Metazoan Gene Expression Research Articles

Conserved and novel enhancers in the Aedes aegypti single-minded locus recapitulate embryonic ventral midline gene expression.

Transcriptional cis-regulatory modules, e.g., enhancers, control the time and location of metazoan gene expression. While changes in enhancers can provide a powerful force for evolution, there is also significant deep conservation of enhancers for developmentally important genes, with function and sequence characteristics maintained over hundreds of millions of years of divergence. Not well understood, however, is how the overall regulatory composition of a locus evolves, with important outstanding questions such as how many enhancers are conserved vs. novel, and to what extent are the locations of conserved enhancers within a locus maintained? We begin here to address these questions with a comparison of the respective single-minded (sim) loci in the two dipteran species Drosophila melanogaster (fruit fly) and Aedes aegypti (mosquito). sim encodes a highly conserved transcription factor that mediates development of the arthropod embryonic ventral midline. We identify two enhancers in the A. aegypti sim locus and demonstrate that they function equivalently in both transgenic flies and transgenic mosquitoes. One A. aegypti enhancer is highly similar to known Drosophila counterparts in its activity, location, and autoregulatory capability. The other differs from any known Drosophila sim enhancers with a novel location, failure to autoregulate, and regulation of expression in a unique subset of midline cells. Our results suggest that the conserved pattern of sim expression in the two species is the result of both conserved and novel regulatory sequences. Further examination of this locus will help to illuminate how the overall regulatory landscape of a conserved developmental gene evolves.

Enhancer selectivity in space and time: from enhancer-promoter interactions to promoter activation.

The primary regulators of metazoan gene expression are enhancers, originally functionally defined as DNA sequences that can activate transcription at promoters in an orientation-independent and distance-independent manner. Despite being crucial for gene regulation in animals, what mechanisms underlie enhancer selectivity for promoters, and more fundamentally, how enhancers interact with promoters and activate transcription, remain poorly understood. In this Review, we first discuss current models of enhancer-promoter interactions in space and time and how enhancers affect transcription activation. Next, we discuss different mechanisms that mediate enhancer selectivity, including repression, biochemical compatibility and regulation of 3D genome structure. Through 3D polymer simulations, we illustrate how the ability of 3D genome folding mechanisms to mediate enhancer selectivity strongly varies for different enhancer-promoter interaction mechanisms. Finally, we discuss how recent technical advances may provide new insights into mechanisms of enhancer-promoter interactions and how technical biases in methods such as Hi-C and Micro-C and imaging techniques may affect their interpretation.

Distinct negative elongation factor conformations regulate RNA polymerase II promoter-proximal pausing

Metazoan gene expression regulation involves pausing of RNA polymerase (Pol II) in the promoter-proximal region of genes and is stabilized by DSIF and NELF. Upon depletion of elongation factors, NELF appears to accompany elongating Pol II past pause sites; however, prior work indicates that NELF prevents Pol II elongation. Here, we report cryoelectron microscopy structures of Pol II-DSIF-NELF complexes with NELF in two distinct conformations corresponding to paused and poised states. The paused NELF state supports Pol II stalling, whereas the poised NELF state enables transcription elongation as it does not support a tilted RNA-DNA hybrid. Further, the poised NELF state can accommodate TFIIS binding to Pol II, allowing for Pol II reactivation at paused or backtracking sites. Finally, we observe that the NELF-A tentacle interacts with the RPB2 protrusion and is necessary for pausing. Our results define how NELF can support pausing, reactivation, and elongation by Pol II.

Role of RNA Polymerase II Promoter-Proximal Pausing in Viral Transcription.

The promoter-proximal pause induced by the binding of the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) to RNAP II is a key step in the regulation of metazoan gene expression. It helps maintain a permissive chromatin landscape and ensures a quick transcriptional response from stimulus-responsive pathways such as the innate immune response. It is also involved in the biology of several RNA viruses such as the human immunodeficiency virus (HIV), the influenza A virus (IAV) and the hepatitis delta virus (HDV). HIV uses the pause as one of its mechanisms to enter and maintain latency, leading to the creation of viral reservoirs resistant to antiretrovirals. IAV, on the other hand, uses the pause to acquire the capped primers necessary to initiate viral transcription through cap-snatching. Finally, the HDV RNA genome is transcribed directly by RNAP II and requires the small hepatitis delta antigen to displace NELF from the polymerase and overcome the transcriptional block caused by RNAP II promoter-proximal pausing. In this review, we will discuss the RNAP II promoter-proximal pause and the roles it plays in the life cycle of RNA viruses such as HIV, IAV and HDV.

Large‐scale analysis of Drosophila core promoter function using synthetic promoters

The core promoter plays a central role in setting metazoan gene expression levels, but how exactly it “computes” expression remains poorly understood. To dissect its function, we carried out a comprehensive structure–function analysis in Drosophila. First, we performed a genome‐wide bioinformatic analysis, providing an improved picture of the sequence motifs architecture. We then measured synthetic promoters’ activities of ~3,000 mutational variants with and without an external stimulus (hormonal activation), at large scale and with high accuracy using robotics and a dual luciferase reporter assay. We observed a strong impact on activity of the different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, nucleosome positioning, and flanking sequences. A linear combination of the individual motif features largely accounts for the combinatorial effects on core promoter activity. These findings shed new light on the quantitative assessment of gene expression in metazoans.

The Integrator Complex Attenuates Promoter-Proximal Transcription at Protein-Coding Genes.

The transition of RNA polymerase II (Pol II) from initiation to productive elongation is a central, regulated step in metazoan gene expression. At many genes, Pol II pauses stably in early elongation, remaining engaged with the 25- to 60-nt-long nascent RNA for many minutes while awaiting signals for release into the gene body. However, 15%-20% of genes display highly unstable promoter Pol II, suggesting that paused polymerase might dissociate from template DNA at these promoters and release a short, non-productive mRNA. Here, we report that paused Pol II can be actively destabilized by the Integrator complex. Specifically, we present evidence that Integrator utilizes its RNA endonuclease activity to cleave nascent RNA and drive termination of paused Pol II. These findings uncover a previously unappreciated mechanism of metazoan gene repression, akin to bacterial transcription attenuation, wherein promoter-proximal Pol II is prevented from entering productive elongation through factor-regulated termination.

Correction: A sequence level model of an intact locus predicts the location and function of nonadditive enhancers.

[This corrects the article DOI: 10.1371/journal.pone.0180861.].

A sequence level model of an intact locus predicts the location and function of nonadditive enhancers.

Metazoan gene expression is controlled through the action of long stretches of noncoding DNA that contain enhancers—shorter sequences responsible for controlling a single aspect of a gene’s expression pattern. Models built on thermodynamics have shown how enhancers interpret protein concentration in order to determine specific levels of gene expression, but the emergent regulatory logic of a complete regulatory locus shows qualitative and quantitative differences from isolated enhancers. Such differences may arise from steric competition limiting the quantity of DNA that can simultaneously influence the transcription machinery. We incorporated this competition into a mechanistic model of gene regulation, generated efficient algorithms for this computation, and applied it to the regulation of Drosophila even-skipped (eve). This model finds the location of enhancers and identifies which factors control the boundaries of eve expression. This model predicts a new enhancer that, when assayed in vivo, drives expression in a non-eve pattern. Incorporation of chromatin accessibility eliminates this inconsistency.

Paused RNA polymerase II inhibits new transcriptional initiation.

RNA polymerase II (Pol II) pauses downstream of the transcription initiation site before beginning productive elongation. This pause is a key component of metazoan gene expression regulation. Some promoters have a strong disposition for Pol II pausing and often mediate faster, more synchronous changes in expression. This requires multiple rounds of transcription and thus cannot rely solely on pause release. However, it is unclear how pausing affects the initiation of new transcripts during consecutive rounds of transcription. Using our recently developed ChIP-nexus method, we find that Pol II pausing inhibits new initiation. We propose that paused Pol II helps prevent new initiation between transcription bursts, which may reduce noise.

Nuclear phosphoinositide regulation of chromatin.

Phospholipid signaling has clear connections to a wide array of cellular processes, particularly in gene expression and in controlling the chromatin biology of cells. However, most of the work elucidating how phospholipid signaling pathways contribute to cellular physiology have studied cytoplasmic membranes, while relatively little attention has been paid to the role of phospholipid signaling in the nucleus. Recent work from several labs has shown that nuclear phospholipid signaling can have important roles that are specific to this cellular compartment. This review focuses on the nuclear phospholipid functions and the activities of phospholipid signaling enzymes that regulate metazoan chromatin and gene expression. In particular, we highlight the roles that nuclear phosphoinositides play in several nuclear-driven physiological processes, such as differentiation, proliferation, and gene expression. Taken together, the recent discovery of several specifically nuclear phospholipid functions could have dramatic impact on our understanding of the fundamental mechanisms that enable tight control of cellular physiology.

Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity.

Histone H3 lysine 36 methylation (H3K36me) is thought to participate in a host of co-transcriptional regulatory events. To study the function of this residue independent from the enzymes that modify it, we used a 'histone replacement' system in Drosophila to generate a non-modifiable H3K36 lysine-to-arginine (H3K36R) mutant. We observed global dysregulation of mRNA levels in H3K36R animals that correlates with the incidence of H3K36me3. Similar to previous studies, we found that mutation of H3K36 also resulted in H4 hyperacetylation. However, neither cryptic transcription initiation, nor alternative pre-mRNA splicing, contributed to the observed changes in expression, in contrast with previously reported roles for H3K36me. Interestingly, knockdown of the RNA surveillance nuclease, Xrn1, and members of the CCR4-Not deadenylase complex, restored mRNA levels for a class of downregulated, H3K36me3-rich genes. We propose a post-transcriptional role for modification of replication-dependent H3K36 in the control of metazoan gene expression.

Gdown1 Associates Efficiently with RNA Polymerase II after Promoter Clearance and Displaces TFIIF during Transcript Elongation.

Pausing during the earliest stage of transcript elongation by RNA polymerase II (Pol II) is a nearly universal control point in metazoan gene expression. The substoichiometric Pol II subunit Gdown1 facilitates promoter proximal pausing in vitro in extract-based transcription reactions, out-competes the initiation/elongation factor TFIIF for binding to free Pol II and co-localizes with paused Pol II in vivo. However, we have shown that Gdown1 cannot functionally associate with the Pol II preinitiation complex (PIC), which contains TFIIF. In the present study, we determined at what point after initiation Gdown1 can associate with Pol II and how rapidly this competition with TFIIF occurs. We show that, as with the PIC, Gdown1 cannot functionally load into open complexes or complexes engaged in abortive synthesis of very short RNAs. Gdown1 can load into early elongation complexes (EECs) with 5–9 nt RNAs, but efficient association with EECs does not take place until the point at which the upstream segment of the long initial transcription bubble reanneals. Tests of EECs assembled on a series of promoter variants confirm that this bubble collapse transition, and not transcript length, modulates Gdown1 functional affinity. Gdown1 displaces TFIIF effectively from all complexes downstream of the collapse transition, but this displacement is surprisingly slow: complete loss of TFIIF stimulation of elongation requires 5 min of incubation with Gdown1. The relatively slow functional loading of Gdown1 in the presence of TFIIF suggests that Gdown1 works in promoter-proximal pausing by locking in the paused state after elongation is already antagonized by other factors, including DSIF, NELF and possibly the first downstream nucleosome.

A novel model of THO/TREX loading onto target RNAs in metazoan gene expression.

The THO/TREX complex consists of several conserved subunits and is required for mRNA export. In metazoans, THO/TREX binds a subset of mRNAs during RNA splicing, and facilitates their nuclear export. How THO/TREX selects RNA targets is, however, incompletely understood. In our recent study, we reported that THO is loaded onto Piwi-interacting RNA (piRNA) precursor transcripts independent of splicing, and facilitates convergent transcription in Drosophila ovary. The precursors are later processed into mature piRNAs, small noncoding RNAs that silence transposable elements (TEs). We observed that piRNAs originating from dual-strand clusters, where precursors are transcribed from both strands, were specifically affected by THO mutation. Analysis of THO-bound RNAs showed enrichment of dual-strand cluster transcripts. Interestingly, THO loading onto piRNA precursors was dependent on Cutoff (Cuff), which comprises the Rhino-Deadlock-Cutoff (RDC) complex that is recruited to dual-strand clusters by recognizing H3K9me3 and licenses convergent transcription from he cluster. We also found that THO mutation affected transcription from dual-strand clusters. Therefore, we concluded that THO/TREX is recruited to dual-strand piRNA clusters, independent of splicing events, via multi-protein interactions with chromatin structure. Then, it facilitates transcription likely by suppressing premature termination to ensure adequate expression of piRNA precursors. [BMB Reports 2016; 49(7): 355-356]

DNA-Encoded Chromatin Structural Intron Boundary Signals Identify Conserved Genes with Common Function

The regulation of metazoan gene expression occurs in part by pre-mRNA splicing into mature RNAs. Signals affecting the efficiency and specificity with which introns are removed have not been completely elucidated. Splicing likely occurs cotranscriptionally, with chromatin structure playing a key regulatory role. We calculated DNA encoded nucleosome occupancy likelihood (NOL) scores at the boundaries between introns and exons across five metazoan species. We found that (i) NOL scores reveal a sequence-based feature at the introns on both sides of the intron-exon boundary; (ii) this feature is not part of any recognizable consensus sequence; (iii) this feature is conserved throughout metazoa; (iv) this feature is enriched in genes sharing similar functions: ATPase activity, ATP binding, helicase activity, and motor activity; (v) genes with these functions exhibit different genomic characteristics; (vi) in vivo nucleosome positioning data confirm ontological enrichment at this feature; and (vii) genes with this feature exhibit unique dinucleotide distributions at the intron-exon boundary. The NOL scores point toward a physical property of DNA that may play a role in the mechanism of pre-mRNA splicing. These results provide a foundation for identification of a new set of regulatory DNA elements involved in splicing regulation.

Functional Impact of RNA editing and ADARs on regulation of gene expression: perspectives from deep sequencing studies.

Cells regulate gene expression at multiple levels leading to a balance between robustness and complexity within their proteome. One core molecular step contributing to this important balance during metazoan gene expression is RNA editing, such as the co-transcriptional recoding of RNA transcripts catalyzed by the adenosine deaminse acting on RNA (ADAR) family of enzymes. Understanding of the adenosine-to-inosine RNA editing process has been broadened considerably by the next generation sequencing (NGS) technology, which allows for in-depth demarcation of an RNA editome at nucleotide resolution. However, critical issues remain unresolved with regard to how RNA editing cooperates with other transcript-associated events to underpin regulated gene expression. Here we review the growing body of evidence, provided by recent NGS-based studies, that links RNA editing to other mechanisms of post-transcriptional RNA processing and gene expression regulation including alternative splicing, transcript stability and localization, and the biogenesis and function of microRNAs (miRNAs). We also discuss the possibility that systematic integration of NGS data may be employed to establish the rules of an “RNA editing code”, which may give us new insights into the functional consequences of RNA editing.

SnapShot-Seq: A Method for Extracting Genome-Wide, In Vivo mRNA Dynamics from a Single Total RNA Sample

mRNA synthesis, processing, and destruction involve a complex series of molecular steps that are incompletely understood. Because the RNA intermediates in each of these steps have finite lifetimes, extensive mechanistic and dynamical information is encoded in total cellular RNA. Here we report the development of SnapShot-Seq, a set of computational methods that allow the determination of in vivo rates of pre-mRNA synthesis, splicing, intron degradation, and mRNA decay from a single RNA-Seq snapshot of total cellular RNA. SnapShot-Seq can detect in vivo changes in the rates of specific steps of splicing, and it provides genome-wide estimates of pre-mRNA synthesis rates comparable to those obtained via labeling of newly synthesized RNA. We used SnapShot-Seq to investigate the origins of the intrinsic bimodality of metazoan gene expression levels, and our results suggest that this bimodality is partly due to spillover of transcriptional activation from highly expressed genes to their poorly expressed neighbors. SnapShot-Seq dramatically expands the information obtainable from a standard RNA-Seq experiment.

Stable Pausing by RNA Polymerase II Provides an Opportunity to Target and Integrate Regulatory Signals

Metazoan gene expression is often regulated after the recruitment of RNA polymerase II (Pol II) to promoters, through the controlled release of promoter-proximally paused Pol II into productive RNA synthesis. Despite the prevalence of paused Pol II, very little is known about the dynamics of these early elongation complexes or the fate of the short transcription start site-associated (tss) RNAs they produce. Here, we demonstrate that paused elongation complexes can be remarkably stable, with half-lives exceeding 15 min at genes with inefficient pause release. Promoter-proximal termination by Pol II is infrequent, and released tssRNAs are targeted for rapid degradation. Further, we provide evidence that the predominant tssRNA species observed are nascent RNAs held within early elongation complexes. We propose that stable pausing of polymerase provides a temporal window of opportunity for recruitment of factors to modulate gene expression and that the nascent tssRNA represents an appealing target for these interactions.

Isolation and functional characterization of P-TEFb-associated factors that control general and HIV-1 transcriptional elongation

Originally identified as a factor crucial for RNA polymerase (Pol) II transcriptional elongation of cellular genes, the P-TEFb kinase was subsequently shown to also serve as a specific host co-factor required for HIV-1 transcription. Recruited by either the bromodomain protein Brd4 to cellular promoters for general transcription or the HIV-1 Tat protein to the viral LTR for activated HIV-1 transcription, P-TEFb stimulates the processivity of Pol II through phosphorylating the C-terminal domain of Pol II and a pair of negative elongation factors, leading to the synthesis of full-length transcripts. However, abundant evidence indicates that P-TEFb does not act alone in the cell and that all of its known biological functions are likely mediated through the interactions with various regulators. Although a number of P-TEFb-associated factors have already been identified, there are likely more yet to be discovered. Given that P-TEFb plays an essential role in HIV-1 transcription, a major challenge facing the field is to identify all the P-TEFb-associated factors and determine how they may modulate Tat-transactivation and HIV-1 replication. Described here is a set of experimental procedures that have not only enabled us to isolate and identify several P-TEFb-associated factors, but also provided the means to characterize their biochemical functions in HIV-1 transcriptional control. In light of the recent demonstrations that transcriptional elongation plays a much more important role in controlling metazoan gene expression than previously thought, the techniques presented here will also be useful for analyzing Pol II elongation of cellular genes.

Coordinated Chromatin Control: Structural and Functional Linkage of DNA and Histone Methylation

One of the most fundamental questions in the control of gene expression in mammals is how epigenetic methylation patterns of DNA and histones are established, erased, and recognized. This central process in controlling metazoan gene expression includes coordinated covalent modifications of DNA and its associated histones. This review focuses on recent developments in characterizing the functional links between the methylation status of the DNA and of two particularly important histone marks. Mammalian DNA methylation is intricately connected to the presence of unmodified lysine 4 and methylated lysine 9 residues in histone H3. An interconnected network of methyltransferases, demethylases, and accessory proteins is responsible for changing or maintaining the modification status of specific regions of chromatin. The structural and functional interactions among members of this network are critical to processes that include imprinting and differentiation, dysregulation of which is associated with disorders ranging from inflammation to cancer.

Lifespan extension and increased pumping rate accompany pharyngeal muscle‐specific expression of nfi‐1 in C. elegans

Caenorhabditis elegans nfi-1 belongs to the Nuclear Factor I (NFI) family of transcription factors known to regulate metazoan gene expression and development. We showed previously that loss of nfi-1 in worms results in multiple behavioral defects; slower pharyngeal pumping rate, impaired egg laying, defective motility, and a shortened life span. Here, we generated cell-type specific transgenic worms to determine the cells in which nfi-1 must be expressed to rescue the pharyngeal pumping defect. Expression of nfi-1 from the pharyngeal muscle-specific myo-2 promoter, but not from the F25B3.3 or myo-3 promoters, rescued the pharyngeal pumping defect of nfi-1 worms. Surprisingly, myo-2-driven nfi-1 expression also rescued the shortened lifespan of nfi-1 worms, demonstrating a possible cell-autonomous role of nfi-1 in pharyngeal muscle for both phenotypes. We propose some relationships between the pharyngeal pumping and lifespan phenotypes and potential mechanisms of nfi-1 function.