Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines

Conditional expression of short hairpin RNAs (shRNAs) to knock down target genes is a powerful tool to study gene function. The most common inducible expression systems are based on tetracycline-regulated RNA polymerase III promoters. During the last years, several tetracycline-inducible U6 and H1 promoter variants have been reported in different experimental settings showing variable efficiencies. In this study, we compare the most common variants of these promoters in several mammalian cell lines. For all cell lines tested, we find that several inducible U6 and H1 promoters containing single tetracycline operator (tetO) sequences show high-transcriptional background in the non-induced state. Promoter variants containing two tetO sequences show tight suppression of transcription in the non-induced state, and high tet responsiveness and high gene knockdown efficiency upon induction in all cell lines tested. We report a variant of the H1 promoter containing two O2-type tetO sequences flanking the TATA box that shows little transcriptional background in the non-induced state and up to 90% target knockdown when the inducer molecule (dox–doxycycline) is added. This inducible system for RNAi-based gene silencing is a good candidate for use both in basic research on gene function and for potential therapeutic applications

DNA Methylation and Gene Expression Changes in Monozygotic Twins Discordant for Psoriasis: Identification of Epigenetically Dysregulated Genes

Monozygotic (MZ) twins do not show complete concordance for many complex diseases; for example, discordance rates for autoimmune diseases are 20%–80%. MZ discordance indicates a role for epigenetic or environmental factors in disease. We used MZ twins discordant for psoriasis to search for genome-wide differences in DNA methylation and gene expression in CD4+ and CD8+ cells using Illumina's HumanMethylation27 and HT-12 expression assays, respectively. Analysis of these data revealed no differentially methylated or expressed genes between co-twins when analyzed separately, although we observed a substantial amount of small differences. However, combined analysis of DNA methylation and gene expression identified genes where differences in DNA methylation between unaffected and affected twins were correlated with differences in gene expression. Several of the top-ranked genes according to significance of the correlation in CD4+ cells are known to be associated with psoriasis. Further, gene ontology (GO) analysis revealed enrichment of biological processes associated with the immune response and clustering of genes in a biological pathway comprising cytokines and chemokines. These data suggest that DNA methylation is involved in an epigenetic dysregulation of biological pathways involved in the pathogenesis of psoriasis. This is the first study based on data from MZ twins discordant for psoriasis to detect epigenetic alterations that potentially contribute to development of the disease

Extensive variation and low heritability of DNA methylation identified in a twin study

Disturbance of DNA methylation leading to aberrant gene expression has been implicated in the etiology of many diseases. Whereas variation at the genetic level has been studied extensively, less is known about the extent and function of epigenetic variation. To explore variation and heritability of DNA methylation, we performed bisulfite sequencing of 1760 CpG sites in 186 regions in the human major histocompatibility complex (MHC) in CD4+ lymphocytes from 49 monozygotic (MZ) and 40 dizygotic (DZ) twin pairs. Individuals show extensive variation in DNA methylation both between and within regions. In addition, many regions also have a complex pattern of variation. Globally, there appears to be a bimodal distribution of DNA methylation in the regions, but a significant fraction of the CpG sites are also heterogeneously methylated. Classification of regions into CpG islands (intragenic and intergenic), 5′ end of genes not associated with a defined CpG island, conserved noncoding regions, and random CpG sites shows region-type differences in variation and heritability. Analyses revealed slightly lower intra-pair differences among MZ than among DZ pairs, suggesting some genetic influences on DNA methylation variation, with most of the variance attributed to nongenetic factors. Overall, heritability estimates of DNA methylation were low. Our heritability estimates are, however, somewhat deflated due to the presence of batch effects that artificially inflate the estimates of shared environment

Gene ontology results of significantly enriched GO terms identified in a combined analysis of DNA methylation and gene expression in CD4⁺ cells.

a<p>Number of genes in the given input gene list which are involved in a specific GO term.</p>b<p>Percentage of the genes in the given input gene list which are involved in a specific GO term.</p>c<p>Adjusted according to Benjamini and Hochberg.</p

Subset of genes with a correlated difference in DNA methylation and gene expression.

<p>This table consists of the top 50 genes ranked according to the significance of the correlation of differences in DNA methylation and gene expression between unaffected and affected MZ co-twins in CD4⁺ cells (genes known to be associated with psoriasis are shown in bold). The magnitude of the mean differences in DNA methylation and gene expression (unaffected versus affected) are presented as deltaBeta and log fold change, respectively.</p

Volcano plots of cell-type specific differences in DNA methylation and gene expression.

<p>(A) Differences in DNA methylation between CD4⁺ and CD8⁺ cells. Each point represents a CpG site, with mean β-value difference across 12 unaffected individuals along the <i>x</i>-axis and −log10 of a corrected <i>P</i>-value from a paired <i>t</i>-test along the <i>y</i>-axis. (B) Differences in gene expression between CD4⁺ and CD8⁺ cells. Each point represents a gene, with mean log2 fold change across 13 unaffected individuals along the <i>x</i>-axis and −log10 of a corrected <i>P</i>-value from a paired <i>t</i>-test along the <i>y</i>-axis. Dashed lines represent the FDR of 5%.</p

Volcano plots of differences in DNA methylation and gene expression in discordant MZ twins.

<p>(A–B) Differences in DNA methylation in CD4⁺ (<i>n</i> = 17 pairs) and CD8⁺ cells (<i>n</i> = 13 pairs), respectively. Each point represents a gene, with mean co-twin β-value difference along the <i>x</i>-axis and −log10 of the uncorrected <i>P</i>-value from a paired <i>t</i>-test along the <i>y</i>-axis. (C–D) Differences in gene expression in CD4⁺ cells (<i>n</i> = 17 pairs) and CD8⁺ cells (<i>n</i> = 14 pairs), respectively. Each point represents a gene, with mean log2 fold change along the <i>x</i>-axis and log odds along the <i>y</i>-axis.</p