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1. Exploiting sequence-based features for predicting enhancer–promoter interactions

   https://doi.org/10.1093/bioinformatics/btx257  

   Yang, Yang ; Zhang, Ruochi ; Singh, Shashank ; Ma, Jian ( July 2017 , Bioinformatics)

   A large number of distal enhancers and proximal promoters form enhancer–promoter interactions to regulate target genes in the human genome. Although recent high-throughput genome-wide mapping approaches have allowed us to more comprehensively recognize potential enhancer–promoter interactions, it is still largely unknown whether sequence-based features alone are sufficient to predict such interactions.

   Here, we develop a new computational method (named PEP) to predict enhancer–promoter interactions based on sequence-based features only, when the locations of putative enhancers and promoters in a particular cell type are given. The two modules in PEP (PEP-Motif and PEP-Word) use different but complementary feature extraction strategies to exploit sequence-based information. The results across six different cell types demonstrate that our method is effective in predicting enhancer–promoter interactions as compared to the state-of-the-art methods that use functional genomic signals. Our work demonstrates that sequence-based features alone can reliably predict enhancer–promoter interactions genome-wide, which could potentially facilitate the discovery of important sequence determinants for long-range gene regulation.

   The source code of PEP is available at: https://github.com/ma-compbio/PEP.

   Supplementary data are available at Bioinformatics online.

    

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2. Illuminating links between cis-regulators and trans-acting variants in the human prefrontal cortex

   https://doi.org/10.1186/s13073-022-01133-8  

   Liu, Shuang ; Won, Hyejung ; Clarke, Declan ; Matoba, Nana ; Khullar, Saniya ; Mu, Yudi ; Wang, Daifeng ; Gerstein, Mark ( November 2022 , Genome Medicine)

   Neuropsychiatric disorders afflict a large portion of the global population and constitute a significant source of disability worldwide. Although Genome-wide Association Studies (GWAS) have identified many disorder-associated variants, the underlying regulatory mechanisms linking them to disorders remain elusive, especially those involving distant genomic elements. Expression quantitative trait loci (eQTLs) constitute a powerful means of providing this missing link. However, most eQTL studies in human brains have focused exclusively on cis-eQTLs, which link variants to nearby genes (i.e., those within 1 Mb of a variant). A complete understanding of disease etiology requires a clearer understanding of trans-regulatory mechanisms, which, in turn, entails a detailed analysis of the relationships between variants and expression changes in distant genes.

   By leveraging large datasets from the PsychENCODE consortium, we conducted a genome-wide survey of trans-eQTLs in the human dorsolateral prefrontal cortex. We also performed colocalization and mediation analyses to identify mediators in trans-regulation and use trans-eQTLs to link GWAS loci to schizophrenia risk genes.

   We identified ~80,000 candidate trans-eQTLs (at FDR<0.25) that influence the expression of ~10K target genes (i.e., “trans-eGenes”). We found that many variants associated with these candidate trans-eQTLs overlap with known cis-eQTLs. Moreover, for >60% of these variants (by colocalization), the cis-eQTL’s target gene acts as a mediator for the trans-eQTL SNP's effect on the trans-eGene, highlighting examples of cis-mediation as essential for trans-regulation. Furthermore, many of these colocalized variants fall into a discernable pattern wherein cis-eQTL’s target is a transcription factor or RNA-binding protein, which, in turn, targets the gene associated with the candidate trans-eQTL. Finally, we show that trans-regulatory mechanisms provide valuable insights into psychiatric disorders: beyond what had been possible using only cis-eQTLs, we link an additional 23 GWAS loci and 90 risk genes (using colocalization between candidate trans-eQTLs and schizophrenia GWAS loci).

   We demonstrate that the transcriptional architecture of the human brain is orchestrated by both cis- and trans-regulatory variants and found that trans-eQTLs provide insights into brain-disease biology.

    

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3. Two-phase differential expression analysis for single cell RNA-seq

   https://doi.org/10.1093/bioinformatics/bty329  

   Wu, Zhijin ; Zhang, Yi ; Stitzel, Michael L. ; Wu, Hao ; Berger, ed., Bonnie ( April 2018 , Bioinformatics)

   Single-cell RNA-sequencing (scRNA-seq) has brought the study of the transcriptome to higher resolution and makes it possible for scientists to provide answers with more clarity to the question of ‘differential expression’. However, most computational methods still stick with the old mentality of viewing differential expression as a simple ‘up or down’ phenomenon. We advocate that we should fully embrace the features of single cell data, which allows us to observe binary (from Off to On) as well as continuous (the amount of expression) regulations.

   We develop a method, termed SC2P, that first identifies the phase of expression a gene is in, by taking into account of both cell- and gene-specific contexts, in a model-based and data-driven fashion. We then identify two forms of transcription regulation: phase transition, and magnitude tuning. We demonstrate that compared with existing methods, SC2P provides substantial improvement in sensitivity without sacrificing the control of false discovery, as well as better robustness. Furthermore, the analysis provides better interpretation of the nature of regulation types in different genes.

   SC2P is implemented as an open source R package publicly available at https://github.com/haowulab/SC2P.

   Supplementary data are available at Bioinformatics online.

    

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4. Smar2C2: A Simple and Efficient Protocol for the Identification of Transcription Start Sites

   https://doi.org/10.1002/cpz1.705  

   Murray, Andrew ; Vollmers, Christopher ; Schmitz, Robert J. ( March 2023 , Current Protocols)

   Promoters and the noncoding sequences that drive their function are fundamental aspects of genes that are critical to their regulation. The transcription preinitiation complex binds and assembles on promoters where it facilitates transcription. The transcription start site (TSS) is located downstream of the promoter sequence and is defined as the location in the genome where polymerase begins transcribing DNA into RNA. Knowing the location of TSSs is useful for annotation of genes, identification of non‐coding sequences important to gene regulation, detection of alternative TSSs, and understanding of 5′ UTR content. Several existing techniques make it possible to accurately identify TSSs, but are often difficult to perform experimentally, require large amounts of input RNA, or are unable to identify a large number of TSSs from a single sample. Many of these protocols take advantage of template switching reverse transcriptases (TSRTs), which reliably place an adaptor at the 5′ end of a first strand synthesis of cDNA. Here, we introduce a protocol that exploits TSRT activity combined with rolling circle amplification to identify TSSs with several unique advantages over existing methods. Sequence adaptors are placed on the 5′ and 3′ end of the full‐length cDNA copy of a transcript. A splint compatible with those adaptors is then used to circularize the full‐length cDNA. Linear DNA containing concatemers of the cDNA are generated using rolling circle amplification, and a sequencing library is formed by fragmenting the concatemers. This protocol is straightforward to execute, requiring limited bench time with relatively stable reagents. Using extremely low amounts of RNA input, this protocol produces large numbers of accurate, deduplicated TSSs genome wide. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

   Basic Protocol 1: Splint generation

   Basic Protocol 2: RNA extraction

   Basic Protocol 3: cDNA synthesis

   Basic Protocol 4: cDNA circularization and amplification

   Basic Protocol 5: Library generation

    

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5. Trans- species microRNA loci in the parasitic plant Cuscuta campestris have a U6-like snRNA promoter

   https://doi.org/10.1093/plcell/koad076  

   Hudzik, Collin ; Maguire, Sean ; Guan, Shengxi ; Held, Jeremy ; Axtell, Michael J ( March 2023 , The Plant Cell)

   Abstract Small regulatory RNAs can move between organisms and regulate gene expression in the recipient. Whether the trans-species small RNAs being exported are distinguished from the normal endogenous small RNAs of the source organism is not known. The parasitic plant Cuscuta campestris (dodder) produces many microRNAs that specifically accumulate at the host–parasite interface, several of which have trans-species activity. We found that induction of C. campestris interface-induced microRNAs is similar regardless of host species and occurs in C. campestris haustoria produced in the absence of any host. The loci-encoding C. campestris interface-induced microRNAs are distinguished by a common cis-regulatory element. This element is identical to a conserved upstream sequence element (USE) used by plant small nuclear RNA loci. The properties of the interface-induced microRNA primary transcripts strongly suggest that they are produced via U6-like transcription by RNA polymerase III. The USE promotes accumulation of interface-induced miRNAs (IIMs) in a heterologous system. This promoter element distinguishes C. campestris IIM loci from other plant small RNAs. Our data suggest that C. campestris IIMs are produced in a manner distinct from canonical miRNAs. All confirmed C. campestris microRNAs with documented trans-species activity are interface-induced and possess these features. We speculate that RNA polymerase III transcription of IIMs may allow these miRNAs to be exported to hosts. 

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