Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces

Jiang, Minghong; Zhang, Yuhao; Jing, Fei; Chang, Xinxia; Fan, Weiwei; Qian, Xueqing; Zhang, Tianbao; Lu, Daru

doi:10.1038/labinvest.2009.132

Cited by 97 publications

(105 citation statements)

References 28 publications

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“…The relative peak percentage was calculated as described 8 using the peak height of 1 allele divided by the sum of the peak heights of both alleles. The relative genomic SNP peak height was compared with the same peak sequenced from full-length cDNA transcripts.…”

Section: Relative Allele Expressionmentioning

confidence: 99%

GATA2 haploinsufficiency caused by mutations in a conserved intronic element leads to MonoMAC syndrome

Hsu

Johnson

Falcone

et al. 2013

Blood

217

201

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Section: Relative Allele Expressionmentioning

confidence: 99%

GATA2 haploinsufficiency caused by mutations in a conserved intronic element leads to MonoMAC syndrome

Hsu

Johnson

Falcone

et al. 2013

Blood

217

201

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“…The methylation levels of CpG dinucleotides were determined by measuring the ratio of each of the cytosine peak heights to the sum of respective cytosine and thymine peak heights in automated DNA sequencing traces, according to a technique published by Jiang and colleagues. 32 …”

Section: Bisulfite Conversion and Sequence Analysismentioning

confidence: 99%

Interplay of Promoter Usage and Intragenic CpG Content: Impact on GFP Reporter Gene Expression

et al. 2015

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Successful therapeutic protein production in vitro and in vivo requires efficient and long-term transgene expression supported by optimized vector and transgene cis-regulatory sequence elements. This study provides a comparative analysis of CpG-rich, highly expressed, versus CpG-depleted, poorly expressed green fluorescent protein (GFP) reporter transgenes, transcribed by various promoters in two different cell systems. Long-term GFP expression from a defined locus in stable Chinese hamster ovary cells was clearly influenced by the combination of transgene CpG content and promoter usage, as shown by differential silencing effects on selection pressure removal among the cytomegalovirus (CMV) promoter and elongation factor (EF)-1α promoter. Whereas a high intragenic CpG content promoted local DNA methylation, CpG depletion rather accelerated transgene loss and increased the local chromatin density. On lentiviral transfer of various expression modules into epigenetically sensitive P19 embryonic pluripotent carcinoma cells, CMV promoter usage led to rapid gene silencing irrespective of the intragenic CpG content. In contrast, EF-1α promoter-controlled constructs showed delayed silencing activity and high-level transgene expression, in particular when the CpG-rich GFP reporter was used. Notably, GFP silencing in P19 cells could be prevented completely by the bidirectional, dual divergently transcribed A2UCOE (ubiquitously acting chromatin-opening element derived from the human HNRPA2B1-CBX3 locus) promoter. Because the level of GFP expression by the A2UCOE promoter was entirely unaffected by the intragenic CpG level, we suggest that A2UCOE can overcome chromatin compaction resulting from intragenic CpG depletion due to its ascribed chromatin-opening abilities. Our analyses provide insights into the interplay of the intragenic CpG content with promoter sequences and regulatory sequence elements, thus contributing toward the design of therapeutic transgene expression cassettes for future gene therapy applications.

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“…Then PCR products were sequenced using a DNA sequencer (ABI PRISM 3730, Foster City, CA, USA). Methylation level was measured according to our previously reported method (Jiang et al, 2008(Jiang et al, , 2009). …”

Section: Quantification Of Methylation Level Of Mir-146a Promotermentioning

confidence: 99%

Dysregulated expression of miR‐146a contributes to age‐related dysfunction of macrophages

Jiang¹,

Xiang²,

Wang

et al. 2011

Aging Cell

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SummaryAge-associated immune dysfunction, characterized by increased systemic levels of cytokines, manifests as an increased susceptibility to infections. Thus, understanding these negative regulators of the immune response has paved the way to delineating signaling pathways that impact immune senescence. In the present study, we found that miR-146a, which negatively regulated the expression of IL-1b and IL-6, was highly expressed in aged mice. However, there was a lack of response to the stimulation of lipopolysaccharide (LPS) and proinflammatory cytokines in macrophages of aged mice. As a result, the negative feedback regulation loop with miR-146a involving down-regulation of inflammation factors was interrupted in aged mice. Aberrant NF-jB binding to the miR-146a promoter was demonstrated to be associated with the abnormal expression of miR-146a in aged mice. The DNA methyltransferase inhibitor (5-aza-2-deoxycytidine) and the histone deacetylase inhibitor [trichostatin A (TSA)] both significantly up-regulated miR-146a transcriptional activation by altering the DNA-binding activity of NF-jB in macrophages isolated from aged mice, which suggests that DNA methylation and histone acetylation are involved in the suppression of age-dependent miR-146a expression. Additionally, high levels of histone deacetylase (HDACs) expressions contributed to the inhibition of miR-146a expression in LPS-stimulated macrophages from aged mice in vitro. While the suppression of HDACs activities by TSA could improve LPS-induced inflammatory responses owing to up-regulation of miR-146a expression in macrophages from aged mice. These data indicate that the dysregulated expression of miR-146a results in the age-associated dysfunction of macrophages, and miR-146a may be a good target for the treatment of age-related inflammatory diseases.

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Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces

Cited by 97 publications

References 28 publications

GATA2 haploinsufficiency caused by mutations in a conserved intronic element leads to MonoMAC syndrome

GATA2 haploinsufficiency caused by mutations in a conserved intronic element leads to MonoMAC syndrome

Interplay of Promoter Usage and Intragenic CpG Content: Impact on GFP Reporter Gene Expression

Dysregulated expression of miR‐146a contributes to age‐related dysfunction of macrophages

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