Dysregulated gene expression of imprinted and X-linked genes: a link to poor development of bovine haploid androgenetic embryos

AbstractMammalian uniparental embryos are efficient models for genome imprinting research and allow studies on the contribution of the paternal and maternal genome to early embryonic development. In this study, we analyzed different methodologies for production of bovine haploid androgenetic embryos (hAE) to elucidate the causes behind their poor developmental potential. The results showed that hAE can be efficiently generated by using intracytoplasmic sperm injection and oocyte enucleation at telophase II. Although haploidy does not disturb early development up to around the 3rd mitotic division, androgenetic development is disturbed after the time of zygote genome activation those that reach the morula stage are less capable to become a blastocyst. Analysis of gene expression indicated abnormal levels of methyltransferase 3B and key long non-coding RNAs involved in X-chromosome inactivation and genomic imprinting of the KCNQ1 locus, which is associated to the methylation status of imprinted control regions of XIST and KCNQ1OT1. Thus, our results seem to exclude micromanipulation consequences and chromosomal abnormalities as major factors in developmental restriction, suggesting that their early developmental constraint is regulated at an epigenetic level.

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Dysregulated Gene Expression of Imprinted and X-Linked Genes: A Link to Poor Development of Bovine Haploid Androgenetic Embryos

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.640712 ◽

2021 ◽

Vol 9 ◽

Author(s):

Luis Aguila ◽

Joao Suzuki ◽

Amanda B. T. Hill ◽

Mónica García ◽

Karine de Mattos ◽

...

Keyword(s):

Gene Expression ◽

Genomic Imprinting ◽

X Chromosome ◽

De Novo ◽

Chromosomal Abnormalities ◽

Methylation Status ◽

Blastocyst Stage ◽

Developmental Potential ◽

Stage Of Development ◽

Androgenetic Embryos

Mammalian uniparental embryos are efficient models for genome imprinting research and allow studies on the contribution of the paternal and maternal genomes to early embryonic development. In this study, we analyzed different methods for production of bovine haploid androgenetic embryos (hAE) to elucidate the causes behind their poor developmental potential. Results indicate that hAE can be efficiently generated by using intracytoplasmic sperm injection and oocyte enucleation at telophase II. Although androgenetic haploidy does not disturb early development up to around the 8-cell stage, androgenetic development is disturbed after the time of zygote genome activation and hAE that reach the morula stage are less capable to reach the blastocyst stage of development. Karyotypic comparisons to parthenogenetic- and ICSI-derived embryos excluded chromosomal segregation errors as causes of the developmental constraints of hAE. However, analysis of gene expression indicated abnormal levels of transcripts for key long non-coding RNAs involved in X chromosome inactivation and genomic imprinting of the KCNQ1 locus, suggesting an association with X chromosome and some imprinted loci. Moreover, transcript levels of methyltransferase 3B were significantly downregulated, suggesting potential anomalies in hAE establishing de novo methylation. Finally, the methylation status of imprinted control regions for XIST and KCNQ1OT1 genes remained hypomethylated in hAE at the morula and blastocyst stages, confirming their origin from spermatozoa. Thus, our results exclude micromanipulation and chromosomal abnormalities as major factors disturbing the normal development of bovine haploid androgenotes. In addition, although the cause of the arrest remains unclear, we have shown that the inefficient development of haploid androgenetic bovine embryos to develop to the blastocyst stage is associated with abnormal expression of key factors involved in X chromosome activity and genomic imprinting.

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164 EPIGENETIC ANALYSIS OF GENOMIC DNA IN PREPUBERAL AND ADULT BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab164 ◽

2011 ◽

Vol 23 (1) ◽

pp. 184

Author(s):

M. Diederich ◽

J. Heinzmann ◽

W. Kues ◽

U. Baulain ◽

T. Haaf ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Methylation Status ◽

Control Group ◽

Specific Gene ◽

In Vitro Production ◽

Adult Cattle ◽

Developmental Potential ◽

Bovine Oocytes

The use of oocytes obtained from prepuberal cattle shortens the generation interval by producing descendants of genetically valuable animals before achieving actual cultivation maturity. However, several studies proved that oocytes derived from prepuberal animals differ significantly from oocytes of adult animals with regard to their developmental capability and therefore reproductive potential. Epigenetic events are taken into consideration as a possible reason for this phenomenon. Particularly DNA methylation, allele specific gene expression in a parent-of-origin-specific manner (imprinting), and certain histone modifications, like acetylations, carboxylations, and phosphorylations, play an important role. This project aims to gain knowledge about the mechanisms involved in attaining of the full developmental potential of bovine oocytes. Using immature and in vitro matured oocytes of prepuberal and adult cattle, a comparative study was conducted by measuring mRNA expression of 4 developmentally relevant, but non-imprinted genes (GDF9, GLUT1, PRDX1, and ZAR1) as well as the general DNA methylation status, performed by bisulfite sequencing of 2 satellite sequences [bovine testis satellite I DNA segment 2 (BTSS2) and Bos taurus α satellite I DNA (BTS)]. After various pretreatments, immature bovine oocytes were collected from prepuberal calves [6–9 months, either left untreated (Ca1) or treated with FSH (Ca2) or FSH+IGF1 (Ca3) or FSH+IGFK (Ca4)] and adult animals [≥2nd lactation, either left untreated (Ad1) or treated with FSH (Ad2)] using the Ovum-pick-up (OPU) technique. The Ad1 group was considered the control group. First results of the qPCR analyses of immature oocytes show differences between treatment groups for GLUT1, PRDX1, and ZAR1 transcripts. Compared with Ad1, GLUT1 expression increased in Ad2 [fold change (FC) 2.2], Ca1 (FC 2.0), Ca2 (FC 1.8), and Ca3 (FC 1.4). The genes PRDX1 and ZAR1 were reduced in all groups by 0.02 to 0.07 in comparison with Ad1. The GDF9 showed generally a very low expression. The methylation analysis shows for BTSS2 and BTS significant differences before and after in vitro maturation in the groups Ad1 (BTSS2: 49.6 v. 64.9%), Ad2 (BTS: 76.7 v. 52.5%), Ca1 (BTSS2: 74.6 v. 53.3%), Ca2 (BTS: 72.8 v. 57.8%) and Ca3 (BTSS2: 60.6 v. 71.7%). Currently, the first experiment and statistical analysis are under way. The preliminary data confirm differences in gene expression between prepuberal and adult animals, and demonstrates the dependence of the methylation pattern on age and maturation status. These results contribute to a better understanding of the developmental potential of prepuberal oocytes in order to optimize their use for in vitro production of embryos. This work was supported by the H. Wilhelm Schaumann Foundation, Hamburg.

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Epigenetic Profiling Identifies Novel Genes for Ascending Aortic Aneurysm Formation with Bicuspid Aortic Valves

The Heart Surgery Forum ◽

10.1532/hsf.1247 ◽

2015 ◽

Vol 18 (4) ◽

pp. 134 ◽

Cited By ~ 12

Author(s):

Asad A Shah

Keyword(s):

Gene Expression ◽

Aortic Aneurysm ◽

Methylation Status ◽

Aortic Aneurysms ◽

Aortic Valves ◽

Ascending Aortic Aneurysm ◽

Novel Genes ◽

Aneurysm Formation ◽

Aortic Replacement ◽

Bicuspid Aortic Valves

Background: Bicuspid aortic valves predispose to ascending aortic aneurysms, but the mechanisms underlying this aortopathy remain incompletely characterized. We sought to identify epigenetic pathways predisposing to aneurysm formation in bicuspid patients.Methods: Ascending aortic aneurysm tissue samples were collected at the time of aortic replacement in subjects with bicuspid and trileaflet aortic valves. Genome-wide DNA methylation status was determined on DNA from tissue using the Illumina 450K methylation chip, and gene expression was profiled on the same samples using Illumina Whole-Genome DASL arrays. Gene methylation and expression were compared between bicuspid and trileaflet individuals using an unadjusted Wilcoxon rank sum test. Results: Twenty-seven probes in 9 genes showed significant differential methylation and expression (P<5.5x10-4). The top gene was protein tyrosine phosphatase, non-receptor type 22 (PTPN22), which was hypermethylated (delta beta range: +15.4 to +16.0%) and underexpressed (log 2 gene expression intensity: bicuspid 5.1 vs. trileaflet 7.9, P=2x10-5) in bicuspid patients, as compared to tricuspid patients. Numerous genes involved in cardiovascular development were also differentially methylated, but not differentially expressed, including ACTA2 (4 probes, delta beta range: -10.0 to -22.9%), which when mutated causes the syndrome of familial thoracic aortic aneurysms and dissectionsConclusions: Using an integrated, unbiased genomic approach, we have identified novel genes associated with ascending aortic aneurysms in patients with bicuspid aortic valves, modulated through epigenetic mechanisms. The top gene was PTPN22, which is involved in T-cell receptor signaling and associated with various immune disorders. These differences highlight novel potential mechanisms of aneurysm development in the bicuspid population.

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Epigenetic Changes During Mechanically Induced Osteogenic Lineage Commitment

Journal of Biomechanical Engineering ◽

10.1115/1.4029551 ◽

2015 ◽

Vol 137 (2) ◽

Cited By ~ 14

Author(s):

Julia C. Chen ◽

Mardonn Chua ◽

Raymond B. Bellon ◽

Christopher R. Jacobs

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Shear Stress ◽

Fluid Shear Stress ◽

Methylation Status ◽

Lineage Commitment ◽

Fluid Shear ◽

Osteogenic Lineage ◽

Osteogenic Markers ◽

Heterochromatin Structure

Osteogenic lineage commitment is often evaluated by analyzing gene expression. However, many genes are transiently expressed during differentiation. The availability of genes for expression is influenced by epigenetic state, which affects the heterochromatin structure. DNA methylation, a form of epigenetic regulation, is stable and heritable. Therefore, analyzing methylation status may be less temporally dependent and more informative for evaluating lineage commitment. Here we analyzed the effect of mechanical stimulation on osteogenic differentiation by applying fluid shear stress for 24 hr to osteocytes and then applying the osteocyte-conditioned medium (CM) to progenitor cells. We analyzed gene expression and changes in DNA methylation after 24 hr of exposure to the CM using quantitative real-time polymerase chain reaction and bisulfite sequencing. With fluid shear stress stimulation, methylation decreased for both adipogenic and osteogenic markers, which typically increases availability of genes for expression. After only 24 hr of exposure to CM, we also observed increases in expression of later osteogenic markers that are typically observed to increase after seven days or more with biochemical induction. However, we observed a decrease or no change in early osteogenic markers and decreases in adipogenic gene expression. Treatment of a demethylating agent produced an increase in all genes. The results indicate that fluid shear stress stimulation rapidly promotes the availability of genes for expression, but also specifically increases gene expression of later osteogenic markers.

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LPS-induced expression of CD14 in the TRIF pathway is epigenetically regulated by sulforaphane in porcine pulmonary alveolar macrophages

Innate Immunity ◽

10.1177/1753425916669418 ◽

2016 ◽

Vol 22 (8) ◽

pp. 682-695 ◽

Cited By ~ 12

Author(s):

Qin Yang ◽

Maren J Pröll ◽

Dessie Salilew-Wondim ◽

Rui Zhang ◽

Dawit Tesfaye ◽

...

Keyword(s):

Gene Expression ◽

Alveolar Macrophages ◽

Methylation Status ◽

Gene Body ◽

Gene Body Methylation ◽

Tnf Α ◽

Pulmonary Alveolar Macrophages ◽

Cluster Of Differentiation ◽

Cd14 Gene

Pulmonary alveolar macrophages (AMs) are important in defense against bacterial lung inflammation. Cluster of differentiation 14 (CD14) is involved in recognizing bacterial lipopolysaccharide (LPS) through MyD88-dependent and TRIF pathways of innate immunity. Sulforaphane (SFN) shows anti-inflammatory activity and suppresses DNA methylation. To identify CD14 epigenetic changes by SFN in the LPS-induced TRIF pathway, an AMs model was investigated in vitro. CD14 gene expression was induced by 5 µg/ml LPS at the time point of 12 h and suppressed by 5 µM SFN. After 12 h of LPS stimulation, gene expression was significantly up-regulated, including TRIF, TRAF6, NF-κB, TRAF3, IRF7, TNF-α, IL-1β, IL-6, and IFN-β. LPS-induced TRAM, TRIF, RIPK1, TRAF3, TNF-α, IL-1β and IFN-β were suppressed by 5 µM SFN. Similarly, DNMT3a expression was increased by LPS but significantly down-regulated by 5 µM SFN. It showed positive correlation of CD14 gene body methylation with in LPS-stimulated AMs, and this methylation status was inhibited by SFN. This study suggests that SFN suppresses CD14 activation in bacterial inflammation through epigenetic regulation of CD14 gene body methylation associated with DNMT3a. The results provide insights into SFN-mediated epigenetic down-regulation of CD14 in LPS-induced TRIF pathway inflammation and may lead to new methods for controlling LPS-induced inflammation in pigs.

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The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7971-7976.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7971-7976

Author(s):

L M Whyatt ◽

A Düwel ◽

A G Smith ◽

P D Rathjen

Keyword(s):

Gene Expression ◽

Inner Cell Mass ◽

Es Cells ◽

Expression System ◽

Cell Mass ◽

Embryonic Stem ◽

Expression Vectors ◽

Developmental Control ◽

Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

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Abstract 40: Disturbed Flow Alters Genomewide DNA Methylation Patterns, Regulating Endothelial Gene Expression and Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.40 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Jessilyn Dunn ◽

Haiwei Qiu ◽

Soyeon Kim ◽

Daudi Jjingo ◽

Ryan Hoffman ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Dna Methyltransferase ◽

Methylation Status ◽

Western Diet ◽

Dependent Manner ◽

Gene Promoters ◽

Genome Wide ◽

Methylation Patterns ◽

Endothelial Gene Expression

Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow (d-flow), which alters gene expression, endothelial function, and atherosclerosis. Here, we show that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase (DNMT)-dependent manner. We found that d-flow induced expression of DNMT1, but not DNMT3a or DNMT3b, in mouse arterial endothelium in vivo and in cultured endothelial cells by oscillatory shear (OS) compared to unidirectional laminar shear in vitro. The DNMT inhibitor 5-Aza-2’deoxycytidine (5Aza) or DNMT1 siRNA significantly reduced OS-induced endothelial inflammation. Moreover, 5Aza reduced lesion formation in two atherosclerosis models using ApoE-/- mice (western diet for 3 months and the partial carotid ligation model with western diet for 3 weeks). To identify the 5Aza mechanisms, we conducted two genome-wide studies: reduced representation bisulfite sequencing (RRBS) and transcript microarray using endothelial-enriched gDNA and RNA, respectively, obtained from the partially-ligated left common carotid artery (LCA exposed to d-flow) and the right contralateral control (RCA exposed to s-flow) of mice treated with 5Aza or vehicle. D-flow induced DNA hypermethylation in 421 gene promoters, which was significantly prevented by 5Aza in 335 genes. Systems biological analyses using the RRBS and the transcriptome data revealed 11 mechanosensitive genes whose promoters were hypermethylated by d-flow but rescued by 5Aza treatment. Of those, five genes contain hypermethylated cAMP-response-elements in their promoters, including the transcription factors HoxA5 and Klf3. Their methylation status could serve as a mechanosensitive master switch in endothelial gene expression. Our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.

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Comparative gene expression profile and DNA methylation status in diabetic patients of Kazak and Han people

Medicine ◽

10.1097/md.0000000000011982 ◽

2018 ◽

Vol 97 (36) ◽

pp. e11982 ◽

Cited By ~ 1

Author(s):

Cuizhe Wang ◽

Xiaodan Ha ◽

Wei Li ◽

Peng Xu ◽

Zhiwei Zhang ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Gene Expression Profile ◽

Expression Profile ◽

Methylation Status ◽

Diabetic Patients

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Arginine Methyltransferases as Regulators of RNA-Binding Protein Activities in Pathogenic Kinetoplastids

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.692668 ◽

2021 ◽

Vol 8 ◽

Author(s):

Gustavo D. Campagnaro ◽

Edward Nay ◽

Michael J. Plevin ◽

Angela K. Cruz ◽

Pegine B. Walrad

Keyword(s):

Gene Expression ◽

Transcriptional Control ◽

Rna Binding ◽

Methylation Status ◽

Regulation Of Gene Expression ◽

Structural Features ◽

Arginine Methylation ◽

Diverse Range ◽

Host Interaction ◽

Intronless Genes

A large number of eukaryotic proteins are processed by single or combinatorial post-translational covalent modifications that may alter their activity, interactions and fate. The set of modifications of each protein may be considered a “regulatory code”. Among the PTMs, arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), can affect how a protein interacts with other macromolecules such as nucleic acids or other proteins. In fact, many RNA-binding (RBPs) proteins are targets of PRMTs. The methylation status of RBPs may affect the expression of their bound RNAs and impact a diverse range of physiological and pathological cellular processes. Unlike most eukaryotes, Kinetoplastids have overwhelmingly intronless genes that are arranged within polycistronic units from which mature mRNAs are generated by trans-splicing. Gene expression in these organisms is thus highly dependent on post-transcriptional control, and therefore on the action of RBPs. These genetic features make trypanosomatids excellent models for the study of post-transcriptional regulation of gene expression. The roles of PRMTs in controlling the activity of RBPs in pathogenic kinetoplastids have now been studied for close to 2 decades with important advances achieved in recent years. These include the finding that about 10% of the Trypanosoma brucei proteome carries arginine methylation and that arginine methylation controls Leishmania:host interaction. Herein, we review how trypanosomatid PRMTs regulate the activity of RBPs, including by modulating interactions with RNA and/or protein complex formation, and discuss how this impacts cellular and biological processes. We further highlight unique structural features of trypanosomatid PRMTs and how it contributes to their singular functionality.

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Title Page Title: The role of DNA methylation in the accumulation of iridoid glycosides in Rehmannia glutinosa

10.21203/rs.3.rs-403607/v1 ◽

2021 ◽

Author(s):

Tianyu Dong ◽

Xiaoyan Wei ◽

Qianting Qi ◽

Peilei Chen ◽

Yanqing Zhou ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Secondary Metabolites ◽

Iridoid Glycosides ◽

Methylation Status ◽

Plant Secondary Metabolites ◽

Dna Demethylation ◽

Terpene Biosynthesis ◽

The Relationship

Abstract Background: Epigenetic regulation plays a significant role in the accumulation of plant secondary metabolites. The terpenoids are the most abundant in the secondary metabolites of plants, iridoid glycosides belong to monoterpenoids which is one of the main medicinal components of R.glutinosa. At present, study on iridoid glycosides mainly focuses on its pharmacology, accumulation and distribution, while the mechanism of its biosynthesis and the relationship between DNA methylation and plant terpene biosynthesis are seldom reports. Results: The research showed that the expression of DXS, DXR, 10HGO, G10H, GPPS and accumulation of iridoid glycosides increased at first and then decreased with the maturity of R.glutinosa, and under different concentrations of 5-azaC, the expression of DXS, DXR, 10HGO, G10H, GPPS and the accumulation of total iridoid glycosides were promoted, the promotion effect of low concentration (15μM-50μM) was more significant, the content of genomic DNA 5mC decreased significantly, the DNA methylation status of R.glutinosa genomes was also changed. DNA demethylation promoted gene expression and increased the accumulation of iridoid glycosides, but excessive demethylation inhibited gene expression and decreased the accumulation of iridoid glycosides. Conclusion: The analysis of DNA methylation, gene expression, and accumulation of iridoid glycoside provides insights into accumulation of terpenoids in R.glutinosa and lays a foundation for future studies on the effects of epigenetics on the synthesis of secondary metabolites.

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