Person:

Liu, Xiaole

Background: Transcription by RNA polymerase II is regulated at many steps including initiation, promoter release, elongation and termination. Accumulation of RNA polymerase II at particular locations across genes can be indicative of sites of regulation. RNA polymerase II is thought to accumulate at the promoter and at sites of co-transcriptional alternative splicing where the rate of RNA synthesis slows. Results: To further understand transcriptional regulation at a global level, we determined the distribution of RNA polymerase II within regions of the human genome designated by the ENCODE project. Hypophosphorylated RNA polymerase II localizes almost exclusively to 5' ends of genes. On the other hand, localization of total RNA polymerase II reveals a variety of distinct landscapes across many genes with 74% of the observed enriched locations at exons. RNA polymerase II accumulates at many annotated constitutively spliced exons, but is biased for alternatively spliced exons. Finally, RNA polymerase II is also observed at locations not in gene regions. Conclusion: Localizing RNA polymerase II across many millions of base pairs in the human genome identifies novel sites of transcription and provides insights into the regulation of transcription elongation. These data indicate that RNA polymerase II accumulates most often at exons during transcription. Thus, a major factor of transcription elongation control in mammalian cells is the coordination of transcription and pre-mRNA processing to define exons.

Histone modifications play important roles in regulating eukaryotic gene expression and have been used to model expression levels. Here, we present a regression model to systematically infer mRNA stability by comparing transcriptome profiles with ChIP-seq of H3K4me3, H3K27me3 and H3K36me3. The results from multiple human and mouse cell lines show that the inferred unstable mRNAs have significantly longer 3′Untranslated Regions (UTRs) and more microRNA binding sites within 3′UTR than the inferred stable mRNAs. Regression residuals derived from RNA-seq, but not from GRO-seq, are highly correlated with the half-lives measured by pulse-labeling experiments, supporting the rationale of our inference. Whereas, the functions enriched in the inferred stable and unstable mRNAs are consistent with those from pulse-labeling experiments, we found the unstable mRNAs have higher cell-type specificity under functional constraint. We conclude that the systematical use of histone modifications can differentiate non-expressed mRNAs from unstable mRNAs, and distinguish stable mRNAs from highly expressed ones. In summary, we represent the first computational model of mRNA stability inference that compares transcriptome and epigenome profiles, and provides an alternative strategy for directing experimental measurements.

We performed a systematic evaluation of how variations in sequencing depth and other parameters influence interpretation of Chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq) experiments. Using Drosophila S2 cells, we generated ChIP-seq datasets for a site-specific transcription factor (Suppressor of Hairy-wing) and a histone modification (H3K36me3). We detected a chromatin state bias, open chromatin regions yielded higher coverage, which led to false positives if not corrected and had a greater effect on detection specificity than any base-composition bias. Paired-end sequencing revealed that single-end data underestimated ChIP library complexity at high coverage. The removal of reads originating at the same base reduced false-positives while having little effect on detection sensitivity. Even at a depth of ~1 read/bp coverage of mappable genome, ~1% of the narrow peaks detected on a tiling array were missed by ChIP-seq. Evaluation of widely-used ChIP-seq analysis tools suggests that adjustments or algorithm improvements are required to handle datasets with deep coverage.

Motivation: With the rapid development of high-throughput sequencing technologies, the genome-wide profiling of nucleosome positioning has become increasingly affordable. Many future studies will investigate the dynamic behaviour of nucleosome positioning in cells that have different states or that are exposed to different conditions. However, a robust method to effectively identify the regions of differential nucleosome positioning (RDNPs) has not been previously available. Results:: We describe a novel computational approach, DiNuP, that compares nucleosome profiles generated by high-throughput sequencing under various conditions. DiNuP provides a statistical P-value for each identified RDNP based on the difference of read distributions. DiNuP also empirically estimates the false discovery rate as a cutoff when two samples have different sequencing depths and differentiate reliable RDNPs from the background noise. Evaluation of DiNuP showed it to be both sensitive and specific for the detection of changes in nucleosome location, occupancy and fuzziness. RDNPs that were identified using publicly available datasets revealed that nucleosome positioning dynamics are closely related to the epigenetic regulation of transcription. Availability and implementation: DiNuP is implemented in Python and is freely available at http://www.tongji.edu.cn/~zhanglab/DiNuP.

MicroRNAs (miRNAs) are a class of 20–23 nucleotide small RNAs that regulate gene expression post-transcriptionally in animals and plants. Annotation of miRNAs by the miRNA database (miRBase) has largely relied on computational approaches. As a result, many miRBase entries lack experimental validation, and discrepancies between miRBase annotation and actual miRNA sequences are often observed. In this study, we integrated the small RNA sequencing (smRNA-seq) datasets in Caenorhabditis elegans and Drosophila melanogaster and devised an analytical pipeline coupled with detailed manual inspection to curate miRNA annotation systematically in miRBase. Our analysis reveals 19 (17.0%) and 51 (31.3%) miRNAs entries with detectable smRNA-seq reads have mature sequence discrepancies in C. elegans and D. melanogaster, respectively. These discrepancies frequently occur either for conserved miRNA families whose mature sequences were predicted according to their homologous counterparts in other species or for miRNAs whose precursor miRNA (pre-miRNA) hairpins produce an abundance of multiple miRNA isoforms or variants. Our analysis shows that while Drosophila pre-miRNAs, on average, produce less than 60% accurate mature miRNA reads in addition to their 5′ and 3′ variant isoforms, the precision of miRNA processing in C. elegans is much higher, at over 90%. Based on the revised miRNA sequences, we analyzed expression patterns of the more conserved (MC) and less conserved (LC) miRNAs and found that, whereas MC miRNAs are often co-expressed at multiple developmental stages, LC miRNAs tend to be expressed specifically at fewer stages.

The androgen receptor (AR) is a key factor that regulates the behavior and fate of prostate cancer cells. The AR-regulated network is activated when AR binds enhancer elements and modulates specific enhancer–promoter looping. Kallikrein-related peptidase 3 (KLK3), which codes for prostate-specific antigen (PSA), is a well-known AR-regulated gene and its upstream enhancers produce bidirectional enhancer RNAs (eRNAs), termed KLK3e. Here, we demonstrate that KLK3e facilitates the spatial interaction of the KLK3 enhancer and the KLK2 promoter and enhances long-distance KLK2 transcriptional activation. KLK3e carries the core enhancer element derived from the androgen response element III (ARE III), which is required for the interaction of AR and Mediator 1 (Med1). Furthermore, we show that KLK3e processes RNA-dependent enhancer activity depending on the integrity of core enhancer elements. The transcription of KLK3e was detectable and its expression is significantly correlated with KLK3 ((R^2 = 0.6213, P < 5 × 10^{−11})) and KLK2 ((R^2 = 0.5893, P < 5 × 10^{−10})) in human prostate tissues. Interestingly, RNAi silencing of KLK3e resulted in a modest negative effect on prostate cancer cell proliferation. Accordingly, we report that an androgen-induced eRNA scaffolds the AR-associated protein complex that modulates chromosomal architecture and selectively enhances AR-dependent gene expression.

We propose a statistical algorithm MethylPurify that uses regions with bisulfite reads showing discordant methylation levels to infer tumor purity from tumor samples alone. MethylPurify can identify differentially methylated regions (DMRs) from individual tumor methylome samples, without genomic variation information or prior knowledge from other datasets. In simulations with mixed bisulfite reads from cancer and normal cell lines, MethylPurify correctly inferred tumor purity and identified over 96% of the DMRs. From patient data, MethylPurify gave satisfactory DMR calls from tumor methylome samples alone, and revealed potential missed DMRs by tumor to normal comparison due to tumor heterogeneity. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0419-x) contains supplementary material, which is available to authorized users.

Human neurons are functional over an entire lifetime, yet the mechanisms that preserve function and protect against neurodegeneration during ageing are unknown. Here we show that induction of the repressor element 1-silencing transcription factor (REST; also known as neuron-restrictive silencer factor, NRSF) is a universal feature of normal ageing in human cortical and hippocampal neurons. REST is lost, however, in mild cognitive impairment and Alzheimer’s disease. Chromatin immunoprecipitation with deep sequencing and expression analysis show that REST represses genes that promote cell death and Alzheimer’s disease pathology, and induces the expression of stress response genes. Moreover, REST potently protects neurons from oxidative stress and amyloid β-protein toxicity, and conditional deletion of REST in the mouse brain leads to age-related neurodegeneration. A functional orthologue of REST, Caenorhabditis elegans SPR-4, also protects against oxidative stress and amyloid β-protein toxicity. During normal ageing, REST is induced in part by cell non-autonomous Wnt signalling. However, in Alzheimer’s disease, frontotemporal dementia and dementia with Lewy bodies, REST is lost from the nucleus and appears in autophagosomes together with pathological misfolded proteins. Finally, REST levels during ageing are closely correlated with cognitive preservation and longevity. Thus, the activation state of REST may distinguish neuroprotection from neurodegeneration in the ageing brain.