoupling DNA- and RNA-directed DNA methylation at Rasgrf1

ABSTRACTLong noncoding RNAs (lncRNAs) have garnered much attention as possible links between DNA sequence and the protein factors that mediate DNA methylation. However, the mechanisms by which DNA methylation is directed to specific genomic locations remain poorly understood. We previously identified a lncRNA in mouse, the pitRNA, that was implicated in the control of DNA methylation at the imprinted Rasgrf1 locus. The pitRNA is transcribed in the developing male germline antisense to the differentially methylated region (DMR) that harbors paternal allele methylation, and is driven by a series of tandem repeats that are necessary for imprinted methylation.MitoPLD, a factor necessary for piRNA biogenesis, both processes piRNAs from the pitRNA, and is necessary for complete methylation at the locus, along with piRNA binding proteins. Using two independent mouse systems where pitRNA transcription is driven by the doxycycline-inducible Tet Operator, we demonstrate that pitRNA transcription across the DMR is insufficient for imprinted methylation, and that the Rasgrf1 repeats have additional, critical cis-acting roles for imparting DNA methylation to Rasgrf1, independently of their control of pitRNA transcription. Furthermore, pitRNA overexpression and oocyte loading of pitRNA is insufficient to induce transallelic and transgenerational effects previously reported for Rasgrf1. Notably, manipulation of the pitRNA with the TetOFF system led to transcriptional perturbations over a broad chromosomal region surrounding the inserted Tet Operator, revealing that the effects of this regulatory tool are not localized to a single target gene.AUTHOR SUMMARYDNA methylation is a heritable genetic modification known to impact vital biological processes. While the proteins that establish, maintain, and remove DNA methylation are well characterized, the mechanisms by which these proteins are directed to specific genetic sequences are poorly understood. We have previously demonstrated that DNA methylation at the imprinted Rasgrf1 locus requires a DNA element with a series of tandem repeats. These repeats act as a promoter for a long noncoding RNA, the pitRNA, which is targeted by a small noncoding RNA pathway known to silence viral elements in the male germline via DNA methylation. We queried the sufficiency of the pitRNA to mediate DNA methylation at Rasgrf1. We show that, in the absence of the repeats, the pitRNA expression is insufficient to establish imprinted methylation. This work supports a pitRNA-independent mechanism for methylation at Rasgrf1, and a critical cis-acting role for the tandem repeats separate from their control of pitRNA transcription.

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Tandem Repeat Hypothesis in Imprinting: Deletion of a Conserved Direct Repeat Element Upstream of H19 Has No Effect on Imprinting in the Igf2-H19 Region

Molecular and Cellular Biology ◽

10.1128/mcb.24.13.5650-5656.2004 ◽

2004 ◽

Vol 24 (13) ◽

pp. 5650-5656 ◽

Cited By ~ 28

Author(s):

Annabelle Lewis ◽

Kohzoh Mitsuya ◽

Miguel Constancia ◽

Wolf Reik

Keyword(s):

Tandem Repeat ◽

Tandem Repeats ◽

Direct Repeat ◽

Regulatory Elements ◽

Repeat Element ◽

Allelic Expression ◽

Differentially Methylated Region ◽

Paternal Allele ◽

Imprinted Genes ◽

Allele Specific

ABSTRACT Igf2 and H19 are reciprocally imprinted genes on mouse distal chromosome 7. They share several regulatory elements, including a differentially methylated region (DMR) upstream of H19 that is paternally methylated throughout development. The cis-acting sequence requirements for targeting DNA methylation to the DMR remain unknown; however, it has been suggested that direct tandem repeats near DMRs could be involved. Previous studies of the imprinted Rasgrf1 locus demonstrate indeed that a direct repeat element adjacent to a DMR is responsible for establishing paternal allele-specific methylation at the DMR and therefore allelic expression of the Rasgrf1 transcript. We identified a prominent and conserved direct tandem repeat 1 kb upstream of the H19 DMR and proposed that it played a similar role in imprinted regulation of H19. To test our hypothesis, we generated mice harboring a 1.7-kb targeted deletion of the direct repeat element and analyzed fetal growth, allelic expression, and methylation within the Igf2-H19 region. Surprisingly the deletion had no effect on imprinting. These results together with deletions of other repeats close to imprinted genes suggest that direct repeats may not be important for the targeting of methylation at the majority of imprinted loci and that the Rasgrf1 locus may be an exception to this rule.

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278 METHYLATION STATUS IN THE INTRAGENIC DIFFERENTIALLY METHYLATED REGION OF THE IGF2 LOCUS IN UNSORTED AND SEX-SORTED SPERM

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab278 ◽

2011 ◽

Vol 23 (1) ◽

pp. 237

Author(s):

J. O. Carvalho ◽

V. A. Michalczechen-Lacerda ◽

F. C. Rodrigues ◽

R. Sartori ◽

M. M. Franco ◽

...

Keyword(s):

Dna Methylation ◽

Flow Cytometry ◽

Sperm Quality ◽

Methylation Status ◽

Methylation Pattern ◽

Vector System ◽

Differentially Methylated Region ◽

Paternal Allele ◽

Igf2 Gene ◽

Exon 10

Methylation is the main coordinator of epigenetic inheritance between generations, influencing the regulation of gene expression. Therefore, changes of its pattern in any cell can cause important alterations affecting its function. The methylation pattern of imprinted genes has been reported to be altered by numerous environmental factors such as nutrition, diseases, and drugs. Sexing by flow cytometry exposes the sperm cells to various procedures that can affect sperm quality and could induce methylation changes, resulting in lower fertilization when compared with conventional sperm. Although many studies have related changes in methylation pattern to in vitro culture of oocytes and embryos, no reports have evaluated these changes in sperm caused by the sexing process. The IGF2 imprinted gene, which is predominantly expressed by the paternal allele with the maternal one silenced, is expected to have its intragenic differentially methylated region (DMR) highly methylated in the sperm. Therefore, it became an excellent candidate region to be used for evaluating changes in methylation status of the sperm. The objective of this study was to evaluate the influence of sexing by flow cytometry on the methylation pattern of the DMR located in exon 10 of the IGF2 gene. Frozen–thawed unsorted and sex-sorted sperm samples from 4 Nellore bulls were used (5–10 years old). Each ejaculate was separated into 3 fractions: nonsexed (NS), sexed for X-sperm (SX), and sexed for Y-sperm (SY). Then, 1 straw of each treatment/bull was thawed, placed in 40:70 Percoll gradient (GE Bioscience®, Uppsala, Sweden) and centrifuged at 700 × g for 45 min to separate the somatic cells from the sperm. Sperm pellets were then used for DNA extraction. After that, DNA was treated with sodium bisulfite using the EZ DNA methylation kit (Zymo Research®, Orange, CA, USA). Bisulfite-treated DNA was amplified in 2-round PCR strategy (nested-PCR) and the amplicons were purified using GenClean III kit (MP Biomedical®, Solon, OH, USA). The purified amplicons were cloned into the pGEM-T easy vector system (Promega®, Madison, WI, USA) and transformed into Escherichia coli cells (XL-1 Blue). The resulting individual clones were sequenced, using a dideoxy fluorescence terminator system (ABI 3130xl, Applied Biosystems, Foster City, CA, USA). The experiment was performed in 3 replicates per bull. Sequences were analysed using the BiQ Analyzer software with a GenBank sequence (X53553) as a reference. At least 60 clones were sequenced per group. The methylation status of the 28 CpG sites was compared among the groups using analysis of variance and Tukey test in the Prophet software. No differences in DNA methylation were found between NS (97.5 ± 1.7%), SX (95.8 ± 1.8%), and SY (97.3 ± 0.2%) (P = 0.36). Moreover, 100% of analysed sequences in all groups were hypermethylated. Therefore, we can conclude that the process of sexing by flow cytometry does not change the methylation pattern in the intragenic DMR located in exon 10 of the IGF2 gene. Financial support: FAPESP and Embrapa.

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DNA Methylation Is Linked to Deacetylation of Histone H3, but Not H4, on the Imprinted Genes Snrpnand U2af1-rs1

Molecular and Cellular Biology ◽

10.1128/mcb.21.16.5426-5436.2001 ◽

2001 ◽

Vol 21 (16) ◽

pp. 5426-5436 ◽

Cited By ~ 92

Author(s):

Richard I. Gregory ◽

Tamzin E. Randall ◽

Colin A. Johnson ◽

Sanjeev Khosla ◽

Izuho Hatada ◽

...

Keyword(s):

Dna Methylation ◽

Es Cells ◽

Histone H3 ◽

Embryonic Stem ◽

Histone Deacetylation ◽

Differentially Methylated Region ◽

Paternal Allele ◽

Maternal Allele ◽

Control Center ◽

Mbd Protein

ABSTRACT The relationship between DNA methylation and histone acetylation at the imprinted mouse genes U2af1-rs1 and Snrpnis explored by chromatin immunoprecipitation (ChIP) and resolution of parental alleles using single-strand conformational polymorphisms. TheU2af1-rs1 gene lies within a differentially methylated region (DMR), while Snrpn has a 5′ DMR (DMR1) with sequences homologous to the imprinting control center of the Prader-Willi/Angelman region. For both DMR1 of Snrpn and the 5′ untranslated region (5′-UTR) and 3′-UTR ofU2af1-rs1, the methylated and nonexpressed maternal allele was underacetylated, relative to the paternal allele, at all H3 lysines tested (K14, K9, and K18). For H4, underacetylation of the maternal allele was exclusively (U2af1-rs1) or predominantly (Snrpn) at lysine 5. Essentially the same patterns of differential acetylation were found in embryonic stem (ES) cells, embryo fibroblasts, and adult liver from F1 mice and in ES cells from mice that were dipaternal or dimaternal for U2af1-rs1. In contrast, in a region within Snrpn that has biallelic methylation in the cells and tissues analyzed, the paternal (expressed) allele showed relatively increased acetylation of H4 but not of H3. The methyl-CpG-binding-domain (MBD) protein MeCP2 was found, by ChIP, to be associated exclusively with the maternal U2af1-rs1 allele. To ask whether DNA methylation is associated with histone deacetylation, we produced mice with transgene-induced methylation at the paternal allele of U2af1-rs1. In these mice, H3 was underacetylated across both the parental U2af1-rs1 alleles whereas H4 acetylation was unaltered. Collectively, these data are consistent with the hypothesis that CpG methylation leads to deacetylation of histone H3, but not H4, through a process that involves selective binding of MBD proteins.

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Faculty Opinions recommendation of Auto-assembly of E. coli DsrA small noncoding RNA: Molecular characteristics and functional consequences.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718145208.793485386 ◽

2013 ◽

Author(s):

Luc Jaeger

Keyword(s):

Noncoding Rna ◽

Molecular Characteristics ◽

Small Noncoding Rna ◽

E Coli ◽

Functional Consequences

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COT-04 Circulating biomarker for glioblastoma and primary central nervous system lymphoma -Next Generation Sequencing of small noncoding RNA-

Neuro-Oncology Advances ◽

10.1093/noajnl/vdaa143.093 ◽

2020 ◽

Vol 2 (Supplement_3) ◽

pp. ii21-ii21

Author(s):

Shumpei Onishi ◽

Fumiyuki Yamasaki ◽

Motoki Takano ◽

Ushio Yonezawa ◽

Kazuhiko Sugiyama ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Noncoding Rna ◽

Central Nervous System Lymphoma ◽

Serum Samples ◽

Small Noncoding Rna ◽

Non Invasive ◽

Diagnostic Models ◽

Healthy Control ◽

Generation Sequencing

Abstract Objective: Glioblastoma (GBM) and Primary Central Nervous System Lymphoma (PCNSL) are common intracranial malignant tumors. They sometimes present similar radiological findings and diagnoses could be difficult without surgical biopsy. For improving the current management, development of non-invasive biomarkers are desired. In this study, we explored the differently expressed circulating small noncoding RNA (sncRNA) in serum for specific diagnostic tool of GBM and PCNSL. Material & Methods: Serum samples were obtained from three groups: 1) GBM patients (N=26), 2) PCNSL patients (N=14) 3) healthy control (N=114). The total small RNAs were extracted from serum. The whole expression profiles of serum sncRNAs were measured using Next-Generation Sequencing System. We analyzed serum levels of sncRNAs (15–55 nt) in each serum samples. The difference of sncRNAs expression profile among three groups were compared. Data analysis was performed by logistic regression analysis followed by leave-one-out cross-validation (LOOCV). The accuracy of diagnostic models of sncRNAs combination were evaluated by receiver operating characteristic (ROC) analysis. Results: We created the combination models using three sncRNA in each models based on the logistic regression analysis. The model 1 (based on sncRNA-X1, X2 and X3) enabled to differentiate GBM patients form healthy control with a sensitivity of 92.3% and a specificity of 99.2% (AUC: 0.993). The model 2 (based on sncRNA-Y1, Y2 and Y3) enabled to differentiate PCNSL patients form healthy control with a sensitivity of 100% and a specificity of 93.9% (AUC: 0.984). The model 3 (based on sncRNA-Z1, Z2 and Z3) enabled to differentiate GBM patients form PCNSL patients with a sensitivity of 92.3% and a specificity of 78.6% (AUC: 0.920). Conclusion: We found three diagnostic models of serum sncRNAs as non-invasive biomarkers potentially useful for detection of GBM and PCNSL from healthy control, and for differentiation GBM from PCNSL.

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Natural Antisense Transcript PEBP1P3 Regulates the RNA Expression, DNA Methylation and Histone Modification of CD45 Gene

Genes ◽

10.3390/genes12050759 ◽

2021 ◽

Vol 12 (5) ◽

pp. 759

Author(s):

Zhongjing Su ◽

Guangyu Liu ◽

Bin Zhang ◽

Ze Lin ◽

Dongyang Huang

Keyword(s):

Dna Methylation ◽

Alternative Splicing ◽

Histone Modification ◽

Antisense Rna ◽

Noncoding Rna ◽

Antisense Transcript ◽

Natural Antisense Transcript ◽

Regulation Of Expression ◽

Splicing Isoforms ◽

Intron 2

The leukocyte common antigen CD45 is a transmembrane phosphatase expressed on all nucleated hemopoietic cells, and the expression levels of its splicing isoforms are closely related to the development and function of lymphocytes. PEBP1P3 is a natural antisense transcript from the opposite strand of CD45 intron 2 and is predicted to be a noncoding RNA. The genotype-tissue expression and quantitative PCR data suggested that PEBP1P3 might be involved in the regulation of expression of CD45 splicing isoforms. To explore the regulatory mechanism of PEBP1P3 in CD45 expression, DNA methylation and histone modification were detected by bisulfate sequencing PCR and chromatin immunoprecipitation assays, respectively. The results showed that after the antisense RNA PEBP1P3 was knocked down by RNA interference, the DNA methylation of CD45 intron 2 was decreased and histone H3K9 and H3K36 trimethylation at the alternative splicing exons of CD45 DNA was increased. Knockdown of PEBP1P3 also increased the binding levels of chromatin conformation organizer CTCF at intron 2 and the alternative splicing exons of CD45. The present results indicate that the natural antisense RNA PEBP1P3 regulated the alternative splicing of CD45 RNA, and that might be correlated with the regulation of histone modification and DNA methylation.

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Sequence Variations of Epstein-Barr Virus-Encoded Small Noncoding RNA and Latent Membrane Protein 1 in Hematologic Tumors in Northern China

Intervirology ◽

10.1159/000510398 ◽

2021 ◽

Vol 64 (2) ◽

pp. 69-80

Author(s):

Hai-Yu Wang ◽

Lingling Sun ◽

Ping Li ◽

Wen Liu ◽

Zhong-Guang Zhang ◽

...

Keyword(s):

Membrane Protein ◽

Nested Pcr ◽

Noncoding Rna ◽

Epstein Barr Virus ◽

Northern China ◽

Latent Membrane Protein ◽

Latent Membrane Protein 1 ◽

Small Noncoding Rna ◽

Barr Virus ◽

Epstein Barr

Objective: To investigate the relationship between hematologic tumors and Epstein-Barr virus (EBV)-encoded small noncoding RNA (EBER) variations as well as latent membrane protein 1 (LMP1) variations. Methods: Patients with leukemia and myelodysplastic syndrome (MDS) were selected as subjects. Genotypes 1/2 and genotypes F/f were analyzed using the nested PCR technology, while EBER and LMP1 subtypes were analyzed by the nested PCR and DNA sequencing. Results: Type 1 was more dominant than type 2, found in 59 out of 82 (72%) leukemia and in 31 out of 35 (88.6%) MDS, while type F was more prevalent than type f in leukemia (83/85, 97.6%) and MDS (29/31, 93.5%) samples. The distribution of EBV genotypes 1/2 was not significantly different among leukemia, MDS, and healthy donor groups, neither was that of EBV genotypes F/f. EB-6m prototype was the dominant subtype of EBER in leukemia and MDS (73.2% [30/41] and 83.3% [10/12], respectively). The frequency of EB-6m was lower than that of healthy people (96.7%, 89/92), and the difference was significant (p < 0.05). China 1 subtype was the dominant subtype of LMP1 in leukemia and MDS (70% [28/40] and 90% [9/10], respectively), and there was no significant difference in the distribution of LMP1 subtypes among the 3 groups (p > 0.05). Conclusion: The distribution of EBV 1/2, F/f, EBER, and LMP1 subtypes in leukemia and MDS was similar to that in the background population in Northern China, which means that these subtypes may be rather region-restricted but not associated with leukemia and MDS pathogenesis.

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Interaction between Host MicroRNAs and the Gut Microbiota in Colorectal Cancer

mSystems ◽

10.1128/msystems.00205-17 ◽

2018 ◽

Vol 3 (3) ◽

Cited By ~ 35

Author(s):

Ce Yuan ◽

Michael B. Burns ◽

Subbaya Subramanian ◽

Ran Blekhman

Keyword(s):

Gene Expression ◽

Colorectal Cancer ◽

Gut Microbiota ◽

Mirna Expression ◽

Gut Microbiome ◽

Noncoding Rna ◽

Microbial Community Composition ◽

Host Cells ◽

Microbiome Composition ◽

Small Noncoding Rna

ABSTRACT Although variation in gut microbiome composition has been linked with colorectal cancer (CRC), the factors that mediate the interactions between CRC tumors and the microbiome are poorly understood. MicroRNAs (miRNAs) are known to regulate CRC progression and are associated with patient survival outcomes. In addition, recent studies suggested that host miRNAs can also regulate bacterial growth and influence the composition of the gut microbiome. Here, we investigated the association between miRNA expression and microbiome composition in human CRC tumor and normal tissues. We identified 76 miRNAs as differentially expressed (DE) in tissue from CRC tumors and normal tissue, including the known oncogenic miRNAs miR-182, miR-503, and mir-17~92 cluster. These DE miRNAs were correlated with the relative abundances of several bacterial taxa, including Firmicutes , Bacteroidetes , and Proteobacteria . Bacteria correlated with DE miRNAs were enriched with distinct predicted metabolic categories. Additionally, we found that miRNAs that correlated with CRC-associated bacteria are predicted to regulate targets that are relevant for host-microbiome interactions and highlight a possible role for miRNA-driven glycan production in the recruitment of pathogenic microbial taxa. Our work characterized a global relationship between microbial community composition and miRNA expression in human CRC tissues. IMPORTANCE Recent studies have found an association between colorectal cancer (CRC) and the gut microbiota. One potential mechanism by which the microbiota can influence host physiology is through affecting gene expression in host cells. MicroRNAs (miRNAs) are small noncoding RNA molecules that can regulate gene expression and have important roles in cancer development. Here, we investigated the link between the gut microbiota and the expression of miRNA in CRC. We found that dozens of miRNAs are differentially regulated in CRC tumors and adjacent normal colon and that these miRNAs are correlated with the abundance of microbes in the tumor microenvironment. Moreover, we found that microbes that have been previously associated with CRC are correlated with miRNAs that regulate genes related to interactions with microbes. Notably, these miRNAs likely regulate glycan production, which is important for the recruitment of pathogenic microbial taxa to the tumor. This work provides a first systems-level map of the association between microbes and host miRNAs in the context of CRC and provides targets for further experimental validation and potential interventions.

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