Promoter-proximal Site Research Articles

Redundant pathways for removal of defective RNA polymerase II complexes at a promoter-proximal pause checkpoint.

The biological purpose of Integrator and RNA polymerase II (RNAPII) promoter-proximal pausing remains uncertain. Here, we show that loss of INTS6 in human cells results in increased interaction of RNAPII with proteins that can mediate its dissociation from the DNA template, including the CRL3ARMC5 E3 ligase, which ubiquitylates CTD serine5-phosphorylated RPB1 for degradation. ARMC5-dependent RNAPII ubiquitylation is activated by defects in factors acting at the promoter-proximal pause, including Integrator, DSIF, and capping enzyme. This ARMC5 checkpoint normally curtails a sizeable fraction of RNAPII transcription, and ARMC5 knockout cells produce more uncapped transcripts. When both the Integrator and CRL3ARMC5 turnover mechanisms are compromised, cell growth ceases and RNAPII with high pausing propensity disperses from the promoter-proximal pause site into the gene body. These data support a model in which CRL3ARMC5 functions alongside Integrator in a checkpoint mechanism that removes faulty RNAPII complexes at promoter-proximal pause sites to safeguard transcription integrity.

Cockayne syndrome B protein is implicated in transcription and associated chromatin dynamics in homeostatic and genotoxic conditions.

The integrity of the actively transcribed genome against helix-distorting DNA lesions relies on a multilayered cellular response that enhances Transcription-Coupled Nucleotide Excision Repair (TC-NER). When defective, TC-NER is causatively associated with Cockayne-Syndrome (CS), a rare severe human progeroid disorder. Although the presence of unresolved transcription-blocking lesions is considered a driver of the aging process, the molecular features of the transcription-driven response to genotoxic stress in CS-B cells remain largely unknown. Here, an in-depth view of the transcriptional and associated chromatin dynamics that occur in CS-B cells illuminates the role of CSB therein. By employing high-throughput genome-wide approaches, we observed that absence of a functional CSB protein results in a delay in transcription progression, more positioned +1 nucleosomes, and less dynamic chromatin structure, compared to normal cells. We found that early after exposure to UV, CS-B cells released RNA polymerase II (RNAPII) from promoter-proximal pause sites into elongation. However, the magnitude of this response and the progression of RNAPII were reduced compared to normal counterparts. Notably, we detected increased post-UV retainment of unprocessed nascent RNA transcripts and chromatin-associated elongating RNAPII molecules. Contrary to the prevailing models, we found that transcription initiation is operational in CS-B fibroblasts early after UV and that chromatin accessibility showed a marginal increase. Our study provides robust evidence for the role of CSB in shaping the transcription and chromatin landscape both in homeostasis and in response to genotoxic insults, which is independent of its known role in TC-NER, and which may underlie major aspects of the CS phenotype.

The role(s) of NF-Y in development and differentiation.

NF-Y is a conserved sequence-specific trimeric Transcription Factor -TF- binding to the CCAAT element. We review here the role(s) in development, from pre-implantation embryo to terminally differentiated tissues, by rationalizing and commenting on genetic, genomic, epigenetic and biochemical studies. This effort brings to light the impact of NF-YA isoforms on stemness and differentiation, as well as binding to distal vs promoter proximal sites and connections with selected TFs.

Promoter-centred chromatin interactions associated with EVI1 expression in EVI1+3q- myeloid leukaemia cells.

EVI1 expression is associated with poor prognosis in myeloid leukaemia, which can result from Chr.3q alterations that juxtapose enhancers to induce EVI1 expression via long-range chromatin interactions. More often, however, EVI1 expression occurs unrelated to 3q alterations, and it remained unclear if, in these cases, EVI1 expression is similarly caused by aberrant enhancer activation. Here, we report that, in EVI1+3q- myeloid leukaemia cells, the EVI1 promoter interacts via long-range chromatin interactions with promoters of distally located, active genes, rather than with enhancer elements. Unlike in 3q+ cells, EVI1 expression and long-range interactions appear to not depend on CTCF/cohesin, though EVI1+3q- cells utilise an EVI1 promoter-proximal site to enhance its expression that is also involved in CTCF-mediated looping in 3q+ cells. Long-range interactions in 3q- cells connect EVI1 to promoters of multiple genes, whose transcription correlates with EVI1 in EVI1+3q- cell lines, suggesting a shared mechanism of transcriptional regulation. In line with this, CRISPR interference-induced silencing of two of these sites minimally, but consistently reduced EVI1 expression. Together, we provide novel evidence of features associated with EVI1 expression in 3q- leukaemia and consolidate the view that EVI1 in 3q- leukaemia is largely promoter-driven, potentially involving long-distance promoter clustering.

Model-based characterization of the equilibrium dynamics of transcription initiation and promoter-proximal pausing in human cells.

In metazoans, both transcription initiation and the escape of RNA polymerase (RNAP) from promoter-proximal pausing are key rate-limiting steps in gene expression. These processes play out at physically proximal sites on the DNA template and appear to influence one another through steric interactions. Here, we examine the dynamics of these processes using a combination of statistical modeling, simulation, and analysis of real nascent RNA sequencing data. We develop a simple probabilistic model that jointly describes the kinetics of transcription initiation, pause-escape, and elongation, and the generation of nascent RNA sequencing read counts under steady-state conditions. We then extend this initial model to allow for variability across cells in promoter-proximal pause site locations and steric hindrance of transcription initiation from paused RNAPs. In an extensive series of simulations, we show that this model enables accurate estimation of initiation and pause-escape rates. Furthermore, we show by simulation and analysis of real data that pause-escape is often strongly rate-limiting and that steric hindrance can dramatically reduce initiation rates. Our modeling framework is applicable to a variety of inference problems, and our software for estimation and simulation is freely available.

Promoter-proximal regulation of gene transcription: Key factors involved and emerging role of general transcription factors in assisting productive elongation

The pausing of RNA polymerase II (Pol II) at the promoter-proximal sites is a key rate-limiting step in gene expression. Cells have dedicated a specific set of proteins that sequentially establish pause and then release the Pol II from promoter-proximal sites. A well-controlled pausing and subsequent release of Pol II is crucial for the fine tuning of expression of genes including signal-responsive and developmentally-regulated ones. The release of paused Pol II broadly involves its transition from initiation to elongation. In this review article, we will discuss the phenomenon of Pol II pausing, the underlying mechanism, and also the role of different known factors, with an emphasis on general transcription factors, involved in this overall regulation. We will further discuss some recent findings suggesting a possible role (underexplored) of initiation factors in assisting the transition of transcriptionally-engaged paused Pol II into productive elongation.

Targeting RNA elongation in endothelial-to-mesenchymal transition

Abstract Background and purpose EndMT describes a process in which endothelial cells lose their endothelial features such as the maintenance of the vascular barrier function and trans-differentiate into mesenchymal like cells. ECs which have underwent EndMT are enriched in vulnerable plaques in humans. Several stress stimuli such as hypoxia or inflammation demand immediate induction of adaptive gene expression to improve cell survival. In contrast to the usual induction of gene expression which requires several hours, release of paused RNA Polymerase II (RNAPII) from promoter allows rapid gene induction via RNA elongation. This rapid response mechanism is mediated by the super elongation complex (SEC), which is built around the scaffold proteins AFF1 and AFF4. Here, we hypothesized that RNA elongation regulates EndMT and therefore investigated on the contribution of the SEC scaffold proteins during the process of EndMT. Methods and results After induction of EndMT in human primary endothelial cells (EC) the elongation factors AFF1 and AFF4 were induced (AFF1: 1.9-fold and AFF4: 2.7-fold, P<0.05). Overexpression of AFF4 resulted in increased expression of the mesenchymal marker genes SM22 (1.8-fold, P<0.05) and VCAN (2.0-fold, P<0.05). Accordingly, treatment of EC with two small molecules inhibiting the SEC reduced the expression of several mesenchymal markers SM22 (−87.6%, P<0.05) and Calponin (−93.8%, P<0.05) during EndMT. After induction of EndMT, a large number of newly synthesized RNA transcripts were upregulated, indicating regulation of gene expression on the level of RNA elongation during EndMT. Sequencing of newly transcribed RNA (4sU-sequencing) after treatment with the SEC inhibitors revealed a lower transcript density downstream of promoter sites, suggesting a decreased RNA elongation rate in ECs. SEC inhibitors decreased the expression of the majority of EndMT-induced transcripts (63.6%). Among these rapid induced genes we found the mRNA of RE1 (+10,8-fold). We validated the regulation of RE1 by qPCR (2.8-fold, P<0.05) and single-cell sequencing. Chromatin immunoprecipitation (ChIP) sequencing revealed a reduced occupation of RNAPII in proximity of promotor sites of RE1 upon induction of EndMT. These data suggest a SEC-mediated regulation on the level of RNA elongation of RE1 during EndMT. Conclusion SEC scaffold proteins AFF1 and AFF4 are upregulated during EndMT. Targeting the SEC during EndMT reduces the expression of common mesenchymal markers, consistent with an overall higher occupancy of paused RNAPII at promoter-proximal sites. We identify RE1 as a rapidly induced gene during EndMT that is mediated by the SEC scaffold proteins AFF1 and AFF4. Our findings suggest that RNA elongation is an inducer of EndMT. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)

Mammalian PERIOD2 regulates H2A.Z incorporation in chromatin to orchestrate circadian negative feedback.

Mammalian circadian oscillators are built on a feedback loop in which the activity of the transcription factor CLOCK-BMAL1 is repressed by the PER-CRY complex. Here, we show that murine Per-/- fibroblasts display aberrant nucleosome occupancy around transcription start sites (TSSs) and at promoter-proximal and distal CTCF sites due to impaired histone H2A.Z deposition. Knocking out H2A.Z mimicked the Per null chromatin state and disrupted cellular rhythms. We found that endogenous mPER2 complexes retained CTCF as well as the specific H2A.Z-deposition chaperone YL1-a component of the ATP-dependent remodeler SRCAP and p400-TIP60 complex. While depleting YL1 or mutating chaperone-binding sites on H2A.Z lengthened the circadian period, H2A.Z deletion abrogated BMAL1 chromatin recruitment and promoted its proteasomal degradation. We propose that a PER2-mediated H2A.Z deposition pathway (1) compacts CLOCK-BMAL1 binding sites to establish negative feedback, (2) organizes circadian chromatin landscapes using CTCF and (3) bookmarks genomic loci for BMAL1 binding to impinge on the positive arm of the subsequent cycle.

MafB, WDR77, and ß-catenin interact with each other and have similar genome association profiles.

MafB (a bZIP transcription factor), ß-catenin (the ultimate target of the Wnt signal transduction pathway that acts as a transcriptional co-activator of LEF/TCF proteins), and WDR77 (a transcriptional co-activator of multiple hormone receptors) are important for breast cellular transformation. Unexpectedly, these proteins interact directly with each other, and they have similar genomic binding profiles. Furthermore, while some of these common target sites coincide with those bound by LEF/TCF, the majority are located just downstream of transcription initiation sites at a position near paused RNA polymerase (Pol II) and the +1 nucleosome. Occupancy levels of these factors at these promoter-proximal sites are strongly correlated with the level of paused Pol II and transcriptional activity.

Dynamic Runx1 chromatin boundaries affect gene expression in hematopoietic development

The transcription factor RUNX1 is a critical regulator of developmental hematopoiesis and is frequently disrupted in leukemia. Runx1 is a large, complex gene that is expressed from two alternative promoters under the spatiotemporal control of multiple hematopoietic enhancers. To dissect the dynamic regulation of Runx1 in hematopoietic development, we analyzed its three-dimensional chromatin conformation in mouse embryonic stem cell (ESC) differentiation cultures. Runx1 resides in a 1.1 Mb topologically associating domain (TAD) demarcated by convergent CTCF motifs. As ESCs differentiate to mesoderm, chromatin accessibility, Runx1 enhancer-promoter (E-P) interactions, and CTCF-CTCF interactions increase in the TAD, along with initiation of Runx1 expression from the P2 promoter. Differentiation to hematopoietic progenitor cells is associated with the formation of tissue-specific sub-TADs over Runx1, a shift in E-P interactions, P1 promoter demethylation, and robust expression from both Runx1 promoters. Deletion of promoter-proximal CTCF sites at the sub-TAD boundaries has no obvious effects on E-P interactions but leads to partial loss of domain structure, mildly affects gene expression, and delays hematopoietic development. Together, our analysis of gene regulation at a large multi-promoter developmental gene reveals that dynamic sub-TAD chromatin boundaries play a role in establishing TAD structure and coordinated gene expression.

Two distinct mechanisms of RNA polymerase II elongation stimulation in vivo

Transcription by RNA polymerase II (RNA Pol II) relies on the elongation factors PAF1 complex (PAF), RTF1, and SPT6. Here, we use rapid factor depletion and multi-omics analysis to investigate how these elongation factors influence RNA Pol II elongation activity in human cells. Whereas depletion of PAF subunits PAF1 and CTR9 has little effect on cellular RNA synthesis, depletion of RTF1 or SPT6 strongly compromises RNA Pol II activity, albeit in fundamentally different ways. RTF1 depletion decreases RNA Pol II velocity, whereas SPT6 depletion impairs RNA Pol II progression through nucleosomes. These results show that distinct elongation factors stimulate either RNA Pol II velocity or RNA Pol II progression through chromatin invivo. Further analysis provides evidence for two distinct barriers to early elongation: the promoter-proximal pause site and the+1 nucleosome. It emerges that the first barrier enables loading of elongation factors that are required to overcome the second and subsequent barriers to transcription.

A Large Insertion Domain in the Rho Factor From a Low G + C, Gram-negative Bacterium is Critical for RNA Binding and Transcription Termination Activity

Rho-dependent termination of transcription (RDTT) is a critical regulatory mechanism specific to bacteria. In a subset of species including most Actinobacteria and Bacteroidetes, the Rho factor contains a large, poorly conserved N-terminal insertion domain (NID) of cryptic function. To date, only two NID-bearing Rho factors from high G + C Actinobacteria have been thoroughly characterized. Both can trigger RDTT at promoter-proximal sites or with structurally constrained transcripts that are unsuitable for the archetypal, NID-less Rho factor of Escherichia coli (EcRho). Here, we provide the first biochemical characterization of a NID-bearing Rho factor from a low G + C bacterium. We show that Bacteroides fragilis Rho (BfRho) is a bona fide RNA-dependent NTPase motor able to unwind long RNA:DNA duplexes and to disrupt transcription complexes. The large NID (~40% of total mass) strongly increases BfRho affinity for RNA, is strictly required for RDTT, but does not promote RDTT at promoter-proximal sites or with a structurally constrained transcript. Furthermore, the NID does not preclude modulation of RDTT by transcription factors NusA and NusG or by the Rho inhibitor bicyclomycin. Although the NID contains a prion-like Q/N-rich motif, it does not spontaneously trigger formation of β-amyloids. Thus, despite its unusually large RNA binding domain, BfRho behaves more like the NID-less EcRho than NID-bearing counterparts from high G + C Actinobacteria. Our data highlight the evolutionary plasticity of Rho’s N-terminal region and illustrate how RDTT is adapted to distinct genomic contents.

Regulation of fiber-specific actin expression by the Drosophila SRF ortholog Blistered.

ABSTRACTSerum response factor (SRF) has an established role in controlling actin homeostasis in mammalian cells, yet its role in non-vertebrate muscle development has remained enigmatic. Here, we demonstrate that the single Drosophila SRF ortholog, termed Blistered (Bs), is expressed in all adult muscles, but Bs is required for muscle organization only in the adult indirect flight muscles. Bs is a direct activator of the flight muscle actin gene Act88F, via a conserved promoter-proximal binding site. However, Bs only activates Act88F expression in the context of the flight muscle regulatory program provided by the Pbx and Meis orthologs Extradenticle and Homothorax, and appears to function in a similar manner to mammalian SRF in muscle maturation. These studies place Bs in a regulatory framework where it functions to sustain the flight muscle phenotype in Drosophila. Our studies uncover an evolutionarily ancient role for SRF in regulating muscle actin expression, and provide a model for how SRF might function to sustain muscle fate downstream of pioneer factors.

Targeting Processive Transcription Elongation via SEC Disruption for MYC-Induced Cancer Therapy

The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.

Fine‐Tuning of an Evolutionarily Conserved Histone Chaperone, FACT, by Ubiquitin‐Proteasome System, and its Targeting to the Active Gene by mRNA Capping Machinery to Regulate Transcriptional Elongation

Facilitates Chromatin Transcription (FACT), an evolutionarily conserved histone chaperone involved in multiple cellular processes, is upregulated in cancers, and its downregulation is associated with cellular death. However, it is not clearly understood how FACT is fine‐tuned for normal cellular functions. Here, we have used a variety of biochemical, genetic, and molecular biological methodologies to understand regulation of FACT and its abundance. We find that the FACT subunit, Spt16, is ubiquitylated by San1 (an E3 ubiquitin ligase) and degraded by the 26S proteasome. Enhanced abundance of Spt16 in the absence of San1 impairs transcriptional elongation. Likewise, decreased abundance of Spt16 also reduces transcription. Thus, an optimal level of Spt16 is required for efficient transcriptional elongation, which is maintained by San1 via ubiquitylation and proteasomal degradation. Consistently, San1 associates with the coding sequences of active genes to regulate Spt16's abundance. Further, our results reveal that enhanced abundance of Spt16 in the absence of San1 impairs chromatin reassembly at the coding sequence, similar to the results seen following inactivation of Spt16. Efficient chromatin reassembly enhances the fidelity of transcriptional elongation. These results demonstrate for the first time the fine‐tuning of FACT by ubiquitin proteasome system in promoting chromatin reassembly in the wake of elongating RNA polymerase II and transcriptional elongation. Further, it is not clear how FACT is targeted to the active gene. Our recent results reveal that FACT is targeted to the active gene by an mRNA capping enzyme, Cet1, independently of its mRNA capping activity. Subsequently, FACT enhances the release of paused RNA polymerase II from promoter proximal site via Paf1 complex (RNA polymerase II‐associated factor 1 complex) to promote transcriptional elongation. Collectively, these results shed much light on the regulation of FACT and its abundance in controlling transcription, thus providing novel regulatory mechanisms of gene expression.Support or Funding InformationNIH 1R15GM088798‐01 and 2R15GM088798‐02This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Herpes Simplex Virus 1 Dramatically Alters Loading and Positioning of RNA Polymerase II on Host Genes Early in Infection.

Herpes simplex virus 1 (HSV-1) transcription is mediated by cellular RNA polymerase II (Pol II). Recent studies investigating how Pol II transcription of host genes is altered after HSV-1 are conflicting. Chromatin immunoprecipitation sequencing (ChIP-seq) studies suggest that Pol II is almost completely removed from host genes at 4 h postinfection (hpi), while 4-thiouridine (4SU) labeling experiments show that host transcription termination is extended at 7 hpi, implying that a significant amount of Pol II remains associated with host genes in infected cells. To address this discrepancy, we used precision nuclear run-on analysis (PRO-seq) to determine the location of Pol II to single-base-pair resolution in combination with quantitative reverse transcription-PCR (qRT-PCR) analysis at 3 hpi. HSV-1 decreased Pol II on approximately two-thirds of cellular genes but increased Pol II on others. For more than 85% of genes for which transcriptional termination could be statistically assessed, Pol II was displaced to positions downstream of the normal termination zone, suggesting extensive termination defects. Pol II amounts at the promoter, promoter-proximal pause site, and gene body were also modulated in a gene-specific manner. qRT-PCR of selected RNAs showed that HSV-1-induced extension of the termination zone strongly correlated with decreased RNA and mRNA accumulation. However, HSV-1-induced increases of Pol II occupancy on genes without termination zone extension correlated with increased cytoplasmic mRNA. Functional grouping of genes with increased Pol II occupancy suggested an upregulation of exosome secretion and downregulation of apoptosis, both of which are potentially beneficial to virus production.IMPORTANCE This study provides a map of RNA polymerase II location on host genes after infection with HSV-1 with greater detail than previous ChIP-seq studies and rectifies discrepancies between ChIP-seq data and 4SU labeling experiments with HSV-1. The data show the effects that a given change in RNA Pol II location on host genes has on the abundance of different RNA types, including nuclear, polyadenylated mRNA and cytoplasmic, polyadenylated mRNA. It gives a clearer understanding of how HSV-1 augments host transcription of some genes to provide an environment favorable to HSV-1 replication.

Tyrosine-1 of RNA Polymerase II CTD Controls Global Termination of Gene Transcription in Mammals

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is composed of a repetition of YSPTSPS heptads and functions as a loading platform for protein complexes that regulate transcription, splicing, and maturation of RNAs. Here, we studied mammalian CTD mutants to analyze the function of tyrosine1 residues in the transcription cycle. Mutation of 3/4 of the tyrosine residues (YFFF mutant) resulted in a massive read-through transcription phenotype in the antisense direction of promoters as well as in the 3' direction several hundred kilobases downstream of genes. The YFFF mutant shows reduced Pol II at promoter-proximal pause sites, a loss of interaction with the Mediator and Integrator complexes, and impaired recruitment of these complexes to chromatin. Consistent with these observations, Pol II loading at enhancers and maturation of snRNAs are altered in the YFFF context genome-wide. We conclude that tyrosine1 residues of the CTD control termination of transcription by Pol II.

Nuclear RNA Decay Pathways Aid Rapid Remodeling of Gene Expression in Yeast

SummaryIn budding yeast, the nuclear RNA surveillance system is active on all pre-mRNA transcripts and modulated by nutrient availability. To test the role of nuclear surveillance in reprogramming gene expression, we identified transcriptome-wide binding sites for RNA polymerase II and the exosome cofactors Mtr4 (TRAMP complex) and Nab3 (NNS complex) by UV crosslinking immediately following glucose withdrawal (0, 4, and 8 min). In glucose, mRNA binding by Nab3 and Mtr4 was mainly restricted to promoter-proximal sites, reflecting early transcription termination. Following glucose withdrawal, many growth-related mRNAs showed reduced transcription but increased Nab3 binding, accompanied by downstream recruitment of Mtr4, and oligo(A) tailing. We conclude that transcription termination is followed by TRAMP-mediated RNA decay. Upregulated transcripts evaded increased surveillance factor binding following glucose withdrawal. Some upregulated genes showed use of alternative transcription starts to bypass strong NNS binding sites. We conclude that nuclear surveillance pathways regulate both positive and negative responses to glucose availability.

The Virulence Regulator Rns Activates the Expression of CS14 Pili.

Although many viral and bacterial pathogens cause diarrhea, enterotoxigenic E. coli (ETEC) is one of the most frequently encountered in impoverished regions where it is estimated to kill between 300,000 and 700,000 children and infants annually. Critical ETEC virulence factors include pili which mediate the attachment of the pathogen to receptors in the intestinal lumen. In this study we show that the ETEC virulence regulator Rns positively regulates the expression of CS14 pili. Three Rns binding sites were identified upstream of the CS14 pilus promoter centered at −34.5, −80.5, and −155.5 relative to the Rns-dependent transcription start site. Mutagenesis of the promoter proximal site significantly decreased expression from the CS14 promoter. In contrast, the contribution of Rns bound at the promoter distal site was negligible and largely masked by occupancy of the promoter proximal site. Unexpectedly, Rns bound at the site centered at −80.5 had a slight but statistically significant inhibitory effect upon the pilin promoter. Nevertheless, this weak inhibitory effect was not sufficient to overcome the substantial promoter activation from Rns bound to the promoter proximal site. Thus, CS14 pili belong to a group of pili that depend upon Rns for their expression.

63 Transcriptome wide profiling of mRNA 3′-ends in preeclampsia using PAS-SEQ

Maternal symptoms of preeclampsia (PE) are a primary consequence of excessive levels of sFlt1 protein in the maternal blood supply 1. sFlt1 is encoded by the gene Fms-related tyrosine kinase 1 (FLT1), which produces multiple protein isoforms: (1) transmembrane bound Flt1 (mFlt1), a tyrosine kinase receptor participating in vascular endothelial growth factor (VEGF) signaling; and (2) truncated and blood circulating (soluble) forms, sFlt1s, which lack the transmembrane anchoring domain and act as antagonists to mFlt1. Relative production of these isoforms results from polyadenylation sites within exon 30 (mFlt1) or within introns 13 and 14 (sFlt1s). The goals of this project are three-fold: (1) Determine whether increased sFlt1 production in PE results from an overall increase in FLT1 gene expression, from preferential use of intronic polyadenylation sites or both; (2) assess general gene expression changes in PE versus non-PE placentae; (3) determine if there is a global change in APA (alternative polyadenylation) site usage in PE. To address these goals we used a next-generation sequencing specifically targeting the 3′-ends of mRNAs: PolyAdenylation Site Sequencing (PAS-Seq). PAS-Seq allows for quantitative gene expression measurements as well as transcriptome-wide assessment of APA 2,3. Using total RNA extracted from the villous tissue of 6 normal (N) and 11 PE patient placentae, we generated and sequenced 17 PAS-Seq libraries. Among the PE patients, 8 were early onset (eoPE = delivery <34 weeks). By combining all counts from individual mRNA isoforms into a single measure (reads per million, RPM) for each gene and performing differential expression analysis (via DESeq2), we identified 288 up-regulated genes and 170 down-regulated genes in PE compared to N (padj ⩽0.05). Of these 458 differentially expressed genes, 200 (44%) overlap with the 1295 differentially expressed genes previously identified for severe PE upon meta-analysis of microarray data 4. However, only 15 (3%) of our set overlap with the 297 differentially expressed genes recently determined by RNA-Seq 5.Given the extreme heterogeneity of this disease [i.e., eoPE vs loPE; intrauterine growth restriction (IUGR) PE versus non-IUGR-PE] such a high degree of variability is perhaps unsurprising. Nonetheless, FLT1 was among the small set of differentially expressed genes common to all three datasets. In our PAS-Seq data, FLT1 was the most up-regulated gene (2.8-fold in all PE samples vs. N; 4.2-fold in eoPE vs N). Among all detectable APA isoforms, the four most abundant were: sFlt1-e15a > sFlt1-i13 long > sFlt1-i13 short > m-Flt1. All four isoforms were significantly higher (p ⩽0.05; t-test) in PE versus N; this suggests an overall increase in Flt1 gene transcription in PE. Along with this increased transcription, there also appears to be a switch toward more promoter-proximal sites. We are currently working to determine whether this switch to more promoter-proximal APA sites is a general phenomenon in PE placentae, or is solely a feature of the FLT1 gene. Our PAS-Seq data indicate that the FLT1 gene is subject to both transcriptional upregulation and a shift toward promoter-proximal polyadenylation in PE. Further data analysis regarding global APA switches and pathway analysis will be presented at the conference. R.J. Levine, et al., N. Engl. J. Med. 350, (2004) 672–683. P.J. Shepard, et al., RNA 17 (2011), 761–772. A. Derti et al., Gen. Res. 22 (2012), 1173–1183. K. Leavey, et al., PLoS One 10 (2015), e0116508. S. Sõber, et al., Sci. Rep. (2015), 1–17.