David Ibberson

Transient cyclical methylation of promoter DNA.

Methylation of CpG dinucleotides is generally associated with epigenetic silencing of transcription and is maintained through cellular division. Multiple CpG sequences are rare in mammalian genomes, but frequently occur at the transcriptional start site of active genes, with most clusters of CpGs being hypomethylated. We reported previously that the proximal region of the trefoil factor 1 (TFF1, also known as pS2) and oestrogen receptor α (ERα) promoters could be partially methylated by treatment with deacetylase inhibitors, suggesting the possibility of dynamic changes in DNA methylation. Here we show that cyclical methylation and demethylation of CpG dinucleotides, with a periodicity of around 100 min, is characteristic for five selected promoters, including the oestrogen (E2)-responsive pS2 gene, in human cells. When the pS2 gene is actively transcribed, DNA methylation occurs after the cyclical occupancy of ERα and RNA polymerase II (polII). Moreover, we report conditions that provoke methylation cycling of the pS2 promoter in cell lines in which pS2 expression is quiescent and the proximal promoter is methylated. This coincides with a low-level re-expression of ERα and of pS2 transcripts.

Direct and indirect effects of H-NS and Fis on global gene expression control in Escherichia coli

Nucleoid-associated proteins (NAPs) are global regulators of gene expression in Escherichia coli, which affect DNA conformation by bending, wrapping and bridging the DNA. Two of these—H-NS and Fis—bind to specific DNA sequences and structures. Because of their importance to global gene expression, the binding of these NAPs to the DNA was previously investigated on a genome-wide scale using ChIP-chip. However, variation in their binding profiles across the growth phase and the genome-scale nature of their impact on gene expression remain poorly understood. Here, we present a genome-scale investigation of H-NS and Fis binding to the E. coli chromosome using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq). By performing our experiments under multiple time-points during growth in rich media, we show that the binding regions of the two proteins are mutually exclusive under our experimental conditions. H-NS binds to significantly longer tracts of DNA than Fis, consistent with the linear spread of H-NS binding from high- to surrounding lower-affinity sites; the length of binding regions is associated with the degree of transcriptional repression imposed by H-NS. For Fis, a majority of binding events do not lead to differential expression of the proximal gene; however, it has a significant indirect effect on gene expression partly through its effects on the expression of other transcription factors. We propose that direct transcriptional regulation by Fis is associated with the interaction of tandem arrays of Fis molecules to the DNA and possible DNA bending, particularly at operon-upstream regions. Our study serves as a proof-of-principle for the use of ChIP-seq for global DNA-binding proteins in bacteria, which should become significantly more economical and feasible with the development of multiplexing techniques.

Multiple mechanisms induce transcriptional silencing of a subset of genes, including oestrogen receptor alpha, in response to deacetylase inhibition by valproic acid and trichostatin A.

Valproate (VPA) and trichostatin A (TSA), inhibitors of zinc-dependent deacetylase activity, induce reduction in the levels of mRNA encoding oestrogen receptor-α (ERα), resulting in subsequent clearance of ERα protein from breast and ovarian cell lines. Inhibition of oestrogen signalling may account for the endocrine disorders, menstrual abnormalities, osteoporosis and weight gain that occur in a proportion of women treated with VPA for epilepsy or for bipolar mood disorder. Transcriptome profiling revealed that VPA and TSA also modulate the expression of, among others, key regulatory components of the cell cycle. Meta-analysis of genes directly responsive to oestrogen indicates that VPA and TSA have a generally antioestrogenic profile in ERα positive cells. Concomitant treatment with cycloheximide prevented most of these changes in gene expression, including downregulation of ERα mRNA, indicating that a limited number of genes signal a hyperacetylated state within cells. Three members of the NAD-dependent deacetylases, the sirtuins, are upregulated by VPA and by TSA and sirtuin activity contributes to loss of ERα expression. However, prolonged inhibition of the sirtuins by sirtinol also induces loss of ERα from cells. Mechanistically, we show that VPA invokes reversible promoter shutoff of the ERα, pS2 and cyclin D1 promoters, by inducing recruitment of methyl cytosine binding protein 2 (MeCP2) with concomitant exclusion of the maintenance methylase DNMT1. Furthermore, we demonstrate that, in the presence of VPA, local DNA methylation, deacetylation and demethylation of activated histones and recruitment of inhibitory complexes occurs on the pS2 promoter.

RNA degradation compromises the reliability of microRNA expression profiling

BackgroundMicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression and their expression is frequently altered in human diseases, including cancer. To correlate clinically relevant parameters with microRNA expression, total RNA is frequently prepared from samples that were archived for various time periods in frozen tissue banks but, unfortunately, RNA integrity is not always preserved in these frozen tissues. Here, we investigate whether experimentally induced RNA degradation affects microRNA expression profiles.ResultsTissue samples were maintained on ice for defined time periods prior to total RNA extraction, which resulted in different degrees of RNA degradation. MicroRNA expression was then analyzed by microarray analysis (miCHIP) or microRNA-specific real-time quantitative PCR (miQPCR). Our results demonstrate that the loss of RNA integrity leads to in unpredictability of microRNA expression profiles for both, array-based and miQPCR assays.ConclusionMicroRNA expression cannot be reliably profiled in degraded total RNA. For the profiling of microRNAs we recommend use of RNA samples with a RNA integrity number equal to or above seven.

Sequencing of a patient with balanced chromosome abnormalities and neurodevelopmental disease identifies disruption of multiple high risk loci by structural variation.

Balanced chromosome abnormalities (BCAs) occur at a high frequency in healthy and diseased individuals, but cost-efficient strategies to identify BCAs and evaluate whether they contribute to a phenotype have not yet become widespread. Here we apply genome-wide mate-pair library sequencing to characterize structural variation in a patient with unclear neurodevelopmental disease (NDD) and complex de novo BCAs at the karyotype level. Nucleotide-level characterization of the clinically described BCA breakpoints revealed disruption of at least three NDD candidate genes (LINC00299, NUP205, PSMD14) that gave rise to abnormal mRNAs and could be assumed as disease-causing. However, unbiased genome-wide analysis of the sequencing data for cryptic structural variation was key to reveal an additional submicroscopic inversion that truncates the schizophrenia- and bipolar disorder-associated brain transcription factor ZNF804A as an equally likely NDD-driving gene. Deep sequencing of fluorescent-sorted wild-type and derivative chromosomes confirmed the clinically undetected BCA. Moreover, deep sequencing further validated a high accuracy of mate-pair library sequencing to detect structural variants larger than 10 kB, proposing that this approach is powerful for clinical-grade genome-wide structural variant detection. Our study supports previous evidence for a role of ZNF804A in NDD and highlights the need for a more comprehensive assessment of structural variation in karyotypically abnormal individuals and patients with neurocognitive disease to avoid diagnostic deception.

iDamIDseq and iDEAR: An improved method and computational pipeline to profile chromatin-binding proteins

DNA adenine methyltransferase identification (DamID) has emerged as an alternative method to profile protein-DNA interactions; however, critical issues limit its widespread applicability. Here, we present iDamIDseq, a protocol that improves specificity and sensitivity by inverting the steps DpnI-DpnII and adding steps that involve a phosphatase and exonuclease. To determine genome-wide protein-DNA interactions efficiently, we present the analysis tool iDEAR (iDamIDseq Enrichment Analysis with R). The combination of DamID and iDEAR permits the establishment of consistent profiles for transcription factors, even in transient assays, as we exemplify using the small teleost medaka (Oryzias latipes). We report that the bacterial Dam-coding sequence induces aberrant splicing when it is used with different promoters to drive tissue-specific expression. Here, we present an optimization of the sequence to avoid this problem. This and our other improvements will allow researchers to use DamID effectively in any organism, in a general or targeted manner. Summary: Critical improvements to the DamID protocol improve specificity and sensitivity in determining genome-wide protein-DNA interactions in transient or stable transgenic animal lines.

Differential gene expression indicates involvement of F-box proteins and E3 ligases in sexual versus apomictic germline specification in Boechera.

Germline specification is the first step during sexual and apomictic plant reproduction. This takes place in a specialized domain of the reproductive flower tissues, the nucellus of the ovule. In each case, a sporophytic cell is determined to initiate germline development. These cells, the megaspore mother cell (MMC) or apomictic initial cell (AIC) in sexual plants and apomicts, respectively, differ in their developmental fate. While the MMC undergoes meiosis, the AIC aborts or omits meiosis to form the female gametophyte. Although these distinct developmental processes have long been described, little is known about the gene regulatory basis involved. To elucidate gene regulatory networks underlying sexual and apomictic germline specification, we conducted tissue-specific transcriptional profiling using laser-assisted microdissection and RNA-Seq. We compared the transcriptomes of the nucellar tissues harbouring the MMC or AIC between different accessions of Boechera. The six accessions we used represented four species and two ploidy levels, allowing us to distinguish between differences in gene expression caused by the genetic background or the reproductive mode. Comparative data analysis revealed widely overlapping gene expression patterns in apomictic versus sexual Boechera accessions. Nevertheless, 45 significantly differentially expressed genes were identified, which potentially play a role for determination of sexual versus apomictic reproductive mode. Interestingly, based on annotations, these include F-box proteins and E3 ligases that might relate to genes previously described as regulators important for sexual or apomictic reproduction. Thus, our findings provide new insight into the transcriptional basis of sexual and apomictic germline specification. One sentence summary A comprehensive tissue type-specific transcriptional analysis using laser-assisted microdissection combined with RNA-Seq identifies 45 genes consistently differentially expressed during germline specification in different sexual versus apomictic Boechera accessions, indicating roles of protein degradation related to cell cycle, transcriptional and post-transcriptional regulatory processes, and stress response for apomixis.

iDamIDseq and iDEAR: An improved method and a computational pipeline to profile chromatin-binding proteins of developing organisms

DNA adenine methyltransferase identification (DamID) has emerged as an alternative for profiling protein-DNA interactions, however critical issues in the method limit its applicability. Here we present iDamlDseq, a protocol that improves specificity and robustness making its use compatible with developing organisms. In addition, we present the analysis tool iDEAR (iDamlDseq Enrichment Analysis with R) to determine protein-DNA interactions genome wide. The combination of both allows establishing highly reliable transcription factor profiles, even in transient assays. For tissue specific expression we improved the Dam coding sequence to overcome predominant aberrant splicing of Dam fusions we discovered with the commonly used sequence.

Erratum: Cyclical DNA methylation of a transcriptionally active promoter

This corrects the article DOI: 10.1038/nature06544