Xiaobei Zhao

An atlas of active enhancers across human cell types and tissues

Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.

JASPAR 2014: an extensively expanded and updated open-access database of transcription factor binding profiles

JASPAR (http://jaspar.genereg.net) is the largest open-access database of matrix-based nucleotide profiles describing the binding preference of transcription factors from multiple species. The fifth major release greatly expands the heart of JASPAR—the JASPAR CORE subcollection, which contains curated, non-redundant profiles—with 135 new curated profiles (74 in vertebrates, 8 in Drosophila melanogaster, 10 in Caenorhabditis elegans and 43 in Arabidopsis thaliana; a 30% increase in total) and 43 older updated profiles (36 in vertebrates, 3 in D. melanogaster and 4 in A. thaliana; a 9% update in total). The new and updated profiles are mainly derived from published chromatin immunoprecipitation-seq experimental datasets. In addition, the web interface has been enhanced with advanced capabilities in browsing, searching and subsetting. Finally, the new JASPAR release is accompanied by a new BioPython package, a new R tool package and a new R/Bioconductor data package to facilitate access for both manual and automated methods.

JASPAR 2010: the greatly expanded open-access database of transcription factor binding profiles

JASPAR (http://jaspar.genereg.net) is the leading open-access database of matrix profiles describing the DNA-binding patterns of transcription factors (TFs) and other proteins interacting with DNA in a sequence-specific manner. Its fourth major release is the largest expansion of the core database to date: the database now holds 457 non-redundant, curated profiles. The new entries include the first batch of profiles derived from ChIP-seq and ChIP-chip whole-genome binding experiments, and 177 yeast TF binding profiles. The introduction of a yeast division brings the convenience of JASPAR to an active research community. As binding models are refined by newer data, the JASPAR database now uses versioning of matrices: in this release, 12% of the older models were updated to improved versions. Classification of TF families has been improved by adopting a new DNA-binding domain nomenclature. A curated catalog of mammalian TFs is provided, extending the use of the JASPAR profiles to additional TFs belonging to the same structural family. The changes in the database set the system ready for more rapid acquisition of new high-throughput data sources. Additionally, three new special collections provide matrix profile data produced by recent alternative high-throughput approaches.

Polyadenylation site–induced decay of upstream transcripts enforces promoter directionality

Active human promoters produce promoter-upstream transcripts (PROMPTs). Why these RNAs are coupled to decay, whereas their neighboring promoter-downstream mRNAs are not, is unknown. Here high-throughput sequencing demonstrates that PROMPTs generally initiate in the antisense direction closely upstream of the transcription start sites (TSSs) of their associated genes. PROMPT TSSs share features with mRNA-producing TSSs, including stalled RNA polymerase II (RNAPII) and the production of small TSS-associated RNAs. Notably, motif analyses around PROMPT 3′ ends reveal polyadenylation (pA)-like signals. Mutagenesis studies demonstrate that PROMPT pA signals are functional but linked to RNA degradation. Moreover, pA signals are under-represented in promoter-downstream versus promoter-upstream regions, thus allowing for more efficient RNAPII progress in the sense direction from gene promoters. We conclude that asymmetric sequence distribution around human gene promoters serves to provide a directional RNA output from an otherwise bidirectional transcription process.

Biogenic mechanisms and utilization of small RNAs derived from human protein-coding genes

Efforts to catalog eukaryotic transcripts have uncovered many small RNAs (sRNAs) derived from gene termini and splice sites. Their biogenesis pathways are largely unknown, but a mechanism based on backtracking of RNA polymerase II (RNAPII) has been suggested. By sequencing transcripts 12–100 nucleotides in length from cells depleted of major RNA degradation enzymes and RNAs associated with Argonaute (AGO1/2) effector proteins, we provide mechanistic models for sRNA production. We suggest that neither splice site–associated (SSa) nor transcription start site–associated (TSSa) RNAs arise from RNAPII backtracking. Instead, SSa RNAs are largely degradation products of splicing intermediates, whereas TSSa RNAs probably derive from nascent RNAs protected by stalled RNAPII against nucleolysis. We also reveal new AGO1/2-associated RNAs derived from 3′ ends of introns and from mRNA 3′ UTRs that appear to draw from noncanonical microRNA biogenesis pathways.

New histone supply regulates replication fork speed and PCNA unloading

Coupling of replication fork speed and PCNA unloading to nucleosome assembly may maintain chromatin integrity during transient histone shortage.

Plasma membrane-associated sialidase (NEU3) regulates progression of prostate cancer to androgen-independent growth through modulation of androgen receptor signaling

Prostate cancers generally become androgen-independent and resistant to hormone therapy with progression. To understand the underlying mechanisms and facilitate the development of novel treatments for androgen-independent prostate cancer, we have investigated plasma membrane-associated sialidase (NEU3), the key enzyme for ganglioside hydrolysis participating in transmembrane signaling. We have discovered NEU3 to be upregulated in human prostate cancer compared with non-cancerous tissue, correlating with the Gleason score. NEU3 silencing with siRNA in prostate cancer PC-3 and LNCaP cells resulted in increased expression of differentiation markers and in cell apoptosis, but decrease in Bcl-2 as well as a progression-related transcription factor, early growth response gene (EGR-1). In androgen-sensitive LNCaP cells, forced overexpression of NEU3 significantly induced expression of EGR-1, androgen receptor (AR) and PSA both with and without androgen, the cells becoming sensitive to androgen. The NEU3-mediated induction was abrogated by inhibitors for PI-3 kinase and MAP kinase and more specifically by their silencing in the absence of androgen, being confirmed by increased phosphorylation of AKT and ERK1/2 in NEU3 overexpressing cells. NEU3 siRNA introduction caused reduction of cell growth of an androgen-independent PC-3 cells in culture and of transplanted tumors in nude mice. These data suggest that NEU3 regulates tumor progression through AR signaling, and thus be a potential tool for diagnosis and therapy of androgen-independent prostate cancer.

Interaction of antidepressants with the serotonin and norepinephrine transporters: Mutational studies of the S1 substrate binding pocket

Background: SERT and NET are important targets for antidepressants. Results: Antidepressants are differentially affected by mutations within the central S1 pocket of SERT and NET. Conclusion: Our data indicate that many antidepressants bind within the S1 pocket, and inhibitor selectivity is determined by residues within this site. Significance: This study provides a framework for modeling of drug binding, which may be used in future structure-based drug design. The serotonin transporter (SERT) and the norepinephrine transporter (NET) are sodium-dependent neurotransmitter transporters responsible for reuptake of released serotonin and norepinephrine, respectively, into nerve terminals in the brain. A wide range of inhibitors of SERT and NET are used as treatment of depression and anxiety disorders or as psychostimulant drugs of abuse. Despite their clinical importance, the molecular mechanisms by which various types of antidepressant drugs bind and inhibit SERT and NET are still elusive for the majority of the inhibitors, including the molecular basis for SERT/NET selectivity. Mutational analyses have suggested that a central substrate binding site (denoted the S1 pocket) also harbors an inhibitor binding site. In this study, we determine the effect of mutating six key S1 residues in human SERT (hSERT) and NET (hNET) on the potency of 15 prototypical SERT/NET inhibitors belonging to different drug classes. Analysis of the resulting drug sensitivity profiles provides novel information on drug binding modes in hSERT and hNET and identifies specific S1 residues as important molecular determinants for inhibitor potency and hSERT/hNET selectivity.

Systematic Clustering of Transcription Start Site Landscapes

Genome-wide, high-throughput methods for transcription start site (TSS) detection have shown that most promoters have an array of neighboring TSSs where some are used more than others, forming a distribution of initiation propensities. TSS distributions (TSSDs) vary widely between promoters and earlier studies have shown that the TSSDs have biological implications in both regulation and function. However, no systematic study has been made to explore how many types of TSSDs and by extension core promoters exist and to understand which biological features distinguish them. In this study, we developed a new non-parametric dissimilarity measure and clustering approach to explore the similarities and stabilities of clusters of TSSDs. Previous studies have used arbitrary thresholds to arrive at two general classes: broad and sharp. We demonstrated that in addition to the previous broad/sharp dichotomy an additional category of promoters exists. Unlike typical TATA-driven sharp TSSDs where the TSS position can vary a few nucleotides, in this category virtually all TSSs originate from the same genomic position. These promoters lack epigenetic signatures of typical mRNA promoters and a substantial subset of them are mapping upstream of ribosomal protein pseudogenes. We present evidence that these are likely mapping errors, which have confounded earlier analyses, due to the high similarity of ribosomal gene promoters in combination with known G addition bias in the CAGE libraries. Thus, previous two-class separations of promoter based on TSS distributions are motivated, but the ultra-sharp TSS distributions will confound downstream analyses if not removed.

BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation

During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger‐containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1 that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1‐binding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45 recruitment, but not MCM2‐7 loading, is impaired in BRPF3‐depleted cells, identifying a BRPF3‐dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3‐HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby replication origins.