Bertrand Chin-Ming Tan

Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome

RNA editing is a post-transcriptional event that recodes hereditary information. Here we describe a comprehensive profile of the RNA editome of a male Han Chinese individual based on analysis of ∼767 million sequencing reads from poly(A)+, poly(A)− and small RNA samples. We developed a computational pipeline that carefully controls for false positives while calling RNA editing events from genome and whole-transcriptome data of the same individual. We identified 22,688 RNA editing events in noncoding genes and introns, untranslated regions and coding sequences of protein-coding genes. Most changes (∼93%) converted A to I(G), consistent with known editing mechanisms based on adenosine deaminase acting on RNA (ADAR). We also found evidence of other types of nucleotide changes; however, these were validated at lower rates. We found 44 editing sites in microRNAs (miRNAs), suggesting a potential link between RNA editing and miRNA-mediated regulation. Our approach facilitates large-scale studies to profile and compare editomes across a wide range of samples.

Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication

Chromatin is suppressive in nature to cellular enzymes that metabolize DNA, mainly due to the inherent inaccessibility of the DNA template. Despite extensive understanding of the involvement of chromatin‐modifying factors in transcription, roles of related activities in DNA replication remain largely elusive. Here, we show that the heterodimeric transcriptional elongation factor FACT (facilitates chromatin transcription) is functionally linked to DNA synthesis. Its involvement in DNA replication is partly mediated by the stable association with the replicative helicase complex, MCM, and further by the coexistence with MCM on replication origin. Furthermore, relying on its nucleosome‐reorganizing activity, FACT can facilitate chromatin unwinding by the MCM complex, which is otherwise inert on the nucleosomal template. As a consequence, the physical and functional interaction between FACT and MCM is an important determinant in the proper initiation of DNA replication and S phase in vivo. Together, our findings identify FACT as an integral and conserved component of the endogenous replication machinery, and support a model in which the concerted action of helicase and chromatin‐modifying activities promotes chromosome replication.

Nucleophosmin acts as a novel AP2α‐binding transcriptional corepressor during cell differentiation

Nucleophosmin (NPM) is an important nucleolar phosphoprotein with pleiotropic functions in various cellular processes. In this study, we have further examined the largely uncharacterized role of NPM in transcriptional regulation by uncovering novel NPM‐binding transcriptional factors. Among potential interactors, we found that activating protein transcription factor 2 (AP2)α forms a complex with NPM during retinoic‐acid‐induced cell differentiation. We show that this complex is recruited to the promoters of certain retinoic‐acid‐responsive genes, including NPM itself. Such binding of AP2α, and consequent recruitment of NPM, is selective and dependent on a consensus AP2α‐binding sequence. Remarkably, suppression of NPM by RNA interference alleviates the repression of gene expression mediated by retinoic acid and AP2α. Our findings further show that, on promoter binding, NPM probably exerts its repressive effect by inducing a change in local chromatin structure that also engages histone deacetylases. This study unveils a hitherto unrecognized transcriptional corepressor function of the NPM protein, and highlights a novel mechanism by which NPM regulates cell growth and differentiation.

Nek9, a Novel FACT-associated Protein, Modulates Interphase Progression

The heterodimeric Spt16-Pob3/DUF/FACT complex is a class of chromatin structure modulators with important roles in replication and transcription. Although regarded as a transcription elongator for chromatin template, little is known about the mode of action and involvement in other molecular processes of the mammalian FACT. Here we report the identification of a novel interacting and functional partner of FACT, Nek9. Nek9 forms a stable, ∼600-kDa complex with FACT in the interphase nuclei. Its active form is characterized by phosphorylation-dependent electrophoretic mobility shift and phosphorylation at a conserved residue within the activation loop (Thr210). When complexed with FACT, Nek9 exhibits markedly elevated phosphorylation on Thr210. Cell cycle analysis on the Nek9dsRNAi cells directly implicated Nek9 in maintaining proper G1 and S progression, a role temporally correlated to the formation of a phospho-Nek9-FACT complex. Collectively, these observations provide evidence that Nek9, potentially as an active enzymatic partner of FACT, mediates certain FACT-associated cellular processes, which are ultimately essential for interphase progression.

RNA Editome in Rhesus Macaque Shaped by Purifying Selection

Understanding of the RNA editing process has been broadened considerably by the next generation sequencing technology; however, several issues regarding this regulatory step remain unresolved – the strategies to accurately delineate the editome, the mechanism by which its profile is maintained, and its evolutionary and functional relevance. Here we report an accurate and quantitative profile of the RNA editome for rhesus macaque, a close relative of human. By combining genome and transcriptome sequencing of multiple tissues from the same animal, we identified 31,250 editing sites, of which 99.8% are A-to-G transitions. We verified 96.6% of editing sites in coding regions and 97.5% of randomly selected sites in non-coding regions, as well as the corresponding levels of editing by multiple independent means, demonstrating the feasibility of our experimental paradigm. Several lines of evidence supported the notion that the adenosine deamination is associated with the macaque editome – A-to-G editing sites were flanked by sequences with the attributes of ADAR substrates, and both the sequence context and the expression profile of ADARs are relevant factors in determining the quantitative variance of RNA editing across different sites and tissue types. In support of the functional relevance of some of these editing sites, substitution valley of decreased divergence was detected around the editing site, suggesting the evolutionary constraint in maintaining some of these editing substrates with their double-stranded structure. These findings thus complement the “continuous probing” model that postulates tinkering-based origination of a small proportion of functional editing sites. In conclusion, the macaque editome reported here highlights RNA editing as a widespread functional regulation in primate evolution, and provides an informative framework for further understanding RNA editing in human.

Differential regulation of CHOP translation by phosphorylated eIF4E under stress conditions

Cells respond to environmental stress by inducing translation of a subset of mRNAs important for survival or apoptosis. CHOP, a downstream transcriptional target of stress-induced ATF4, is also regulated translationally in a uORF-dependent manner under stress. Low concentration of anisomycin induces CHOP expression at both transcriptional and translational levels. To study specifically the translational aspect of CHOP expression, and further clarify the regulatory mechanisms underlying stress-induced translation initiation, we developed a CMV promoter-regulated, uORFchop-driven reporter platform. Here we show that anisomycin-induced CHOP expression depends on phosphorylated eIF4E/S209 and eIF2α/S51. Contrary to phospho-eIF2α/S51, phospho-eIF4E/S209 is not involved in thapsigargin-induced CHOP expression. Studies using various kinase inhibitors and mutants uncovered that both the p38MAPK-Mnk and mTOR signaling pathways contribute to stress-responsive reporter and CHOP expression. We also demonstrated that anisomycin-induced translation is tightly regulated by partner binding preference of eIF4E. Furthermore, mutating the uORF sequence abolished the anisomycin-induced association of chop mRNA with phospho-eIF4E and polysomes, thus demonstrating the significance of this cis-regulatory element in conferring on the transcript a stress-responsive translational inducibility. Strikingly, although insulin treatment activated ERK-Mnk and mTOR pathways, and consequently eIF4E/S209 phosphorylation, it failed to induce phospho-eIF2α/S51 and reporter translation, thus pinpointing a crucial determinant in stress-responsive translation.

Dephosphorylation of Nucleophosmin by PP1β Facilitates pRB Binding and Consequent E2F1-dependent DNA Repair

We report a new pathway through which PP1β signals to nucleophosmin (NPM) in response to DNA damage. UV induces dephosphorylation of NPM at multiple sites, leading to enhancement of complex formation between NPM and retinoblastoma tumor suppressor protein and the subsequent upregulation of E2F1. Consequently, such signaling pathway potentiates the cellular DNA repair capacity.

WDHD1 modulates the post-transcriptional step of the centromeric silencing pathway

The centromere is a highly specialized chromosomal element that is essential for chromosome segregation during mitosis. Centromere integrity must therefore be properly preserved and is strictly dependent upon the establishment and maintenance of surrounding chromatin structure. Here we identify WDHD1, a WD40-domain and HMG-domain containing protein, as a key regulator of centromere function. We show that WDHD1 associates with centromeres in a cell cycle-dependent manner, coinciding with mid-to-late S phase. WDHD1 down-regulation compromises HP1α localization to pericentric heterochromatin and leads to altered expression of epigenetic markers associated with this chromatin region. As a consequence, such reduced epigenetic silencing is manifested in disrupted heterochromatic state of the centromere and a defective mitosis. Moreover, we demonstrate that a possible underlying mechanism of WDHD1’s involvement lies in the proper generation of the small non-coding RNAs encoded by the centromeric satellite repeats. This role is mediated at the post-transcriptional level and likely through stabilizing Dicer association with centromeric RNA. Collectively, these findings suggest that WDHD1 may be a critical component of the RNA-dependent epigenetic control mechanism that sustains centromere integrity and genomic stability.

Evolutionary Interrogation of Human Biology in Well-Annotated Genomic Framework of Rhesus Macaque

With genome sequence and composition highly analogous to human, rhesus macaque represents a unique reference for evolutionary studies of human biology. Here, we developed a comprehensive genomic framework of rhesus macaque, the RhesusBase2, for evolutionary interrogation of human genes and the associated regulations. A total of 1,667 next-generation sequencing (NGS) data sets were processed, integrated, and evaluated, generating 51.2 million new functional annotation records. With extensive NGS annotations, RhesusBase2 refined the fine-scale structures in 30% of the macaque Ensembl transcripts, reporting an accurate, up-to-date set of macaque gene models. On the basis of these annotations and accurate macaque gene models, we further developed an NGS-oriented Molecular Evolution Gateway to access and visualize macaque annotations in reference to human orthologous genes and associated regulations (www.rhesusbase.org/molEvo). We highlighted the application of this well-annotated genomic framework in generating hypothetical link of human-biased regulations to human-specific traits, by using mechanistic characterization of the DIEXF gene as an example that provides novel clues to the understanding of digestive system reduction in human evolution. On a global scale, we also identified a catalog of 9,295 human-biased regulatory events, which may represent novel elements that have a substantial impact on shaping human transcriptome and possibly underpin recent human phenotypic evolution. Taken together, we provide an NGS data-driven, information-rich framework that will broadly benefit genomics research in general and serves as an important resource for in-depth evolutionary studies of human biology.

Functional cooperation between FACT and MCM is coordinated with cell cycle and differential complex formation

BackgroundFunctional cooperation between FACT and the MCM helicase complex constitutes an integral step during DNA replication initiation. However, mode of regulation that underlies the proper functional interaction of FACT and MCM is poorly understood.Methods & ResultsHere we present evidence indicating that such interaction is coordinated with cell cycle progression and differential complex formation. We first demonstrate the existence of two distinct FACT-MCM subassemblies, FACT-MCM2/4/6/7 and FACT-MCM2/3/4/5. Both complexes possess DNA unwinding activity and are subject to cell cycle-dependent enzymatic regulation. Interestingly, analysis of functional attributes further suggests that they act at distinct, and possibly sequential, steps during origin establishment and replication initiation. Moreover, we show that the phosphorylation profile of the FACT-associated MCM4 undergoes a cell cycle-dependent change, which is directly correlated with the catalytic activity of the FACT-MCM helicase complexes. Finally, at the quaternary structure level, physical interaction between FACT and MCM complexes is generally dependent on persistent cell cycle and further stabilized upon S phase entry. Cessation of mitotic cycle destabilizes the complex formation and likely leads to compromised coordination and activities.ConclusionsTogether, our results correlate FACT-MCM functionally and temporally with S phase and DNA replication. They further demonstrate that enzymatic activities intrinsically important for DNA replication are tightly controlled at various levels, thereby ensuring proper progression of, as well as exit from, the cell cycle and ultimately euploid gene balance.