scholarly journals Analysis of gene expression and DNA methylation in a cognitive phenotype model of schizophrenia

2021 ◽  
Author(s):  
◽  
Saskia Rutherford-Ymker
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Candidate Genes ◽  
Cognitive Deficits ◽  
Whole Genome ◽  
Cognitive Symptoms ◽  
Saline Condition ◽  
Maternal Immune Activation ◽  
Cognitive Phenotype ◽  
Phenotype Model

<p>The underpinning hypothesis of this study is that the environmental insults implicated in schizophrenia cause epigenetic changes that trigger deleterious gene expression, resulting in deviations from normal neurodevelopment. The behavioural abnormalities in schizophrenia can be grouped into the three common classes of symptoms: positive, negative, and cognitive. Cognitive symptoms are symptoms that impair cognitive processing and have detrimental effects on individuals with schizophrenia. Maternal immune activation refers to a rat model that stimulates a maternal immune system with an infection or infectious-like stimulus resulting in adverse phenotypes. A cognitive phenotype, maternal immune activation (MIA) model of schizophrenia was employed to use epigenetic markers to discover what deleterious genes drive the cognitive deficits phenotype.  Previous work has discerned many changes in gene expression that are implicated in schizophrenia. A hypothesis-driven approach was utilized to determine whether previously studied candidate genes are relevant in the cognitive symptoms of schizophrenia in this cognitive-phenotype model. It was found that prenatal treatment of lipopolysaccharide (LPS) (which is the major outer membrane component of gram-negative bacteria and mimics bacterial infection) on prenatal day 10 and 11 led to changes in mRNA levels in the prefrontal cortex of adolescent rats. Typically, an increase in the amount of transcript in the LPS condition compared to the saline condition, or a greater variability in the amount of transcript between replicates in the LPS condition than the saline condition, was observed. Statistical analysis revealed that these changes did not met statistical significance.  To build towards a whole genome DNA methylation analysis, two discrete approaches were used. The first utilized bisulfite modification and investigated changes in candidate genes as a precursor to genome-wide BS-sequencing. DNA methylation was measured across CpG rich regions and an absence of DNA methylation was detected in these regions in both the LPS and saline conditions in the candidate genes.   The second approach utilized a long-read sequencing platform to establish the feasibility of a bisulfite conversion-free method for whole-genome DNA methylation approach within our lab. Through the establishment of this method factors that affect the reliability, quality, and accuracy of the final sequencing product were explored. Many of which were in the downstream-from-sequencing, data analysis component of the process. Discoveries were also made regarding how much data would be needed to make direct DNA methylation detection feasible.   The data presented here demonstrated that the cognitive-phenotype MIA model had altered gene expression correlating with previously measured behavioural cognitive deficits in the prefrontal cortex in genes that were known to be associated with schizophrenia. To extend this further, a whole genome approach would be needed to discover novel drivers of the phenotype. In the current study, headway was made towards the development and establishment of a whole genome DNA methylation detection method to further this continued aim.</p>

scholarly journals Analysis of gene expression and DNA methylation in a cognitive phenotype model of schizophrenia

2021 ◽  
Author(s):  
◽  
Saskia Rutherford-Ymker
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Candidate Genes ◽  
Cognitive Deficits ◽  
Whole Genome ◽  
Cognitive Symptoms ◽  
Saline Condition ◽  
Maternal Immune Activation ◽  
Cognitive Phenotype ◽  
Phenotype Model

<p>The underpinning hypothesis of this study is that the environmental insults implicated in schizophrenia cause epigenetic changes that trigger deleterious gene expression, resulting in deviations from normal neurodevelopment. The behavioural abnormalities in schizophrenia can be grouped into the three common classes of symptoms: positive, negative, and cognitive. Cognitive symptoms are symptoms that impair cognitive processing and have detrimental effects on individuals with schizophrenia. Maternal immune activation refers to a rat model that stimulates a maternal immune system with an infection or infectious-like stimulus resulting in adverse phenotypes. A cognitive phenotype, maternal immune activation (MIA) model of schizophrenia was employed to use epigenetic markers to discover what deleterious genes drive the cognitive deficits phenotype.  Previous work has discerned many changes in gene expression that are implicated in schizophrenia. A hypothesis-driven approach was utilized to determine whether previously studied candidate genes are relevant in the cognitive symptoms of schizophrenia in this cognitive-phenotype model. It was found that prenatal treatment of lipopolysaccharide (LPS) (which is the major outer membrane component of gram-negative bacteria and mimics bacterial infection) on prenatal day 10 and 11 led to changes in mRNA levels in the prefrontal cortex of adolescent rats. Typically, an increase in the amount of transcript in the LPS condition compared to the saline condition, or a greater variability in the amount of transcript between replicates in the LPS condition than the saline condition, was observed. Statistical analysis revealed that these changes did not met statistical significance.  To build towards a whole genome DNA methylation analysis, two discrete approaches were used. The first utilized bisulfite modification and investigated changes in candidate genes as a precursor to genome-wide BS-sequencing. DNA methylation was measured across CpG rich regions and an absence of DNA methylation was detected in these regions in both the LPS and saline conditions in the candidate genes.   The second approach utilized a long-read sequencing platform to establish the feasibility of a bisulfite conversion-free method for whole-genome DNA methylation approach within our lab. Through the establishment of this method factors that affect the reliability, quality, and accuracy of the final sequencing product were explored. Many of which were in the downstream-from-sequencing, data analysis component of the process. Discoveries were also made regarding how much data would be needed to make direct DNA methylation detection feasible.   The data presented here demonstrated that the cognitive-phenotype MIA model had altered gene expression correlating with previously measured behavioural cognitive deficits in the prefrontal cortex in genes that were known to be associated with schizophrenia. To extend this further, a whole genome approach would be needed to discover novel drivers of the phenotype. In the current study, headway was made towards the development and establishment of a whole genome DNA methylation detection method to further this continued aim.</p>

Global Epigenomic Profiling Demonstrates That Myelodysplasia Is Characterized by a Distinct Epigenetic Signature with Aberrant DNA Methylation Changes Involving Various Malignant and Hematopoietic Pathways.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2436-2436
Author(s):  
L. Zhou ◽  
J. Opalinska ◽  
D. Sohal ◽  
R. Thompson ◽  
Y. Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Expression Analysis ◽  
Peripheral Blood ◽  
Gene Expression Analysis ◽  
Whole Genome ◽  
Gene Promoters ◽  
Global Methylation ◽  
Peripheral Blood Leucocyte ◽  
Peripheral Blood Leucocytes

Abstract Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. DNMT inhibitors such as azacytidine have led to clinical responses in patients, though the genes affected by epigenetic alterations are not well known. Whole genome DNA methylation was analyzed by a recently described novel method, The HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Peripheral blood leucocytes from 13 patients with MDS were compared to 9 age matched normal and anemic controls. Gene expression analysis was performed using 37K oligo maskless arrays on cDNA obtained from the same samples. Analysis showed that whole genome methylation profiling has greater discriminatory power in separating clusters of MDS samples from normal and anemic controls when compared to gene expression analysis. Unsupervised clustering based on epigenetic profiling demonstrated that only two cases of early MDS clustered with normals as compared to absolutely no separation between MDS and normals with clustering based on gene expression patterns. A high correlation (r=0.88–0.96) was observed between global methylation profiles of matched sets of bone marrow and peripheral blood leucocyte samples from selected patients demonstrating that peripheral blood leucocytes can be a valid surrogate for epigenomic analysis. Further analysis showed that genes consistently aberrantly methylated in MDS included Syk kinase, HOXB3, several histone acetyltranferases and others. Functional analysis by Ingenuity showed that cancer and cell signaling pathways were the most affected by epigenetic silencing. Most interestingly, a large proportion of gene promoters were also aberrantly hypomethylated. These included genes from Ras oncogene family, the CDC42 GTPase, various methyl binding proteins and other proteins mainly encoding for cancer and hematopoiesis functional pathways, thus biologically validating our analysis. Therefore, our data demonstrates that MDS is characterized by distinct epigenetic aberrations that are preserved in peripheral blood leucocytes. These can be the basis of future studies on pathogenesis and diagnosis for this disease and can potentially uncover a new set of therapeutic gene targets.

Uncovering the Epigenetic Pathomechanism in 13q14.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 487-487
Author(s):  
Daniel Mertens ◽  
Melanie Ruppel ◽  
Angela Philippen ◽  
Verena Fleig ◽  
Bianca Brakel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Tumor Suppressor ◽  
Candidate Genes ◽  
Critical Region ◽  
Replication Timing ◽  
Complete Loss ◽  
Gene Copy ◽  
Active Copy ◽  
Suppressor Mechanism

Abstract INTRODUCTION: Deletions in chromosomal band 13q14.3 distal to RB1 occur in a variety of human neoplasms like B-cell chronic lymphocytic leukaemia (CLL), indicating a tumor suppressor mechanism in this region. Intriguingly, several characteristics of the region of interest point to an epigenetic pathomechanism: candidate genes lack point mutations, yet these genes are downregulated in tumors, the presence of large non-coding RNA genes in 13q14.3 is reminiscent of imprinted regions where only one gene copy is active. The data we show here led us to propose a novel oncogenic mechanism where already in healthy tissue only one gene copy is active while one gene copy is randomly chosen for silencing. Loss of the single active copy is then sufficient for complete loss of gene function in tumor cells. Currently we are trying to identify the (epi-)genetic element that controls the whole locus. AIM: Identification of the epigenetic regulatory mechanism localized in 13q14.3. METHODS and RESULTS: We performed FISH analyses of hematopoietic and non-hematopoietic cell lines to assess replication timing and chromatin packaging of the critical region. In line with an imprinting mechanism, we find that the two copies of the critical region replicate asynchronously and/or show delayed chromatid segregation, suggesting differential chromatin packaging of the two copies of 13q14.3. Next, we found by sequencing of SNPs that 13q14.3 candidate genes are expressed from one copy only in healthy probands. However, expression originated from either the maternal or paternal copy, excluding an imprinting mechanism. We could also show a functional interconnection of DNA methylation and gene expression, as demethylating agents and histone hyperacetylation induced biallelic expression. However, replication timing was not affected. Currently we are employing array- and capillary electrophoresis-based analysis of DNA-methylation (aPRIMES and bioCOBRA) and chromatin-immunoprecipitation on arrayed CpG-libraries (chIP on chip) with antibodies specific for histone modifications in order to identify the epigenetic element regulating the critical region. CONCLUSIONS: We propose that differential replication timing represents an early epigenetic mark that distinguishes the two copies of 13q14.3, resulting in differential chromatin packaging and monoallelic expression. This has profound effects for the tumor suppressor mechanism localized in 13q14.3: Deletion of the single active copy of the region at 13q14.3, which is detected in more than 50% of CLL tumors, will suffice for complete loss of tumor suppressor function, as the remaining gene copies are epigentically silenced. In addition, we are currently identifying the locus control region that orchestrates gene expression in the critical region. Thus, we provide a model for the pathomechanism of 13q14.3 in CLL by the interaction of genetic lesions and epigenetic silencing.

Genetic Profiling of Orbital Fibroblasts from Patients with Graves’ Orbitopathy

2021 ◽  
Author(s):  
Giovanna Rotondo Dottore ◽  
Ilaria Bucci ◽  
Giulia Lanzolla ◽  
Iacopo Dallan ◽  
Angela Sframeli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Cell Proliferation ◽  
Differential Gene Expression ◽  
Global Dna Methylation ◽  
Whole Genome ◽  
Graves Orbitopathy ◽  
Differential Gene ◽  
And Control ◽  
Orbital Fibroblasts

Abstract Context Graves’ orbitopathy (GO) is an autoimmune disease that persists when immunosuppression is achieved. Orbital fibroblasts from GO patients display peculiar phenotypes even if not exposed to autoimmunity, possibly reflecting genetic or epigenetic mechanisms, which we investigated here. Objective We aimed to explore potential genetic or epigenetic differences using primary cultures of orbital fibroblasts from GO and control patients. Methods Cell proliferation, hyaluronic acid (HA) secretion, and HA synthases (HAS) were measured. Next-generation sequencing and gene expression analysis of the whole genome were performed, as well as real-time-PCR of selected genes and global DNA methylation assay on orbital fibroblasts from 6 patients with GO and 6 control patients from a referral center. Results Cell proliferation was higher in GO than in control fibroblasts. Likewise, HA in the cell medium was higher in GO fibroblasts. HAS-1 and HAS-2 did not differ between GO and control fibroblasts, whereas HAS-3 was more expressed in GO fibroblasts. No relevant gene variants were detected by whole-genome sequencing. However, 58 genes were found to be differentially expressed in GO compared with control fibroblasts, and RT-PCR confirmed the findings in 10 selected genes. We postulated that the differential gene expression was related to an epigenetic mechanism, reflecting diverse DNA methylation, which we therefore measured. In support of our hypothesis, global DNA methylation was significantly higher in GO fibroblasts. Conclusions We propose that, following an autoimmune insult, DNA methylation elicits differential gene expression and sustains the maintenance of GO.

High Resolution Epigenomic Profiling of Loss of Heterozygosity in MDS Reveals an Important Role of DNA Methylation in Regulating Expression of Genes in the Deleted Regions of Chromosomes 5q, 7q and 20q.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2120-2120
Author(s):  
D. Sohal ◽  
J. Opalinska ◽  
L. Zhou ◽  
P. Pahanish ◽  
E. Friedman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Loss Of Heterozygosity ◽  
Comparative Genomic ◽  
Type I ◽  
Whole Genome ◽  
Gene Promoters ◽  
Methylation Analysis ◽  
Expression Of Genes ◽  
Lower Expression

Abstract Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. MDS is characterized by large mono-allelic deletions that lead to loss of heterozygosity. It is unclear how the loss of one allele affects the expression of genes on the remaining allele and the role epigenetic silencing plays in this process. To study this directly, we performed whole genome methylation analysis on 3 selected MDS cases with deletions of chromosomes 5q, 7q and 20q. DNA methylation was analyzed by a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction enzyme digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter CpG island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of genes by calculating HpaII/MspI cut fragment intensity ratio. Array Comparative Genomic Hybridization (aCGH) was used to map the exact breakpoints in peripheral blood leucocytes obtained from these 3 MDS cases. Gene expression analysis of the cDNA from the same cells was performed using 37K oligo maskless arrays and integrated with methylation analysis. Promoter methylation status and gene expression of corresponding genes of six age-matched healthy and anemic controls were used as controls. Our results showed that the 5q14.2–31.3 deletion encoded for a total of 248 genes. On comparison with healthy controls, 73 gene promoters were hypomethylated and 143 were methylated. Promoters with lower methylation levels were associated with a greater proportion of genes with higher expression values, thus suggesting that epigenetic modification is a regulator of their transcription. On the other hand, hypermethylation of the remaining allele led to underexpression of important genes such as APC, Cyclin H, CD14 antigen, protein phosphatase 2 (PPP2CA), heat shock 70kDa protein 4, H2A histone family members and others. Similarly, the 7q11.2–36.3 deletion encoded for a total of 497 genes. Out of these, 130 promoters were methylated and resulted in lower expression of genes not previously implicated in MDS such as Alpha 2 Type I Collagen, Schwachman-Bodian-Diamond syndrome protein, MEF3 and others. Lastly, the 20q11.21–13.13 segment encoded for 173 genes out of which 67 were methylated when compared to controls. In these large deleted regions, only 8–25% of the genes had lower expression when compared to controls; suggesting that large segmental mono-allelic deletions themselves do not result in decreased gene expression. Most importantly, epigenetic modifications of the remaining allele seems to be a very important regulator of gene expression in these cases and can be a potential target for therapeutic strategies.

scholarly journals Epigenomic Regulatory Mechanism of Flowering in Apple Demonstrated in Two Varieties with Contrasted Flowering Behaviors

2021 ◽  
Author(s):  
Hua Zhou ◽  
Chenguang Zhang ◽  
Mengxue Wang ◽  
Wei Zhang ◽  
Juanjuan Ma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Cytosine Methylation ◽  
Floral Induction ◽  
Expression Patterns ◽  
Axillary Bud ◽  
Necessary Condition ◽  
Whole Genome ◽  
Transcriptional Responses ◽  
Flowering Genes

Abstract Background: Flowering is the necessary condition and yield basis for woody fruits in their life cycle. Although there has been considerable interest in the regulatory mechanisms underlying floral induction and flowering, the associated epigenetic modifications remain relatively uncharacterized. Results: We identified the genome-wide of DNA methylation changes and the transcriptional responses in axillary bud of ‘Qinguan’ (QA) and ‘Fuji’ (FA) varieties with contrasted flowering behaviors. The DNA methylations were19.35%, 62.96% and 17.68% for FA, and 19.64%, 62.49% and 17.86% for QA in the CG, CHG and CHH contexts, respectively. Number of hypermethylated or hypomethylated DMRs in different regions were contributed to significantly up/downregulated gene expression. DNA methylation can positively or negatively regulate gene expression based on the CG, CHG and CHH contexts in different regions. Additionally, the huge differences in transcription of MIKCc-Type MADS-box genes, and multiple flowering genes in multiple flowering pathways (i.e., light, age, GA and sugar) by changing DNA methylation, contributed to contrasted flowering behaviors in both QA and FA. Specifically, the floral meristem identify genes (i.e., FT, LEAFY, AP1 and SOC1) were significantly higher expression in QA than FA, but the floral repressor (i.e., SVP, AGL15, and AGL18) had an opposite result. Significant differences in multiple hormone levels were due to DEGs and their DMRs in their synthesis pathways, leading to both contrasted axillary bud outgrowth and flowering behaviors. Conclusions: The whole-genome bisulfite sequencing (BS) libraries of QA and FA with diverse flowering capabilities have been constructed for finding whole-genome cytosine methylation profiles. The RNA sequencing of QA and FA and diverse flowering capabilities have been combined together to identify the gene expression patterns and the correlation with their methylation states so that we can better understand the epigenetic regulation mechanisms of floral induction and formation in apple.

scholarly journals DNA methylation affects pre-mRNA transcriptional initiation and processing in Arabidopsis

2021 ◽  
Author(s):  
Qiuhui Li ◽  
Shenjie Chen ◽  
Amy Wing-Sze Leung ◽  
Yaqin Liu ◽  
Yan Xin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Regulation Of Gene Expression ◽  
Mrna Processing ◽  
Intergenic Sequence ◽  
Whole Genome ◽  
Transcriptional Initiation ◽  
Whole Genome Bisulfite Sequencing ◽  
Genome Bisulfite Sequencing

Background: DNA methylation may regulate pre-mRNA transcriptional initiation and processing, thus affecting gene expression. Unlike animal cells, plants, especially Arabidopsis thaliana, have relatively low DNA methylation levels, limiting our ability to observe any correlation between DNA methylation and pre-mRNA processing using typical short-read sequencing. However, with newly developed long-read sequencing technologies, such as Oxford Nanopore Technology Direct RNA sequencing (ONT DRS), combined with whole-genome bisulfite sequencing, we were able to precisely analyze the relationship between DNA methylation and pre-mRNA transcriptional initiation and processing using DNA methylation-related mutants. Results: Using ONT DRS, we generated more than 2 million high-quality full-length long reads of native mRNA for each of the wild type Col-0 and mutants defective in DNA methylation, identifying a total of 117,474 isoforms. We found that low DNA methylation levels around splicing sites tended to prevent splicing events from occurring. The lengths of the poly(A) tail of mRNAs were positively correlated with DNA methylation. DNA methylation before transcription start sites or around transcription termination sites tended to result in gene-silencing or read-through events. Furthermore, using ONT DRS, we identified novel transcripts that we could not have otherwise, since transcripts with intron retention and fusion transcripts containing the uncut intergenic sequence tend not to be exported to the cytoplasm. Using the met1-3 mutant with activated constitutive heterochromatin regions, we confirmed the effects of DNA methylation on pre-mRNA processing. Conclusion: The combination of ONT DRS with whole-genome bisulfite sequencing was a powerful tool for studying the effects of DNA methylation on splicing site selection and pre-mRNA processing, and therefore regulation of gene expression.

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