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TATA and paused promoters active in differentiated tissues have distinct expression characteristics

2021; Springer Nature; Volume: 17; Issue: 2 Linguagem: Inglês

10.15252/msb.20209866

1744-4292

Vivekanandan Ramalingam, Malini Natarajan, Jeffrey Johnston, Julia Zeitlinger,

CAR-T cell therapy research

Article5 February 2021Open Access Transparent process TATA and paused promoters active in differentiated tissues have distinct expression characteristics Vivekanandan Ramalingam Vivekanandan Ramalingam orcid.org/0000-0002-3631-8913 Stowers Institute for Medical Research, Kansas City, MO, USA Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USAThese authors contributed equally to this work Search for more papers by this author Malini Natarajan Malini Natarajan orcid.org/0000-0001-7703-7168 Stowers Institute for Medical Research, Kansas City, MO, USAThese authors contributed equally to this work Search for more papers by this author Jeff Johnston Jeff Johnston orcid.org/0000-0002-8962-6186 Stowers Institute for Medical Research, Kansas City, MO, USA Search for more papers by this author Julia Zeitlinger Corresponding Author Julia Zeitlinger [email protected] orcid.org/0000-0002-5172-3335 Stowers Institute for Medical Research, Kansas City, MO, USA Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA Search for more papers by this author Vivekanandan Ramalingam Vivekanandan Ramalingam orcid.org/0000-0002-3631-8913 Stowers Institute for Medical Research, Kansas City, MO, USA Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USAThese authors contributed equally to this work Search for more papers by this author Malini Natarajan Malini Natarajan orcid.org/0000-0001-7703-7168 Stowers Institute for Medical Research, Kansas City, MO, USAThese authors contributed equally to this work Search for more papers by this author Jeff Johnston Jeff Johnston orcid.org/0000-0002-8962-6186 Stowers Institute for Medical Research, Kansas City, MO, USA Search for more papers by this author Julia Zeitlinger Corresponding Author Julia Zeitlinger [email protected] orcid.org/0000-0002-5172-3335 Stowers Institute for Medical Research, Kansas City, MO, USA Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA Search for more papers by this author Author Information Vivekanandan Ramalingam1,2,3, Malini Natarajan1,4, Jeff Johnston1,5 and Julia Zeitlinger *,1,2 1Stowers Institute for Medical Research, Kansas City, MO, USA 2Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA 3Present address: Department of Genetics, Stanford University, Stanford, CA, USA 4Present address: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA 5Present address: Center for Pediatric Genomic Medicine, Children's Mercy, Kansas City, MO, USA *Corresponding author. E-mail: [email protected] Molecular Systems Biology (2021)17:e9866https://doi.org/10.15252/msb.20209866 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Core promoter types differ in the extent to which RNA polymerase II (Pol II) pauses after initiation, but how this affects their tissue-specific gene expression characteristics is not well understood. While promoters with Pol II pausing elements are active throughout development, TATA promoters are highly active in differentiated tissues. We therefore used a genomics approach on late-stage Drosophila embryos to analyze the properties of promoter types. Using tissue-specific Pol II ChIP-seq, we found that paused promoters have high levels of paused Pol II throughout the embryo, even in tissues where the gene is not expressed, while TATA promoters only show Pol II occupancy when the gene is active. The promoter types are associated with different chromatin accessibility in ATAC-seq data and have different expression characteristics in single-cell RNA-seq data. The two promoter types may therefore be optimized for different properties: paused promoters show more consistent expression when active, while TATA promoters have lower background expression when inactive. We propose that tissue-specific genes have evolved to use two different strategies for their differential expression across tissues. SYNOPSIS This study characterizes expression properties of different promoter types of effector genes in late-stage Drosophila embryos, using scRNA-seq combined with profiles of Pol II occupancy, chromatin accessibility and nucleosome occupancy. Paused promoters with high levels of Pol II pausing throughout the embryo have low expression variability but high background expression. TATA promoters with tissue-specific Pol II recruitment and low chromatin accessibility have low background expression but high expression variability. No promoter type shows both low background expression and low expression variability, suggesting a tradeoff between these characteristics. Introduction The core promoter is the ~ 100 bp sequence surrounding a gene's transcription start site (TSS) that facilitates the assembly of the transcription machinery and Pol II transcription (Smale & Kadonaga, 2003; Haberle & Stark, 2018). Pol II transcription may be stimulated in a tissue-specific manner by activation signals from enhancer sequences (Banerji et al, 1981; Spitz & Furlong, 2012), but a core promoter may also produce basal or background levels of transcription in the absence of an activation signal (Kim et al, 1994; Verrijzer & Tjian, 1996; Juven-Gershon et al, 2006). Ideally, a promoter produces only minimal background expression in the inactive state, is highly responsive to enhancers, and reliably produces the desired level of transcription in the active state. A core promoter element that strongly promotes Pol II initiation is the TATA box (Patikoglou et al, 1999; Reeve, 2003), an ancient core promoter element present in archaea, fungi, plants, and animals (Patikoglou et al, 1999; Reeve, 2003). TATA is bound by TATA-binding protein (TBP) and helps assemble the pre-initiation complex (Nikolov et al, 1992; Kim et al, 1993; Patikoglou et al, 1999). After initiating transcription, Pol II may then pause 30–50 bp downstream of the TSS, before being released into productive elongation (Adelman & Lis, 2012). Based on work in Drosophila, core promoter elements not only influence Pol II initiation, but also Pol II pausing. Promoters with very stably paused Pol II are enriched for downstream pausing elements such as the Pause Button (PB) and the Downstream Promoter Element (DPE) (Burke & Kadonaga, 1997; Lim et al, 2004; Hendrix et al, 2008; Gaertner et al, 2012; Shao & Zeitlinger, 2017). TATA promoters often show minimal Pol II pausing but the evidence is conflicting and predominantly based on cultured cells (Gilchrist et al, 2010; Chen et al, 2013; Shao & Zeitlinger, 2017; Krebs et al, 2017). Swapping core promoter elements or the entire promoter alters the amount and duration of Pol II pausing in Drosophila embryos and cultured cells (Lagha et al, 2013; Shao et al, 2019). The amount of Pol II pausing at a promoter appears to influence the expression characteristics. Promoters with high occupancy of paused Pol II are prevalent among genes that are highly regulated during development (Muse et al, 2007; Zeitlinger et al, 2007; Gaertner et al, 2012) and mediate more synchronous gene expression between cells (Boettiger & Levine, 2009; Lagha et al, 2013). Without well-timed gene activation, coordinated cellular behaviors such as gastrulation may not proceed properly (Lagha et al, 2013). TATA promoters on the other hand are often associated with higher expression variability (Raser & O'Shea, 2004; Blake et al, 2006; Tirosh et al, 2006; Lehner, 2010; Hornung et al, 2012; Li & Gilmour, 2013; Sigalova et al, 2020). However, the expression characteristics of TATA promoters in developing embryos are less understood. Across metazoans, genes with TATA elements are particularly enriched among effector genes, the genes responsible for the structure and function of differentiated tissues (Schug et al, 2005; Carninci et al, 2006; FitzGerald et al, 2006; Engström et al, 2007; Lenhard et al, 2012; FANTOM Consortium et al, 2014). Effector genes start to be expressed primarily at later stages of embryogenesis when cells begin differentiation into morphologically distinct tissues (Erwin & Davidson, 2009). These stages are typically not well studied, and thus, whether TATA promoters confer effector genes different expression characteristics is not clear. Here, we systematically analyzed the relationship between promoter types and gene expression in differentiated tissues of the late Drosophila embryo, where both TATA and paused promoters are active. We mapped the gene expression programs of all cell types using single-cell RNA-seq (scRNA-seq) and determined the occupancy of Pol II in a tissue-specific fashion. Our analysis revealed large differences in Pol II pausing between the promoters of effector genes and showed that TATA promoters are strongly enriched among effector genes with minimal Pol II pausing. Notably, scRNA-seq revealed that TATA genes have higher expression variability but lower background expression than paused promoters and that this property correlates with lower chromatin accessibility. We propose that different promoter types are optimal for different expression properties and discuss the mechanisms by which these differences in promoter function occur. Results Characterization of the tissue-specific expression programs in the late Drosophila embryo using single-cell RNA-seq To obtain an unbiased global view of the gene expression programs in differentiated tissues, we performed scRNA-seq on dissociated cells from late Drosophila embryos (Fig 1A). We chose embryos at stage 16 (14–14.5 h after egg deposition) when the tissues are fully formed but the outside cuticle is not yet developed enough to hamper the dissociation of the cells. After processing the cells through a 10× Genomics Chromium instrument (Klein et al, 2015; Macosko et al, 2015; Zheng et al, 2017), we obtained the expression profiles of approximately 3,500 cells. Cells prepared and sequenced from two separate batches yielded results that were highly similar with regard to data quality and results from clustering (Appendix Fig S1). Figure 1. scRNA-seq captures the expression profiles of effectors genes in the late stages of Drosophila embryogenesis Single cells were isolated from Drosophila embryos 14–14.5 h after egg deposition (AED). Isolated cells were processed through a 10× Genomics instrument. After sequencing the resulting libraries, the reads were aligned and processed using the standard pipeline from 10× Genomics. The single-cell gene expression profiles were used to map the cells to known cell types by comparing against the available in situ hybridization patterns from the Berkeley Drosophila Genome Project. A tSNE projection of the scRNA-seq data is shown in the middle, and the known tissues to which the clusters were assigned to are graphically illustrated outside. Marker genes for each tissue type are shown in parentheses. Download figure Download PowerPoint To identify the tissues to which each scRNA-seq cluster belongs, we correlated the scRNA-seq data with the large-scale in situ hybridization data from the Berkeley Drosophila Genome Project (BDGP) (Tomancak et al, 2002; Tomancak et al, 2007; Hammonds et al, 2013) (Fig 1A). For ambiguous clusters, we analyzed the occurrence of known tissue markers and manually merged or separated clusters such that they better matched anatomical structures. In this manner, we obtained scRNA-seq data for 16 tissues of the late Drosophila embryo: central nervous system (CNS), peripheral nervous system (PNS), glia, germ cells, epidermis, trachea, muscle, dorsal vessel, fat body, plasmocytes, crystal cells, ring gland, salivary gland, gastric cecum, midgut and malpighian tubules (Fig 1B; Dataset EV3). In order to make this classification useful for future studies, we also identified marker genes for each tissue, some of which were previously known (Fig 1B and Appendix Fig S2). Effector genes have different Pol II occupancy patterns across tissues We next asked what promoter type is used by effector genes in the late Drosophila embryo. To distinguish effector genes from housekeeping genes and developmental genes, we defined effector genes by their late upregulation during embryogenesis (> 5×, P < 0.05 from 2–4 h to 14–17 h), which yielded 1,527 genes (Datasets EV1 and EV2, Materials and Methods). As control groups, we also defined ubiquitously expressed housekeeping genes (647 genes), as well as developmental genes that are highly paused throughout embryogenesis (772 genes; Dataset EV1) as defined previously (Gaertner et al, 2012). These late-induced genes were enriched for GO terms of tissue-specific biological functions, e.g., synaptic transmission and chitin-based cuticle development (Fig EV1A), consistent with them being effector genes. They were also under-represented for sequence motifs found in housekeeping genes and enriched for TATA consensus motifs (Fig EV1C). However, sequence motifs typically found in paused developmental promoters such as DPE and PB were also significantly enriched, suggesting that these genes may also be induced by the paused promoter type (Fig EV1C; Dataset EV2). Click here to expand this figure. Figure EV1. Functional categories and categorization of late-induced effector genes A, B. GO term enrichments were calculated for Biological Processes. For each gene group and each GO term, the enrichment was calculated as the fraction of genes in a group associated with a GO term over the fraction of all genes with the same GO term. The statistical significance was calculated using the hypergeometric test. GO terms associated with less than five genes were not included in the analysis. Top representative terms are shown in the bar plot. (A) Identified effector genes are enriched for expected GO terms. (B) GO term enrichments for the TATA group and the highly paused group effector genes were calculated separately. While there are some differences between the groups, both groups have functional categories expected for effector genes. C. The identified effector genes were enriched for promoter elements found at tissue-specific genes (focused promoter elements) and are depleted for motifs associated with housekeeping genes (broad promoter elements). A star denotes significance with a Fisher's exact test, (*P < 0.05). D. Pausing indices (log2), the ratio of Pol II at the promoter vs the gene body, are shown for the different effector gene groups. The pausing indices of genes from the TATA group are significantly lower than that of the highly paused group (Wilcoxon two-sided test, *P < 10−15). E. RNA levels (log2 TPM) from 14 to 17 h whole embryos at the different effector gene groups are shown. The TATA genes are expressed at levels comparable to the paused genes. F. The sequence heat map plot shows motif differences between the different gene groups. Information content at each position is plotted as a sequence logo. G. Pausing index and total gene lengths of genes, of TATA and highly paused effector genes, developmentally paused and housekeeping groups are shown. The pausing indices of genes from the TATA group are significantly lower than that of the highly paused group (Wilcoxon two-sided test, *P < 10−15; left panel). TATA genes are generally shorter than highly paused genes (Wilcoxon two-sided test, *P < 10−15; right panel). H. Promoter shape values are based on the CAGE data (Sigalova et al, 2020). Promoters of TATA-enriched, highly paused and developmentally paused genes groups are primarily narrow, while the housekeeping gene promoters are primarily broad. Box plots in all panels show the median as the central line, the first and the third quartiles as the box, and the upper and lower whiskers extend from the quartile box to the largest/smallest value within 1.5 times of the interquartile range. Download figure Download PowerPoint We therefore set out to characterize these promoters experimentally by performing Pol II ChIP-seq experiments on a variety of tissues isolated from the late Drosophila embryo. Using the INTACT method (Deal & Henikoff, 2011; Bonn et al, 2012), nuclei from the tissue of interest were genetically tagged for biotin labeling and isolated from fixed embryos (14–17 h) with the help of streptavidin-coupled magnetic beads (Fig 2A). The following six tissues were analyzed: neurons (using elav-Gal4), glia (using repo-Gal4), muscle (using mef2-Gal4), trachea (using btl-gal4), and epidermis and gut (using enhancer trap-Gal4 lines 7021 and 110394, respectively, see Materials and Methods; Fig 2B). Figure 2. Tissue-specific Pol II ChIP-seq shows differences in Pol II occupancy patterns at effector genes Tissue-specific ChIP-seq was done by isolating nuclei from specific tissues (shown in red) by expressing the Escherichia coli biotin ligase (BirA) and the biotin ligase recognition peptide (BLRP) fused with a nuclear envelope-targeting sequence in the tissue of interest. This allows the isolation of nuclei from the tissue of interest using streptavidin magnetic beads. Pol II ChIP-seq was performed in six different tissues shown in the left panel (scale bar - 100 µm). The middle and the right panels show the read-count normalized Pol II ChIP-seq tracks (RPM) from the six tissues at individual genes. For each gene, gray and red tracks indicate non-expressing tissues and expressing tissue, respectively. The middle panel shows the Pol II profile at two non-paused genes, which have Pol II only in the expressing tissues. The expression is limited to specific tissues as shown in the in situ images from BDGP. The right panel shows the Pol II profile at a paused gene, which has Pol II in all observed tissues, although the expression is limited to specific tissues as shown in the in situ images from BDGP. Paused Pol II is generally highest in the tissue with the highest expression. We also found systematic differences between samples; thus, some tissues have generally higher enrichments than others, presumably because they are easier to cross-link. Identified effector genes were grouped into seven groups based on Pol II penetrance, i.e. the number of tissues in which Pol II enrichment is above background (calculated in a window starting from the TSS and ending 200 bp downstream). Genes that are highly paused throughout embryogenesis (developmental paused) (Gaertner et al, 2012) and housekeeping genes are shown as a control. Core promoter elements are differentially enriched across the groups (Fisher's exact test with multiple-testing correction, *P < 0.05), allowing us to classify promoter classes based on Pol II penetrance (highly paused, paused, dual TATA, TATA enriched). The highly paused group is defined by Pol II enrichments in 5–6 tissues and is similar to the developmental paused genes. TATA enrichment is found in the groups with Pol II enrichment in 0 or 1 tissues. Sequence heat map plots show clear and consistent motif differences between the TATA-enriched and highly paused gene groups. The information content of the motifs is plotted as a sequence logo below, revealing a degenerative TATA box, an Inr with or without G, and downstream pausing elements all of which are consistent with previous results (Shao et al, 2019). Download figure Download PowerPoint The Pol II ChIP-seq tracks from the six tissues confirmed that the ChIP-seq data are tissue-specific. For example, the tracheal gene Osi20 and the muscle gene Mlp60A showed high Pol II occupancy in the trachea and muscle samples, respectively, but not in the other tissues (Fig 2B middle panel). Global analyses were also consistent with a high concordance between Pol II occupancy and scRNA-seq (Appendix Fig S3). The Pol II occupancy was however not always tissue-specific. Some genes showed high Pol II occupancy at the promoter in many or all tissues, despite being expressed in a very tissue-restricted fashion. For example, expression of Ace is restricted to neuronal populations but showed very high Pol II promoter occupancy in all tissues (Fig 2B right panel). Moreover, the Pol II pattern along the gene was indicative of Pol II pausing since the Pol II occupancy peaks at the pausing position (30–50 bp downstream of the TSS) and is not detected at the gene body (Fig 2B right panel). These results suggest that there are two types of tissue-specific promoters that are regulated in a fundamentally different fashion. At one type of promoter, Pol II is recruited only in tissues where the gene is expressed and proceeds toward productive elongation without detectable pausing (Fig 2B middle panel). On the other end of the spectrum is a promoter type where Pol II is widely recruited and found paused across all tissues, and Pol II only proceeds toward productive elongation in the tissues where the gene is expressed (Fig 2B right panel). Pol II penetrance across tissues separates TATA and paused effector genes We next asked whether the different Pol II occupancy patterns across tissues could distinguish between promoter types. We classified genes based on their Pol II penetrance (Fig 2C), defined as the number of tissues (from 0 to 6 tissues) in which Pol II is detected around the TSS above background (> 2-fold signal over input; Dataset EV2). Genes with the highest Pol II penetrance (5–6 tissues) were strongly enriched for pausing elements such as GAGA, DPE, or PB (362 highly paused genes), similar to developmental paused genes (Fig 2C; Dataset EV2). On the other hand, genes with the lowest Pol II penetrance (0 tissues) were highly enriched for TATA elements (527 TATA-enriched genes). Gene groups with intermediate Pol II penetrance had weaker enrichments for both types of elements (222 paused genes, 415 dual TATA genes that had both TATA and pausing elements). Although TATA-enriched genes did not have detectable Pol II occupancy, they were nevertheless transcribed according to RNA-seq data (Fig EV1E). This suggests that Pol II may be hard to detect at some TATA genes, presumably because Pol II does not pause and significant levels can only be detected with high levels of transcription. These results suggest that Pol II penetrance across tissues is another measurement for Pol II pausing that can be used to classify promoter types. Consistent with this, the Pol II penetrance correlates with Pol II pausing when measured by the pausing index (Fig EV1D; Dataset EV2). Furthermore, we confirmed that Pol II penetrance was not biased by expression levels (Fig EV1E; Dataset EV2), although slight differences in the timing of the induction of the TATA-enriched and paused gene groups were detected (Appendix Fig S4; Dataset EV2). To analyze the promoter groups in more detail, we aligned the promoter groups based on the TSS identified by CAGE data from late Drosophila embryos (Hoskins et al, 2011). We then visualized the sequence composition using color plots and generated consensus motifs for different promoter groups (Fig 2D; Dataset EV2). This revealed that the majority of promoters in the TATA-enriched group indeed showed TATA-like elements at the expected position of −30 bp upstream of the TSS, as well as a weak Initiator sequence (CA), consistent with previous data (Shao et al, 2019). In contrast, the highly paused group showed a strong Initiator sequence (TCAGT) with a G at the +2 position, which has been shown to promote Pol II pausing (Shao et al, 2019). In addition, these promoters had a well-positioned G-rich sequence pattern downstream of the TSS around the site of Pol II pausing (Fig 2D). When we performed the same analysis on the previously identified developmental paused genes, the pattern was strikingly similar (Fig EV1F). This indicates that the highly paused promoters during development and in differentiated tissues are functionally equivalent. The functional equivalence is further supported by their similarity in pausing index, gene length, and promoter shape (Fig EV1G and H; Dataset EV2). Since effector genes have previously been associated with TATA promoters (Schug et al, 2005; Carninci et al, 2006; FitzGerald et al, 2006; Engström et al, 2007; Lenhard et al, 2012; FANTOM Consortium et al, 2014), we asked whether the functions of the highly paused genes also point to them being effector genes. GO analysis revealed functional categories such as chitin metabolic process and amino sugar metabolic process, which are also enriched among TATA genes (Fig EV1B; Dataset EV6). Furthermore, typical tissue-specific functions were identified among highly paused genes, including rhabdomere development, respiratory system development, and generation of neurons. Although categories such as multicellular organism development and signaling were also enriched, these genes were not well-studied developmental genes. In summary, while some of these paused genes might be classified as developmental genes by other methods, we conclude that paused promoters clearly contribute to the specific structure and function of differentiated tissues and thus fulfill the criteria for being effector genes. We did however notice differences between the TATA and paused effector genes. The TATA genes were often short genes found in clusters of gene families (e.g., the Osi gene family, Appendix Fig S5), and many of them were expressed in tissues such as the epidermis, gut, and trachea, which are exposed to the environment and may require adaptation (Dorer et al, 2003; Cornman, 2009; Shah et al, 2012). This indicates that effector genes with different promoter types may have different structural and functional properties. TATA genes are expressed with high variability but low background expression We next analyzed whether the different promoter types might display different expression characteristics across tissues. Using the scRNA-seq data, we analyzed their expression noise, measured by the coefficient of variation across different expression bins. We found that TATA genes had consistently higher expression variation than paused genes in all expression bins (Figs 3A and, EV2A and B; Datasets EV4 and EV5). Figure 3. scRNA-seq reveals differences in expression characteristics of TATA and paused genes A. The differences in cell-to-cell gene expression variability for the TATA and the paused effector gene groups were analyzed using the scRNA-seq data. The coefficient of variation (standard deviation/mean) of gene expression was calculated for all genes in the tissue with the highest expression for each gene. The median coefficient of variation was consistently lower for the paused genes compared with the TATA genes. B. The frequency of cells with any detectable expression (> one read) was calculated in tissues with the highest expression for each gene (expressing tissue) and in five other tissues with the least expression for each gene (other tissues). The frequency of cells with detectable expression in the expressing tissues, a measure of expression robustness, is lower for the TATA genes compared with the highly paused genes (left) (Wilcoxon two-sided test, *P < 10−15). The frequency of cells with detectable expression in the other tissues, a measure of background expression, is also lower for the TATA genes compared with the paused genes (right; Wilcoxon two-sided test, *P < 10−15). C, D. Normalized gene expression levels (read-count normalized for each cell, log2) in different tissues, from the scRNA-seq experiment, for a TATA group gene, and a highly paused group gene, are shown. (C) The TATA gene, Ccp84Aa, shows noisy expression in the epidermis, without detectable background expression in non-expressing tissues. (D) The highly paused gene, Gip, shows very robust expression in crystal cells but has high background expression in the non-expressing tissues. E. The number of annotation terms associated with each gene in the BDGP in situ database. This is a measure of whether the expression of a gene is restricted to specific subsets of tissue (Wilcoxon two-sided test, *P < 10−15). F. The coefficient of variation across different isogenic lines of Drosophila from DGRP, after being corrected for dependence on expression (loess regression), is plotted for different effector gene groups (Sigalova et al, 2020). Genes from the TATA-enriched group show high variability compared with the paused genes (Wilcoxon two-sided test, *P < 10−15). Box plots in all panels show the median as the central line, the first and the third quartiles as the box, and the upper and lower whiskers extend from the quartile box to the largest/smallest value within 1.5 times of the interquartile range. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Differences in the scRNA-seq expression profiles of late-induced effector gene groups Since the coefficient of variation of gene expression (standard deviation/mean) shows a dependency on mean expression (left), it was corrected by loess regression (right). The coefficient of variation after correcting for mean expression is lower for the paused genes compared with the TATA genes (Wilcoxon two-sided test, *P < 10−15). The loess-corrected coefficient of variation of gene expression across different isogenic lines of Drosophila melanogaster from the DGRP collection (Sigalova et al, 2020) also shows that genes from the TATA-enriched group show higher variability compar