Deconvolution of Nascent Sequencing Data Using Transcriptional Regulatory Elements.

The problem of microdissection of heterogeneous tissue samples is of great interest for both fundamental biology and biomedical research. Until now, microdissection in the form of supervised deconvolution of mixed sequencing samples has been limited to assays measuring gene expression (RNA-seq) or chromatin accessibility (ATAC-seq). We present here the first attempt at solving the supervised deconvolution problem for run-on nascent sequencing data (GRO-seq and PRO-seq), a readout of active transcription. Then, we develop a novel filtering method suited to the mixed set of promoter and enhancer regions provided by nascent sequencing, and apply best-practice standards from the RNA-seq literature, using in-silico mixtures of cells. Using these methods, we find that enhancer RNAs are highly informative features for supervised deconvolution. In most cases, simple deconvolution methods perform better than more complex ones for solving the nascent deconvolution problem. Furthermore, undifferentiated cell types confound deconvolution of nascent sequencing data, likely as a consequence of transcriptional activity over the highly open chromatin regions of undifferentiated cell types. Our results suggest that while the problem of nascent deconvolution is generally tractable, stronger approaches integrating other sequencing protocols may be required to solve mixtures containing undifferentiated celltypes.

eRNA co-expression network uncovers TF dependency and convergent cooperativity.

Enhancer RNAs (eRNAs) are non-coding RNAs produced by transcriptional enhancers that are highly correlated with their activity. Using a capped nascent RNA sequencing (PRO-cap) dataset in human lymphoblastoid cell lines across 67 individuals, we identified inter-individual variation in the expression of over 80 thousand transcribed transcriptional regulatory elements (tTREs), in both enhancers and promoters. Co-expression analysis of eRNAs from tTREs across individuals revealed how enhancers are associated with each other and with promoters. Mid- to long-range co-expression showed a distance-dependent decay that was modified by TF occupancy. In particular, we found a class of "bivalent" TFs, including Cohesin, that both facilitate and isolate the interaction between enhancers and/or promoters, depending on their topology. At short distances, we observed strand-specific correlations between nearby eRNAs in both convergent and divergent orientations. Our results support a cooperative model of convergent eRNAs, consistent with eRNAs facilitating adjacent enhancers rather than interfering with each other. Therefore, our approach to infer functional interactions from co-expression analyses provided novel insights into the principles of enhancer interactions as a function of distance, orientation, and binding landscapes of TFs.

Widespread priming of transcriptional regulatory elements by incipient accessibility or RNA polymerase II pause in early embryos of the sea urchin Strongylocentrotus purpuratus.

Transcriptional regulatory elements (TREs) are the primary nodes that control developmental gene regulatory networks. In embryo stages, larvae, and adult differentiated red spherule cells of the sea urchin Strongylocentrotus purpuratus, transcriptionally engaged TREs are detected by Precision Run-On Sequencing (PRO-seq), which maps genome-wide at base pair resolution the location of paused or elongating RNA polymerase II (Pol II). In parallel, TRE accessibility is estimated by the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq). Our analysis identifies surprisingly early and widespread TRE accessibility in 4-cell cleavage embryos that is not necessarily followed by concurrent or subsequent transcription. TRE transcriptional differences identified by PRO-seq provide more contrast among embryonic stages than ATAC-seq accessibility differences, in agreement with the apparent excess of accessible but inactive TREs during embryogenesis. Global TRE accessibility reaches a maximum around the 20-hour late blastula stage, which coincides with the consolidation of major embryo regionalizations and peak histone variant H2A.Z expression. A transcriptional potency model based on labile nucleosome TRE occupancy driven by DNA sequences and the prevalence of histone variants is proposed in order to explain the basal accessibility of transcriptionally inactive TREs during embryogenesis. However, our results would not reconcile well with labile nucleosome models based on simple A/T sequence enrichment. In addition, a large number of distal TREs become transcriptionally disengaged during developmental progression, in support of an early Pol II paused model for developmental gene regulation that eventually resolves in transcriptional activation or silencing. Thus, developmental potency in early embryos may be facilitated by incipient accessibility and transcriptional pause at TREs.

Mammalian evolution of human cis-regulatory elements and transcription factor binding sites.

Understanding the regulatory landscape of the human genome is a long-standing objective of modern biology. Using the reference-free alignment across 241 mammalian genomes produced by the Zoonomia Consortium, we charted evolutionary trajectories for 0.92 million human candidate cis-regulatory elements (cCREs) and 15.6 million human transcription factor binding sites (TFBSs). We identified 439,461 cCREs and 2,024,062 TFBSs under evolutionary constraint. Genes near constrained elements perform fundamental cellular processes, whereas genes near primate-specific elements are involved in environmental interaction, including odor perception and immune response. About 20% of TFBSs are transposable element-derived and exhibit intricate patterns of gains and losses during primate evolution whereas sequence variants associated with complex traits are enriched in constrained TFBSs. Our annotations illuminate the regulatory functions of the human genome.

Functional annotation of the animal genomes: An integrated annotation resource for the horse.

The genomic sequence of the horse has been available since 2009, providing critical resources for discovering important genomic variants regarding both animal health and population structures. However, to fully understand the functional implications of these variants, detailed annotation of the horse genome is required. Due to the limited availability of functional data for the equine genome, as well as the technical limitations of short-read RNA-seq, existing annotation of the equine genome contains limited information about important aspects of gene regulation, such as alternate isoforms and regulatory elements, which are either not transcribed or transcribed at a very low level. To solve above problems, the Functional Annotation of the Animal Genomes (FAANG) project proposed a systemic approach to tissue collection, phenotyping, and data generation, adopting the blueprint laid out by the Encyclopedia of DNA Elements (ENCODE) project. Here we detail the first comprehensive overview of gene expression and regulation in the horse, presenting 39,625 novel transcripts, 84,613 candidate cis-regulatory elements (CRE) and their target genes, 332,115 open chromatin regions genome wide across a diverse set of tissues. We showed substantial concordance between chromatin accessibility, chromatin states in different genic features and gene expression. This comprehensive and expanded set of genomics resources will provide the equine research community ample opportunities for studies of complex traits in the horse.

Functional validation of transposable element-derived cis-regulatory elements in Atlantic salmon.

Transposable elements (TEs) are hypothesized to play important roles in shaping genome evolution following whole-genome duplications (WGDs), including rewiring of gene regulation. In a recent analysis, duplicate gene copies that had evolved higher expression in liver following the salmonid WGD ∼100 million years ago were associated with higher numbers of predicted TE-derived cis-regulatory elements (TE-CREs). Yet, the ability of these TE-CREs to recruit transcription factors (TFs) in vivo and impact gene expression remains unknown. Here, we evaluated the gene-regulatory functions of 11 TEs using luciferase promoter reporter assays in Atlantic salmon (Salmo salar) primary liver cells. Canonical Tc1-Mariner elements from intronic regions showed no or small repressive effects on transcription. However, other TE-CREs upstream of transcriptional start sites increased expression significantly. Our results question the hypothesis that TEs in the Tc1-Mariner superfamily, which were extremely active following WGD in salmonids, had a major impact on regulatory rewiring of gene duplicates, but highlights the potential of other TEs in post-WGD rewiring of gene regulation in the Atlantic salmon genome.

Kinetic networks identify TWIST2 as a key regulatory node in adipogenesis.

Adipocytes contribute to metabolic disorders such as obesity, diabetes, and atherosclerosis. Prior characterizations of the transcriptional network driving adipogenesis have overlooked transiently acting transcription factors (TFs), genes, and regulatory elements that are essential for proper differentiation. Moreover, traditional gene regulatory networks provide neither mechanistic details about individual regulatory element-gene relationships nor temporal information needed to define a regulatory hierarchy that prioritizes key regulatory factors. To address these shortcomings, we integrate kinetic chromatin accessibility (ATAC-seq) and nascent transcription (PRO-seq) data to generate temporally resolved networks that describe TF binding events and resultant effects on target gene expression. Our data indicate which TF families cooperate with and antagonize each other to regulate adipogenesis. Compartment modeling of RNA polymerase density quantifies how individual TFs mechanistically contribute to distinct steps in transcription. The glucocorticoid receptor activates transcription by inducing RNA polymerase pause release, whereas SP and AP-1 factors affect RNA polymerase initiation. We identify Twist2 as a previously unappreciated effector of adipocyte differentiation. We find that TWIST2 acts as a negative regulator of 3T3-L1 and primary preadipocyte differentiation. We confirm that Twist2 knockout mice have compromised lipid storage within subcutaneous and brown adipose tissue. Previous phenotyping of Twist2 knockout mice and Setleis syndrome Twist2 -/- patients noted deficiencies in subcutaneous adipose tissue. This network inference framework is a powerful and general approach for interpreting complex biological phenomena and can be applied to a wide range of cellular processes.

The abscisic acid-responsive element binding factors MAPKKK18 module regulates abscisic acid-induced leaf senescence in Arabidopsis.

The mitogen-activated protein kinase kinase kinase 18 (MAPKKK18) has been reported to play a role in abiotic stress priming in long-term abscisic acid (ABA) response including drought tolerance and leaf senescence. However, the upstream transcriptional regulators of MAPKKK18 remain to be determined. Here, we report ABA-responsive element binding factors (ABFs) as upstream transcription factors of MAPKKK18 expression. Mutants of abf2, abf3, abf4, and abf2abf3abf4 dramatically reduced the transcription of MAPKKK18. Our electrophoresis mobility shift assay and dual-luciferase reporter assay demonstrated that ABF2, ABF3, and ABF4 bound to ABA-responsive element cis-elements within the promoter of MAPKKK18 to transactivate its expression. Furthermore, enrichments of the promoter region of MAPKKK18 by ABF2, ABF3, and ABF4 were confirmed by in vivo chromatin immunoprecipitation coupled with quantitative PCR. In addition, we found that mutants of mapkkk18 exhibited obvious delayed leaf senescence. Moreover, a genetic study showed that overexpression of ABF2, ABF3, and ABF4 in the background of mapkkk18 mostly phenocopied the stay-green phenotype of mapkkk18 and, expression levels of five target genes of ABFs, that is, NYE1, NYE2, NYC1, PAO, and SAG29, were attenuated as a result of MAPKKK18 mutation. These findings demonstrate that ABF2, ABF3, and ABF4 act as transcription regulators of MAPKKK18 and also suggest that, at least in part, ABA acts in priming leaf senescence via ABF-induced expression of MAPKKK18.

Human Cancer Genomes Display Recurrent Repeat Expansions.

Recurrent repeat expansions were identified near known regulatory elements across seven cancer types.

Recurrent repeat expansions in human cancer genomes.

Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases1,2. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs, a phenomenon termed microsatellite instability; however, larger repeat expansions have not been systematically analysed in cancer3-8. Here we identified TR expansions in 2,622 cancer genomes spanning 29 cancer types. In seven cancer types, we found 160 recurrent repeat expansions (rREs), most of which (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with enrichment near candidate cis-regulatory elements, suggesting a potential role in gene regulation. One rRE, a GAAA-repeat expansion, located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, in preliminary experiments, treating cells that harbour this rRE with a GAAA-targeting molecule led to a dose-dependent decrease in cell proliferation. Overall, our results suggest that rREs may be an important but unexplored source of genetic variation in human cancer, and we provide a comprehensive catalogue for further study.

Mapping Transcription Regulation with Run-on and Sequencing Data Using the Web-Based tfTarget Gateway.

Run-on and sequencing assays like GRO-seq, PRO-seq, and ChRO-seq allow for joint profiling of transcription activity of transcriptional regulatory elements (TREs), i.e., promoters and active enhancers, and target genes. Variation in biological conditions, such as treated vs. control, results in changes in the activity of transcription factors (TFs), which induces concerted changes in TREs and target genes. By modeling the differences between two biological conditions, we developed the computational pipeline known as tfTarget that predicts a set of putative TREs and target genes responding to each TF under the biological condition of interest. In this chapter, we demonstrate the use of the new web-based tfTarget in mapping transcription regulation using run-on sequencing data.

Role of Genetic Variation in Transcriptional Regulatory Elements in Heart Rhythm.

Genetic predisposition to cardiac arrhythmias has been a field of intense investigation. Research initially focused on rare hereditary arrhythmias, but over the last two decades, the role of genetic variation (single nucleotide polymorphisms) in heart rate, rhythm, and arrhythmias has been taken into consideration as well. In particular, genome-wide association studies have identified hundreds of genomic loci associated with quantitative electrocardiographic traits, atrial fibrillation, and less common arrhythmias such as Brugada syndrome. A significant number of associated variants have been found to systematically localize in non-coding regulatory elements that control the tissue-specific and temporal transcription of genes encoding transcription factors, ion channels, and other proteins. However, the identification of causal variants and the mechanism underlying their impact on phenotype has proven difficult due to the complex tissue-specific, time-resolved, condition-dependent, and combinatorial function of regulatory elements, as well as their modest conservation across different model species. In this review, we discuss research efforts aimed at identifying and characterizing-trait-associated variant regulatory elements and the molecular mechanisms underlying their impact on heart rate or rhythm.

Develop an efficient and specific AAV-based labeling system for Muller glia in mice.

Reprogramming Müller glia (MG) into functional cells is considered a promising therapeutic strategy to treat ocular diseases and vision loss. However, current AAV-based system for MG-tracing was reported to have high leakage in recent studies. Here, we focused on reducing the leakage of AAV-based labeling systems and found that different AAV serotypes showed a range of efficiency and specificity in labeling MG, leading us to optimize a human GFAP-Cre reporter system packaged in the AAV9 serotype with the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) removed. The leakage ratio of the AAV9-hGFAP-Cre-ΔWPRE decreased by an approximate 40-fold compared with the AAV9-hGFAP-Cre-WPRE labeling system. In addition, we validated the specificity of the AAV-ΔWPRE system for tracing MG reprogramming under Ptbp1-suppression and observed strict non-MG-conversion, similar to previous studies using genetic lineage tracking mouse models. Thus, the AAV9-hGFAP-Cre-ΔWPRE system showed high efficiency and specificity for MG labeling, providing a promising tool for tracing cell fate in vivo.

A map of cis-regulatory modules and constituent transcription factor binding sites in 80% of the mouse genome.

BACKGROUND: Mouse is probably the most important model organism to study mammal biology and human diseases. A better understanding of the mouse genome will help understand the human genome, biology and diseases. However, despite the recent progress, the characterization of the regulatory sequences in the mouse genome is still far from complete, limiting its use to understand the regulatory sequences in the human genome. RESULTS: Here, by integrating binding peaks in ~ 9,000 transcription factor (TF) ChIP-seq datasets that cover 79.9% of the mouse mappable genome using an efficient pipeline, we were able to partition these binding peak-covered genome regions into a cis-regulatory module (CRM) candidate (CRMC) set and a non-CRMC set. The CRMCs contain 912,197 putative CRMs and 38,554,729 TF binding sites (TFBSs) islands, covering 55.5% and 24.4% of the mappable genome, respectively. The CRMCs tend to be under strong evolutionary constraints, indicating that they are likely cis-regulatory; while the non-CRMCs are largely selectively neutral, indicating that they are unlikely cis-regulatory. Based on evolutionary profiles of the genome positions, we further estimated that 63.8% and 27.4% of the mouse genome might code for CRMs and TFBSs, respectively. CONCLUSIONS: Validation using experimental data suggests that at least most of the CRMCs are authentic. Thus, this unprecedentedly comprehensive map of CRMs and TFBSs can be a good resource to guide experimental studies of regulatory genomes in mice and humans.

Notch-dependent DNA cis-regulatory elements and their dose-dependent control of C. elegans stem cell self-renewal.

A long-standing biological question is how DNA cis-regulatory elements shape transcriptional patterns during metazoan development. Reporter constructs, cell culture assays and computational modeling have made major contributions to answering this question, but analysis of elements in their natural context is an important complement. Here, we mutate Notch-dependent LAG-1 binding sites (LBSs) in the endogenous Caenorhabditis elegans sygl-1 gene, which encodes a key stem cell regulator, and analyze the consequences on sygl-1 expression (nascent transcripts, mRNA, protein) and stem cell maintenance. Mutation of one LBS in a three-element cluster approximately halved both expression and stem cell pool size, whereas mutation of two LBSs essentially abolished them. Heterozygous LBS mutant clusters provided intermediate values. Our results lead to two major conclusions. First, both LBS number and configuration impact cluster activity: LBSs act additively in trans and synergistically in cis. Second, the SYGL-1 gradient promotes self-renewal above its functional threshold and triggers differentiation below the threshold. Our approach of coupling CRISPR/Cas9 LBS mutations with effects on both molecular and biological readouts establishes a powerful model for in vivo analyses of DNA cis-regulatory elements.

PCRMS: a database of predicted cis-regulatory modules and constituent transcription factor binding sites in genomes.

More accurate and more complete predictions of cis-regulatory modules (CRMs) and constituent transcription factor (TF) binding sites (TFBSs) in genomes can facilitate characterizing functions of regulatory sequences. Here, we developed a database predicted cis-regulatory modules (PCRMS) (https://cci-bioinfo.uncc.edu) that stores highly accurate and unprecedentedly complete maps of predicted CRMs and TFBSs in the human and mouse genomes. The web interface allows the user to browse CRMs and TFBSs in an organism, find the closest CRMs to a gene, search CRMs around a gene and find all TFBSs of a TF. PCRMS can be a useful resource for the research community to characterize regulatory genomes. Database URL: https://cci-bioinfo.uncc.edu/.

Conservation of dynamic characteristics of transcriptional regulatory elements in periodic biological processes.

BACKGROUND: Cell and circadian cycles control a large fraction of cell and organismal physiology by regulating large periodic transcriptional programs that encompass anywhere from 15 to 80% of the genome despite performing distinct functions. In each case, these large periodic transcriptional programs are controlled by gene regulatory networks (GRNs), and it has been shown through genetics and chromosome mapping approaches in model systems that at the core of these GRNs are small sets of genes that drive the transcript dynamics of the GRNs. However, it is unlikely that we have identified all of these core genes, even in model organisms. Moreover, large periodic transcriptional programs controlling a variety of processes certainly exist in important non-model organisms where genetic approaches to identifying networks are expensive, time-consuming, or intractable. Ideally, the core network components could be identified using data-driven approaches on the transcriptome dynamics data already available. RESULTS: This study shows that a unified set of quantified dynamic features of high-throughput time series gene expression data are more prominent in the core transcriptional regulators of cell and circadian cycles than in their outputs, in multiple organism, even in the presence of external periodic stimuli. Additionally, we observe that the power to discriminate between core and non-core genes is largely insensitive to the particular choice of quantification of these features. CONCLUSIONS: There are practical applications of the approach presented in this study for network inference, since the result is a ranking of genes that is enriched for core regulatory elements driving a periodic phenotype. In this way, the method provides a prioritization of follow-up genetic experiments. Furthermore, these findings reveal something unexpected-that there are shared dynamic features of the transcript abundance of core components of unrelated GRNs that control disparate periodic phenotypes.

Impact of Androgen Receptor Activity on Prostate-Specific Membrane Antigen Expression in Prostate Cancer Cells.

Prostate-specific membrane antigen (PSMA) is an essential molecular regulator of prostate cancer (PCa) progression coded by the FOLH1 gene. The PSMA protein has become an important factor in metastatic PCa diagnosis and radioligand therapy. However, low PSMA expression is suggested to be a resistance mechanism to PSMA-based imaging and therapy. Clinical studies revealed that androgen receptor (AR) inhibition increases PSMA expression. The mechanism has not yet been elucidated. Therefore, this study investigated the effect of activation and inhibition of androgen signaling on PSMA expression levels in vitro and compared these findings with PSMA levels in PCa patients receiving systemic therapy. To this end, LAPC4, LNCaP, and C4-2 PCa cells were treated with various concentrations of the synthetic androgen R1881 and antiandrogens. Changes in FOLH1 mRNA were determined using qPCR. Open access databases were used for ChIP-Seq and tissue expression analysis. Changes in PSMA protein were determined using western blot. For PSMA staining in patients' specimens, immunohistochemistry (IHC) was performed. Results revealed that treatment with the synthetic androgen R1881 led to decreased FOLH1 mRNA and PSMA protein. This effect was partially reversed by antiandrogen treatment. However, AR ChIP-Seq analysis revealed no canonical AR binding sites in the regulatory elements of the FOLH1 gene. IHC analysis indicated that androgen deprivation only resulted in increased PSMA expression in patients with low PSMA levels. The data demonstrate that AR activation and inhibition affects PSMA protein levels via a possible non-canonical mechanism. Moreover, analysis of PCa tissue reveals that low PSMA expression rates may be mandatory to increase PSMA by androgen deprivation.

Three classes of epigenomic regulators converge to hyperactivate the essential maternal gene deadhead within a heterochromatin mini-domain.

The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. Here, we identified Snr1 and Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.