Complementary sequence-mediated exon circularization.

Exon circularization has been identified from many loci in mammals, but the detailed mechanism of its biogenesis has remained elusive. By using genome-wide approaches and circular RNA recapitulation, we demonstrate that exon circularization is dependent on flanking intronic complementary sequences. Such sequences and their distribution exhibit rapid evolutionary changes, showing that exon circularization is evolutionarily dynamic. Strikingly, exon circularization efficiency can be regulated by competition between RNA pairing across flanking introns or within individual introns. Importantly, alternative formation of inverted repeated Alu pairs and the competition between them can lead to alternative circularization, resulting in multiple circular RNA transcripts produced from a single gene. Collectively, exon circularization mediated by complementary sequences in human introns and the potential to generate alternative circularization products extend the complexity of mammalian posttranscriptional regulation.

Expanded encyclopaedias of DNA elements in the human and mouse genomes.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

TE-TSS: an integrated data resource of human and mouse transposable element (TE)-derived transcription start site (TSS).

Transposable elements (TEs) are abundant in the genome and serve as crucial regulatory elements. Some TEs function as epigenetically regulated promoters, and these TE-derived transcription start sites (TSSs) play a crucial role in regulating genes associated with specific functions, such as cancer and embryogenesis. However, the lack of an accessible database that systematically gathers TE-derived TSS data is a current research gap. To address this, we established TE-TSS, an integrated data resource of human and mouse TE-derived TSSs (http://xozhanglab.com/TETSS). TE-TSS has compiled 2681 RNA sequencing datasets, spanning various tissues, cell lines and developmental stages. From these, we identified 5768 human TE-derived TSSs and 2797 mouse TE-derived TSSs, with 47% and 38% being experimentally validated, respectively. TE-TSS enables comprehensive exploration of TSS usage in diverse samples, providing insights into tissue-specific gene expression patterns and transcriptional regulatory elements. Furthermore, TE-TSS compares TE-derived TSS regions across 15 mammalian species, enhancing our understanding of their evolutionary and functional aspects. The establishment of TE-TSS facilitates further investigations into the roles of TEs in shaping the transcriptomic landscape and offers valuable resources for comprehending their involvement in diverse biological processes.

Integration of high-resolution promoter profiling assays reveals novel, cell type-specific transcription start sites across 115 human cell and tissue types.

Accurate transcription start site (TSS) annotations are essential for understanding transcriptional regulation and its role in human disease. Gene collections such as GENCODE contain annotations for tens of thousands of TSSs, but not all of these annotations are experimentally validated nor do they contain information on cell type-specific usage. Therefore, we sought to generate a collection of experimentally validated TSSs by integrating RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression (RAMPAGE) data from 115 cell and tissue types, which resulted in a collection of approximately 50 thousand representative RAMPAGE peaks. These peaks are primarily proximal to GENCODE-annotated TSSs and are concordant with other transcription assays. Because RAMPAGE uses paired-end reads, we were then able to connect peaks to transcripts by analyzing the genomic positions of the 3' ends of read mates. Using this paired-end information, we classified the vast majority (37 thousand) of our RAMPAGE peaks as verified TSSs, updating TSS annotations for 20% of GENCODE genes. We also found that these updated TSS annotations are supported by epigenomic and other transcriptomic data sets. To show the utility of this RAMPAGE rPeak collection, we intersected it with the NHGRI/EBI genome-wide association study (GWAS) catalog and identified new candidate GWAS genes. Overall, our work shows the importance of integrating experimental data to further refine TSS annotations and provides a valuable resource for the biological community.

Epigenetic and chromosomal features drive transposon insertion in Drosophila melanogaster.

Transposons are mobile genetic elements prevalent in the genomes of most species. The distribution of transposons within a genome reflects the actions of two opposing processes: initial insertion site selection, and selective pressure from the host. By analyzing whole-genome sequencing data from transposon-activated Drosophila melanogaster, we identified 43 316 de novo and 237 germline insertions from four long-terminal-repeat (LTR) transposons, one LINE transposon (I-element), and one DNA transposon (P-element). We found that all transposon types favored insertion into promoters de novo, but otherwise displayed distinct insertion patterns. De novo and germline P-element insertions preferred replication origins, often landing in a narrow region around transcription start sites and in regions of high chromatin accessibility. De novo LTR transposon insertions preferred regions with high H3K36me3, promoters and exons of active genes; within genes, LTR insertion frequency correlated with gene expression. De novo I-element insertion density increased with distance from the centromere. Germline I-element and LTR transposon insertions were depleted in promoters and exons, suggesting strong selective pressure to remove transposons from functional elements. Transposon movement is associated with genome evolution and disease; therefore, our results can improve our understanding of genome and disease biology.

Investigating the Potential Roles of SINEs in the Human Genome.

Short interspersed nuclear elements (SINEs) are nonautonomous retrotransposons that occupy approximately 13% of the human genome. They are transcribed by RNA polymerase III and can be retrotranscribed and inserted back into the genome with the help of other autonomous retroelements. Because they are preferentially located close to or within gene-rich regions, they can regulate gene expression by various mechanisms that act at both the DNA and the RNA levels. In this review, we summarize recent findings on the involvement of SINEs in different types of gene regulation and discuss the potential regulatory functions of SINEs that are in close proximity to genes, Pol III-transcribed SINE RNAs, and embedded SINE sequences within Pol II-transcribed genes in the human genome. These discoveries illustrate how the human genome has exapted some SINEs into functional regulatory elements.

Arih2 regulates Hedgehog signaling through smoothened ubiquitylation and ER-associated degradation.

During Hedgehog signaling, the ciliary levels of Ptch1 and Smo are regulated by the pathway. At the basal state, Ptch1 localizes to cilia and prevents the ciliary accumulation and activation of Smo. Upon binding a Hedgehog ligand, Ptch1 exits cilia, relieving inhibition of Smo. Smo then concentrates in cilia, becomes activated and activates downstream signaling. Loss of the ubiquitin E3 ligase Arih2 elevates basal Hedgehog signaling, elevates the cellular level of Smo and increases basal levels of ciliary Smo. Mice express two isoforms of Arih2 with Arih2α found primarily in the nucleus and Arih2ß found on the cytoplasmic face of the endoplasmic reticulum (ER). Re-expression of ER-localized Arih2ß but not nuclear-localized Arih2α rescues the Arih2 mutant phenotypes. When Arih2 is defective, protein aggregates accumulate in the ER and the unfolded protein response is activated. Arih2ß appears to regulate the ER-associated degradation (ERAD) of Smo preventing excess and potentially misfolded Smo from reaching the cilium and interfering with pathway regulation.

Allele-specific binding (ASB) analyzer for annotation of allele-specific binding SNPs.

BACKGROUND: Allele-specific binding (ASB) events occur when transcription factors (TFs) bind more favorably to one of the two parental alleles at heterozygous single nucleotide polymorphisms (SNPs). Evidence suggests that ASB events could reveal the impact of sequence variations on TF binding and may have implications for the risk of diseases. RESULTS: Here we present ASB-analyzer, a software platform that enables the users to quickly and efficiently input raw sequencing data to generate individual reports containing the cytogenetic map of ASB SNPs and their associated phenotypes. This interactive tool thereby combines ASB SNP identification, biological annotation, motif analysis, phenotype associations and report summary in one pipeline. With this pipeline, we identified 3772 ASB SNPs from thirty GM12878 ChIP-seq datasets and demonstrated that the ASB SNPs were more likely to be enriched at important sites in TF-binding domains. CONCLUSIONS: ASB-analyzer is a user-friendly tool that enables the detection, characterization and visualization of ASB SNPs. It is implemented in Python, R and bash shell and packaged in the Conda environment. It is available as an open-source tool on GitHub at https://github.com/Liying1996/ASBanalyzer .

Genetic and epigenetic features of promoters with ubiquitous chromatin accessibility support ubiquitous transcription of cell-essential genes.

Gene expression is controlled by regulatory elements within accessible chromatin. Although most regulatory elements are cell type-specific, a subset is accessible in nearly all the 517 human and 94 mouse cell and tissue types assayed by the ENCODE consortium. We systematically analyzed 9000 human and 8000 mouse ubiquitously-accessible candidate cis-regulatory elements (cCREs) with promoter-like signatures (PLSs) from ENCODE, which we denote ubi-PLSs. These are more CpG-rich than non-ubi-PLSs and correspond to genes with ubiquitously high transcription, including a majority of cell-essential genes. ubi-PLSs are enriched with motifs of ubiquitously-expressed transcription factors and preferentially bound by transcriptional cofactors regulating ubiquitously-expressed genes. They are highly conserved between human and mouse at the synteny level but exhibit frequent turnover of motif sites; accordingly, ubi-PLSs show increased variation at their centers compared with flanking regions among the ∼186 thousand human genomes sequenced by the TOPMed project. Finally, ubi-PLSs are enriched in genes implicated in Mendelian diseases, especially diseases broadly impacting most cell types, such as deficiencies in mitochondrial functions. Thus, a set of roughly 9000 mammalian promoters are actively maintained in an accessible state across cell types by a distinct set of transcription factors and cofactors to ensure the transcriptional programs of cell-essential genes.

Genome-wide analysis of polymerase III-transcribed Alu elements suggests cell-type-specific enhancer function.

Alu elements are one of the most successful families of transposons in the human genome. A portion of Alu elements is transcribed by RNA Pol III, whereas the remaining ones are part of Pol II transcripts. Because Alu elements are highly repetitive, it has been difficult to identify the Pol III-transcribed elements and quantify their expression levels. In this study, we generated high-resolution, long-genomic-span RAMPAGE data in 155 biosamples all with matching RNA-seq data and built an atlas of 17,249 Pol III-transcribed Alu elements. We further performed an integrative analysis on the ChIP-seq data of 10 histone marks and hundreds of transcription factors, whole-genome bisulfite sequencing data, ChIA-PET data, and functional data in several biosamples, and our results revealed that although the human-specific Alu elements are transcriptionally repressed, the older, expressed Alu elements may be exapted by the human host to function as cell-type-specific enhancers for their nearby protein-coding genes.

YAP1 Withdrawal in Hepatoblastoma Drives Therapeutic Differentiation of Tumor Cells to Functional Hepatocyte-Like Cells.

BACKGROUND AND AIMS: Despite surgical and chemotherapeutic advances, the 5-year survival rate for stage IV hepatoblastoma (HB), the predominant pediatric liver tumor, remains at 27%. Yes-associated protein 1 (YAP1) and ß-catenin co-activation occurs in 80% of children's HB; however, a lack of conditional genetic models precludes tumor maintenance exploration. Thus, the need for a targeted therapy remains unmet. Given the predominance of YAP1 and ß-catenin activation in HB, we sought to evaluate YAP1 as a therapeutic target in HB. APPROACH AND RESULTS: We engineered the conditional HB murine model using hydrodynamic injection to deliver transposon plasmids encoding inducible YAP1S127A , constitutive ß-cateninDelN90 , and a luciferase reporter to murine liver. Tumor regression was evaluated using bioluminescent imaging, tumor landscape characterized using RNA and ATAC sequencing, and DNA footprinting. Here we show that YAP1S127A withdrawal mediates more than 90% tumor regression with survival for 230+ days in mice. YAP1S127A withdrawal promotes apoptosis in a subset of tumor cells, and in remaining cells induces a cell fate switch that drives therapeutic differentiation of HB tumors into Ki-67-negative hepatocyte-like HB cells ("HbHeps") with hepatocyte-like morphology and mature hepatocyte gene expression. YAP1S127A withdrawal drives the formation of hbHeps by modulating liver differentiation transcription factor occupancy. Indeed, tumor-derived hbHeps, consistent with their reprogrammed transcriptional landscape, regain partial hepatocyte function and rescue liver damage in mice. CONCLUSIONS: YAP1S127A withdrawal, without silencing oncogenic ß-catenin, significantly regresses hepatoblastoma, providing in vivo data to support YAP1 as a therapeutic target for HB. YAP1S127A withdrawal alone sufficiently drives long-term regression in HB, as it promotes cell death in a subset of tumor cells and modulates transcription factor occupancy to reverse the fate of residual tumor cells to mimic functional hepatocytes.

Comprehensive identification of alternative back-splicing in human tissue transcriptomes.

Circular RNAs (circRNAs) are covalently closed RNAs derived from back-splicing of genes across eukaryotes. Through alternative back-splicing (ABS), a single gene produces multiple circRNAs sharing the same back-splice site. Although many ABS events have recently been discovered, to what extent ABS involves in circRNA biogenesis and how it is regulated in different human tissues still remain elusive. Here, we reported an in-depth analysis of ABS events in 90 human tissue transcriptomes. We observed that ABS occurred for about 84% circRNAs. Interestingly, alternative 5' back-splicing occurs more prevalently than alternative 3' back-splicing, and both of them are tissue-specific, especially enriched in brain tissues. In addition, the patterns of ABS events in different brain regions are similar to each other and are more complex than the patterns in non-brain tissues. Finally, the intron length and abundance of Alu elements positively correlated with ABS event complexity, and the predominant circRNAs had longer flanking introns and more Alu elements than other circRNAs in the same ABS event. Together, our results represent a resource for circRNA research-we expanded the repertoire of ABS events of circRNAs in human tissue transcriptomes and provided insights into the complexity of circRNA biogenesis, expression, and regulation.

An organ-on-a-chip model for pre-clinical drug evaluation in progressive non-genetic cardiomyopathy.

Angiotensin II (Ang II) presents a critical mediator in various pathological conditions such as non-genetic cardiomyopathy. Osmotic pump infusion in rodents is a commonly used approach to model cardiomyopathy associated with Ang II. However, profound differences in electrophysiology and pharmacokinetics between rodent and human cardiomyocytes may limit predictability of animal-based experiments. This study investigates the application of an Organ-on-a-chip (OOC) system in modeling Ang II-induced progressive cardiomyopathy. The disease model is constructed to recapitulate myocardial response to Ang II in a temporal manner. The long-term tissue cultivation and non-invasive functional readouts enable monitoring of both acute and chronic cardiac responses to Ang II stimulation. Along with mapping of cytokine secretion and proteomic profiles, this model presents an opportunity to quantitatively measure the dynamic pathological changes that could not be otherwise identified in animals. Further, we present this model as a testbed to evaluate compounds that target Ang II-induced cardiac remodeling. Through assessing the effects of losartan, relaxin, and saracatinib, the drug screening data implicated multifaceted cardioprotective effects of relaxin in restoring contractile function and reducing fibrotic remodeling. Overall, this study provides a controllable platform where cardiac activities can be explicitly observed and tested over the pathological process. The facile and high-content screening can facilitate the evaluation of potential drug candidates in the pre-clinical stage.

Depletion of TRRAP Induces p53-Independent Senescence in Liver Cancer by Down-Regulating Mitotic Genes.

Hepatocellular carcinoma (HCC) is an aggressive subtype of liver cancer with few effective treatments, and the underlying mechanisms that drive HCC pathogenesis remain poorly characterized. Identifying genes and pathways essential for HCC cell growth will aid the development of new targeted therapies for HCC. Using a kinome CRISPR screen in three human HCC cell lines, we identified transformation/transcription domain-associated protein (TRRAP) as an essential gene for HCC cell proliferation. TRRAP has been implicated in oncogenic transformation, but how it functions in cancer cell proliferation is not established. Here, we show that depletion of TRRAP or its co-factor, histone acetyltransferase KAT5, inhibits HCC cell growth through induction of p53-independent and p21-independent senescence. Integrated cancer genomics analyses using patient data and RNA sequencing identified mitotic genes as key TRRAP/KAT5 targets in HCC, and subsequent cell cycle analyses revealed that TRRAP-depleted and KAT5-depleted cells are arrested at the G2/M phase. Depletion of topoisomerase II alpha (TOP2A), a mitotic gene and TRRAP/KAT5 target, was sufficient to recapitulate the senescent phenotype of TRRAP/KAT5 knockdown. Conclusion: Our results uncover a role for TRRAP/KAT5 in promoting HCC cell proliferation by activating mitotic genes. Targeting the TRRAP/KAT5 complex is a potential therapeutic strategy for HCC.

Identification of multipotent mammary stem cells by protein C receptor expression.

The mammary gland is composed of multiple types of epithelial cells, which are generated by mammary stem cells (MaSCs) residing at the top of the hierarchy. However, the existence of these multipotent MaSCs remains controversial and the nature of such cells is unknown. Here we demonstrate that protein C receptor (Procr), a novel Wnt target in the mammary gland, marks a unique population of multipotent mouse MaSCs. Procr-positive cells localize to the basal layer, exhibit epithelial-to-mesenchymal transition characteristics, and express low levels of basal keratins. Procr-expressing cells have a high regenerative capacity in transplantation assays and differentiate into all lineages of the mammary epithelium by lineage tracing. These results define a novel multipotent mammary stem cell population that could be important in the initiation of breast cancer.

Circular intronic long noncoding RNAs.

We describe the identification and characterization of circular intronic long noncoding RNAs in human cells, which accumulate owing to a failure in debranching. The formation of such circular intronic RNAs (ciRNAs) can be recapitulated using expression vectors, and their processing depends on a consensus motif containing a 7 nt GU-rich element near the 5' splice site and an 11 nt C-rich element close to the branchpoint site. In addition, we show that ciRNAs are abundant in the nucleus and have little enrichment for microRNA target sites. Importantly, knockdown of ciRNAs led to the reduced expression of their parent genes. One abundant such RNA, ci-ankrd52, largely accumulates to its sites of transcription, associates with elongation Pol II machinery, and acts as a positive regulator of Pol II transcription. This study thus suggests a cis-regulatory role of noncoding intronic transcripts on their parent coding genes.

The temporal landscape of recursive splicing during Pol II transcription elongation in human cells.

Recursive splicing (RS) is an evolutionarily conserved process of removing long introns via multiple steps of splicing. It was first discovered in Drosophila and recently proven to occur also in humans. The detailed mechanism of recursive splicing is not well understood, in particular, whether it is kinetically coupled with transcription. To investigate the dynamic process that underlies recursive splicing, we systematically characterized 342 RS sites in three human cell types using published time-series data that monitored synchronized Pol II elongation and nascent RNA production with 4-thiouridine labeling. We found that half of the RS events occurred post-transcriptionally with long delays. For at least 18-47% RS introns, we detected RS junction reads only after detecting canonical splicing junction reads, supporting the notion that these introns were removed by both recursive splicing and canonical splicing. Furthermore, the choice of which splicing mechanism was used showed cell type specificity. Our results suggest that recursive splicing supplements, rather than replaces, canonical splicing for removing long introns.

Inhibition of protein arginine methyltransferase 5 enhances hepatic mitochondrial biogenesis.

Protein arginine methyltransferase 5 (PRMT5) regulates gene expression either transcriptionally by symmetric dimethylation of arginine residues on histones H4R3, H3R8, and H2AR3 or at the posttranslational level by methylation of nonhistone target proteins. Although emerging evidence suggests that PRMT5 functions as an oncogene, its role in metabolic diseases is not well-defined. We investigated the role of PRMT5 in promoting high-fat-induced hepatic steatosis. A high-fat diet up-regulated PRMT5 levels in the liver but not in other metabolically relevant tissues such as skeletal muscle or white and brown adipose tissue. This was associated with repression of master transcription regulators involved in mitochondrial biogenesis. In contrast, lentiviral short hairpin RNA-mediated reduction of PRMT5 significantly decreased phosphatidylinositol 3-kinase/AKT signaling in mouse AML12 liver cells. PRMT5 knockdown or knockout decreased basal AKT phosphorylation but boosted the expression of peroxisome proliferator-activated receptor α (PPARα) and PGC-1α with a concomitant increase in mitochondrial biogenesis. Moreover, by overexpressing an exogenous WT or enzyme-dead mutant PRMT5 or by inhibiting PRMT5 enzymatic activity with a small-molecule inhibitor, we demonstrated that the enzymatic activity of PRMT5 is required for regulation of PPARα and PGC-1α expression and mitochondrial biogenesis. Our results suggest that targeting PRMT5 may have therapeutic potential for the treatment of fatty liver.

Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.

Circular RNAs (circRNAs) derived from back-spliced exons have been widely identified as being co-expressed with their linear counterparts. A single gene locus can produce multiple circRNAs through alternative back-splice site selection and/or alternative splice site selection; however, a detailed map of alternative back-splicing/splicing in circRNAs is lacking. Here, with the upgraded CIRCexplorer2 pipeline, we systematically annotated different types of alternative back-splicing and alternative splicing events in circRNAs from various cell lines. Compared with their linear cognate RNAs, circRNAs exhibited distinct patterns of alternative back-splicing and alternative splicing. Alternative back-splice site selection was correlated with the competition of putative RNA pairs across introns that bracket alternative back-splice sites. In addition, all four basic types of alternative splicing that have been identified in the (linear) mRNA process were found within circRNAs, and many exons were predominantly spliced in circRNAs. Unexpectedly, thousands of previously unannotated exons were detected in circRNAs from the examined cell lines. Although these novel exons had similar splice site strength, they were much less conserved than known exons in sequences. Finally, both alternative back-splicing and circRNA-predominant alternative splicing were highly diverse among the examined cell lines. All of the identified alternative back-splicing and alternative splicing in circRNAs are available in the CIRCpedia database (http://www.picb.ac.cn/rnomics/circpedia). Collectively, the annotation of alternative back-splicing and alternative splicing in circRNAs provides a valuable resource for depicting the complexity of circRNA biogenesis and for studying the potential functions of circRNAs in different cells.