Kaifu Chen

DANPOS: Dynamic analysis of nucleosome position and occupancy by sequencing

Recent developments in next-generation sequencing have enabled whole-genome profiling of nucleosome organizations. Although several algorithms for inferring nucleosome position from a single experimental condition have been available, it remains a challenge to accurately define dynamic nucleosomes associated with environmental changes. Here, we report a comprehensive bioinformatics pipeline, DANPOS, explicitly designed for dynamic nucleosome analysis at single-nucleotide resolution. Using both simulated and real nucleosome data, we demonstrated that bias correction in preliminary data processing and optimal statistical testing significantly enhances the functional interpretation of dynamic nucleosomes. The single-nucleotide resolution analysis of DANPOS allows us to detect all three categories of nucleosome dynamics, such as position shift, fuzziness change, and occupancy change, using a uniform statistical framework. Pathway analysis indicates that each category is involved in distinct biological functions. We also analyzed the influence of sequencing depth and suggest that even 200-fold coverage is probably not enough to identify all the dynamic nucleosomes. Finally, based on nucleosome data from the human hematopoietic stem cells (HSCs) and mouse embryonic stem cells (ESCs), we demonstrated that DANPOS is also robust in defining functional dynamic nucleosomes, not only in promoters, but also in distal regulatory regions in the mammalian genome.

Nucleosome loss leads to global transcriptional up-regulation and genomic instability during yeast aging

All eukaryotic cells divide a finite number of times, although the mechanistic basis of this replicative aging remains unclear. Replicative aging is accompanied by a reduction in histone protein levels, and this is a cause of aging in budding yeast. Here we show that nucleosome occupancy decreased by 50% across the whole genome during replicative aging using spike-in controlled micrococcal nuclease digestion followed by sequencing. Furthermore, nucleosomes became less well positioned or moved to sequences predicted to better accommodate histone octamers. The loss of histones during aging led to transcriptional induction of all yeast genes. Genes that are normally repressed by promoter nucleosomes were most induced, accompanied by preferential nucleosome loss from their promoters. We also found elevated levels of DNA strand breaks, mitochondrial DNA transfer to the nuclear genome, large-scale chromosomal alterations, translocations, and retrotransposition during aging.

3′ UTR shortening represses tumor-suppressor genes in trans by disrupting ceRNA crosstalk

Widespread mRNA 3′ UTR shortening through alternative polyadenylation1 promotes tumor growth in vivo2. A prevailing hypothesis is that it induces proto-oncogene expression in cis through escaping microRNA-mediated repression. Here we report a surprising enrichment of 3′UTR shortening among transcripts that are predicted to act as competing-endogenous RNAs (ceRNAs) for tumor-suppressor genes. Our model-based analysis of the trans effect of 3′ UTR shortening (MAT3UTR) reveals a significant role in altering ceRNA expression. MAT3UTR predicts many trans-targets of 3′ UTR shortening, including PTEN, a crucial tumor-suppressor gene3 involved in ceRNA crosstalk4 with nine 3′UTR-shortening genes, including EPS15 and NFIA. Knockdown of NUDT21, a master 3′ UTR-shortening regulator2, represses tumor-suppressor genes such as PHF6 and LARP1 in trans in a miRNA-dependent manner. Together, the results of our analysis suggest a major role of 3′ UTR shortening in repressing tumor-suppressor genes in trans by disrupting ceRNA crosstalk, rather than inducing proto-oncogenes in cis.Shortening of mRNA 3′ UTRs is often observed in cancer. A combination of model-based analysis and experiments suggests that 3′ UTR shortening disrupts competing endogenous RNA crosstalk, thus influencing tumor-suppressor expression in trans.

A matter of access. Nucleosome disassembly from gene promoters is the central goal of transcriptional activators.

The mechanism whereby transcriptional activators facilitate transcription activation has been debated. Our recent genome-wide profiling of gene expression during aging, where nucleosomes are depleted, indicates that the function of seemingly all transcriptional activators is to trigger nucleosome disassembly from promoters to allow the general transcription machinery access to the DNA.

Sparse conserved under-methylated CpGs are associated with high-order chromatin structure

BackgroundWhole-genome bisulfite sequencing (WGBS) is the gold standard for studying landscape DNA methylation. Current computational methods for WGBS are mainly designed for gene regulatory regions with multiple under-methylated CpGs (UMCs), such as promoters and enhancers.ResultsTo reliably predict the functional importance of single isolated UMCs across the genome, which is usually not achievable using traditional methods, we develop a multi-sample-based method. We identified 9421 sparse conserved under-methylated CpGs (scUMCs) from 31 high-quality methylomes, which are enriched in distal interacting anchor regions co-occupied by multiple chromatin-loop factors and are flanked by highly methylated CpGs. Moreover, cell lineage-specific scUMCs are associated with essential developmental genes, regulators of cell differentiation, and chromatin remodeling enzymes. Dynamic methylation levels of scUMCs correlate with the intensity of chromatin interactions and binding of looping factors as well as patterns of gene expression.ConclusionsWe introduce an innovative computational method for the identification of scUMCs, which are novel epigenetic features associated with high-order chromatin structure, opening new directions in the study of the inter-relationships between DNA methylation and chromatin structure.

Homeobox oncogene activation by pan-cancer DNA hypermethylation

BackgroundCancers have long been recognized to be not only genetically but also epigenetically distinct from their tissues of origin. Although genetic alterations underlying oncogene upregulation have been well studied, to what extent epigenetic mechanisms, such as DNA methylation, can also induce oncogene expression remains unknown.ResultsHere, through pan-cancer analysis of 4174 genome-wide profiles, including whole-genome bisulfite sequencing data from 30 normal tissues and 35 solid tumors, we discover a strong correlation between gene-body hypermethylation of DNA methylation canyons, defined as broad under-methylated regions, and overexpression of approximately 43% of homeobox genes, many of which are also oncogenes. To gain insights into the cause-and-effect relationship, we use a newly developed dCas9-SunTag-DNMT3A system to methylate genomic sites of interest. The locus-specific hypermethylation of gene-body canyon, but not promoter, of homeobox oncogene DLX1, can directly increase its gene expression.ConclusionsOur pan-cancer analysis followed by functional validation reveals DNA hypermethylation as a novel epigenetic mechanism for homeobox oncogene upregulation.

Broad H3K4me3 as A Novel Epigenetic Signature for Normal Development and Disease

The breadth of the enrichment site for post-translational trimethylation of histone H3 at lysine 4 (H3K4me3) on chromatin has attracted great attention recently. H3K4me3, an extensively-studied histone modification, is reported to promote gene transcription by directing preinitiation complex assembly through interaction with effector proteins, e.g., the transcription factor IID (TFIID) complex [1]. Scientists have been interested in the biological implications of signal density, but know little about the importance of the breadth of an H3K4me3 enrichment site. H3K4me3 is usually restricted within a narrow genomic region of approximately one thousand base pairs, with high signal density around the transcription start site (TSS) [2]. However, several groups recently revealed that this is not always the case. A considerable number of H3K4me3 enrichment sites can each be broad while having low signal density. Such broad H3K4me3 domains each spans a genomic region of at least several thousands of base pairs [3–7]. H3K4me3 domain that is as wide as hundreds of thousands of base pairs was reproducibly observed. The upper limit of H3K4me3 domain width is not clear yet. Results from multiple cell types in different species consistently provide strong evidence showing that H3K4me3 breadth has its own biological implications. These findings suggest that the breadth of H3K4me3 is an unexplored functional epigenetic signal.

Ssd1 and Gcn2 suppress global translation efficiency in replicatively aged yeast while their activation extends lifespan

Translational efficiency correlates with longevity, yet its role in lifespan determination remains unclear. Using ribosome profiling, translation efficiency is globally reduced during replicative aging in budding yeast by at least two mechanisms: Firstly, Ssd1 is induced during aging, sequestering mRNAs to P-bodies. Furthermore, Ssd1 overexpression in young cells reduced translation and extended lifespan, while loss of Ssd1 reduced the translational deficit of old cells and shortened lifespan. Secondly, phosphorylation of eIF2α, mediated by the stress kinase Gcn2, was elevated in old cells, contributing to the global reduction in translation without detectable induction of the downstream Gcn4 transcriptional activator. tRNA overexpression activated Gcn2 in young cells and extended lifespan in a manner dependent on Gcn4. Moreover, overexpression of Gcn4 sufficed to extend lifespan in an autophagy-dependent manner in the absence of changes in global translation, indicating that Gcn4-mediated autophagy induction is the ultimate downstream target of activated Gcn2, to extend lifespan.

Transcriptional and genomic mayhem due to aging-induced nucleosome loss in budding yeast

All eukaryotic genomes are assembled into a nucleoprotein structure termed chromatin, which is comprised of regular arrays of nucleosomes. Each nucleosome consists of eight core histone protein molecules around which the DNA is wrapped 1.75 times. The ultimate consequence of packaging the genome into chromatin is that the DNA sequences are relatively inaccessible. This allows the cell to use a comprehensive toolbox of chromatin-altering machineries to reveal access to the DNA sequence at the right time and right place in order to allow genomic processes, such as DNA repair, transcription and replication, to occur in a tightly-regulated manner. In other words, chromatin provides the framework that allows the regulation of all genomic processes, because the machineries that mediate transcription, repair and DNA replication themselves are relatively non-sequence specific and if the genome were naked, they would presumably perform their tasks in a random and unregulated manner. We recently provided support for this prediction in Zheng et al., [Genes and Development (2014) 28: 396-408] by investigating a physiologically relevant scenario in which we had found that cells lose half of the core histone proteins, that is, during the mitotic aging (also called replicative aging) of budding yeast. Using new spike-in normalization techniques, we found that the occupancy of nucleosomes at most DNA sequences is reduced by 50%, leading to transcriptional induction of every single gene. This loss of histones during aging was also accompanied by a significantly-increased frequency of genomic instability including DNA breaks, chromosomal translocations, retrotransposition, and transfer of mitochondrial DNA into the nuclear genome (Figure 1).