2019 journal article
Enhancer Histone Acetylation Modulates Transcriptional Bursting Dynamics of Neuronal Activity-Inducible Genes
CELL REPORTS, 26(5), 1174-+.
doi.org (publisher)
Source: Web Of Science
Added: February 18, 2019
•We quantify transcriptional bursting dynamics of Fos and Npas4 in neurons•Bidirectional regulation of enhancer acetylation tunes Fos transcriptional bursts•Fos driven by enhancer epigenome editing alters neuronal physiology•Enhancer-recruited dCas9-HDAC8 impairs Fos induction in neurons in vivo Neuronal activity-inducible gene transcription correlates with rapid and transient increases in histone acetylation at promoters and enhancers of activity-regulated genes. Exactly how histone acetylation modulates transcription of these genes has remained unknown. We used single-cell in situ transcriptional analysis to show that Fos and Npas4 are transcribed in stochastic bursts in mouse neurons and that membrane depolarization increases mRNA expression by increasing burst frequency. We then expressed dCas9-p300 or dCas9-HDAC8 fusion proteins to mimic or block activity-induced histone acetylation locally at enhancers. Adding histone acetylation increased Fos transcription by prolonging burst duration and resulted in higher Fos protein levels and an elevation of resting membrane potential. Inhibiting histone acetylation reduced Fos transcription by reducing burst frequency and impaired experience-dependent Fos protein induction in the hippocampus in vivo. Thus, activity-inducible histone acetylation tunes the transcriptional dynamics of experience-regulated genes to affect selective changes in neuronal gene expression and cellular function. Neuronal activity-inducible gene transcription correlates with rapid and transient increases in histone acetylation at promoters and enhancers of activity-regulated genes. Exactly how histone acetylation modulates transcription of these genes has remained unknown. We used single-cell in situ transcriptional analysis to show that Fos and Npas4 are transcribed in stochastic bursts in mouse neurons and that membrane depolarization increases mRNA expression by increasing burst frequency. We then expressed dCas9-p300 or dCas9-HDAC8 fusion proteins to mimic or block activity-induced histone acetylation locally at enhancers. Adding histone acetylation increased Fos transcription by prolonging burst duration and resulted in higher Fos protein levels and an elevation of resting membrane potential. Inhibiting histone acetylation reduced Fos transcription by reducing burst frequency and impaired experience-dependent Fos protein induction in the hippocampus in vivo. Thus, activity-inducible histone acetylation tunes the transcriptional dynamics of experience-regulated genes to affect selective changes in neuronal gene expression and cellular function. Transient sensory experiences are transduced into long-lasting changes in synaptic connectivity and neuronal function through the activity-dependent regulation of new gene transcription (Chen et al., 2017Chen L.-F. Zhou A.S. West A.E. Transcribing the connectome: roles for transcription factors and chromatin regulators in activity-dependent synapse development.J. Neurophysiol. 2017; 118: 755-770Crossref PubMed Scopus (16) Google Scholar). Synaptic activity regulates gene transcription by activating intracellular calcium-dependent signaling cascades that modify the function and/or expression of activity-dependent DNA-binding transcription factors and chromatin regulatory proteins (Greer and Greenberg, 2008Greer P.L. Greenberg M.E. From synapse to nucleus: calcium-dependent gene transcription in the control of synapse development and function.Neuron. 2008; 59: 846-860Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). The targets of these activity-regulated signaling pathways in neurons include both immediate-early gene transcription factors and neural-specific programs of gene expression, which directly alter aspects of neuron and synapse structure and function (Leslie and Nedivi, 2011Leslie J.H. Nedivi E. Activity-regulated genes as mediators of neural circuit plasticity.Prog. Neurobiol. 2011; 94: 223-237Crossref PubMed Scopus (85) Google Scholar). In this manner, stimulus-induced transcription provides a compelling mechanism of activity-dependent neuronal plasticity. Genome-level sequencing studies have revealed important roles for chromatin state and structure in the control of gene transcription. In addition to gene promoters, distal enhancers contribute to the activation of gene transcription because of conformational loops that bring them physically close to gene promoters (Heintzman et al., 2009Heintzman N.D. Hon G.C. Hawkins R.D. Kheradpour P. Stark A. Harp L.F. Ye Z. Lee L.K. Stuart R.K. Ching C.W. et al.Histone modifications at human enhancers reflect global cell-type-specific gene expression.Nature. 2009; 459: 108-112Crossref PubMed Scopus (1788) Google Scholar). Enhancers are characterized by their accessibility to transcription factor binding, as well as their enrichment for specific epigenomic marks, including methylation (me) and acetylation (ac) on specific histone H3 lysine (K) residues (H3K4me1 and H3K27ac). Enhancers have been best studied for their role in controlling cell-type-specific programs of gene expression, for which the differential recruitment of the histone acetyltransferases p300 and CREB binding protein (CBP), as well as the presence of H3K27ac, are strong predictors of regulatory elements that are sufficient to drive cell-type-specific gene transcription (Blow et al., 2010Blow M.J. McCulley D.J. Li Z. Zhang T. Akiyama J.A. Holt A. Plajzer-Frick I. Shoukry M. Wright C. Chen F. et al.ChIP-seq identification of weakly conserved heart enhancers.Nat. Genet. 2010; 42: 806-810Crossref PubMed Scopus (333) Google Scholar, Nord et al., 2013Nord A.S. Blow M.J. Attanasio C. Akiyama J.A. Holt A. Hosseini R. Phouanenavong S. Plajzer-Frick I. Shoukry M. Afzal V. et al.Rapid and pervasive changes in genome-wide enhancer usage during mammalian development.Cell. 2013; 155: 1521-1531Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, Visel et al., 2013Visel A. Taher L. Girgis H. May D. Golonzhka O. Hoch R.V. McKinsey G.L. Pattabiraman K. Silberberg S.N. Blow M.J. et al.A high-resolution enhancer atlas of the developing telencephalon.Cell. 2013; 152: 895-908Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). However, neurons undergo dynamic changes in their gene expression repertoires long after they have committed to a postmitotic identity; thus, neurons serve as an ideal substrate for studying the biological functions of the epigenome beyond its role in establishing cellular identity. Membrane depolarization of embryonic mouse cortical neurons induces CBP binding and H3K27ac at a subset of putative enhancers near activity-regulated genes, and regulatory elements that show activity-dependent increases in H3K27ac are highly likely to be sufficient to drive activity-dependent transcription of a reporter gene (Kim et al., 2010Kim T.K. Hemberg M. Gray J.M. Costa A.M. Bear D.M. Wu J. Harmin D.A. Laptewicz M. Barbara-Haley K. Kuersten S. et al.Widespread transcription at neuronal activity-regulated enhancers.Nature. 2010; 465: 182-187Crossref PubMed Scopus (1709) Google Scholar, Malik et al., 2014Malik A.N. Vierbuchen T. Hemberg M. Rubin A.A. Ling E. Couch C.H. Stroud H. Spiegel I. Farh K.K. Harmin D.A. Greenberg M.E. Genome-wide identification and characterization of functional neuronal activity-dependent enhancers.Nat. Neurosci. 2014; 17: 1330-1339Crossref PubMed Scopus (165) Google Scholar). Yet despite widespread correlations between histone modifications and enhancer function, whether these modifications play causative roles in enhancer activity is not always clear (Dorighi et al., 2017Dorighi K.M. Swigut T. Henriques T. Bhanu N.V. Scruggs B.S. Nady N. Still 2nd, C.D. Garcia B.A. Adelman K. Wysocka J. Mll3 and Mll4 facilitate enhancer RNA synthesis and transcription from promoters independently of H3K4 monomethylation.Mol. Cell. 2017; 66: 568-576Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Furthermore, although biochemical studies have shown steady-state increases in both H3K27ac and mRNA at specific time points following neuronal activation, the temporal relationship between these two events is poorly understood. Transcription is an inherently stochastic process determined by the kinetics of the biochemical events that mediate the synthesis of RNA (Symmons and Raj, 2016Symmons O. Raj A. What’s luck got to do with it: single cells, multiple fates, and biological nondeterminism.Mol. Cell. 2016; 62: 788-802Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). As a consequence, the transcription of most genes when observed at the single-cell level stochastically occurs at a higher rate during long intervals of time called transcriptional bursts, followed by variable periods of transcriptional inactivity (Dar et al., 2012Dar R.D. Razooky B.S. Singh A. Trimeloni T.V. McCollum J.M. Cox C.D. Simpson M.L. Weinberger L.S. Transcriptional burst frequency and burst size are equally modulated across the human genome.Proc. Natl. Acad. Sci. USA. 2012; 109: 17454-17459Crossref PubMed Scopus (296) Google Scholar). Bursting can be described by the frequency, duration, and size of the active intervals, which reflect dynamic promoter transitions between inactive and active states. Burst kinetics are highly gene specific and tuned by the diverse array of molecular regulatory mechanisms that control transcription (Suter et al., 2011Suter D.M. Molina N. Gatfield D. Schneider K. Schibler U. Naef F. Mammalian genes are transcribed with widely different bursting kinetics.Science. 2011; 332: 472-474Crossref PubMed Scopus (598) Google Scholar). Transcription factor binding, enhancer function, and chromatin features have all been linked to effects on burst kinetics in a context-specific manner (Fukaya et al., 2016Fukaya T. Lim B. Levine M. Enhancer control of transcriptional bursting.Cell. 2016; 166: 358-368Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, Wu et al., 2017Wu S. Li K. Li Y. Zhao T. Li T. Yang Y.-F. Qian W. Independent regulation of gene expression level and noise by histone modifications.PLoS Comput. Biol. 2017; 13: e1005585Crossref PubMed Scopus (27) Google Scholar). Emerging evidence also suggests that dynamic changes in chromatin state can modulate burst properties to control gene expression levels. For example, inducible histone acetylation at gene promoters covaries with increased burst frequency across the circadian cycle for several mammalian circadian genes (Nicolas et al., 2018Nicolas D. Zoller B. Suter D.M. Naef F. Modulation of transcriptional burst frequency by histone acetylation.Proc. Natl. Acad. Sci. USA. 2018; 115: 7153-7158Crossref PubMed Scopus (65) Google Scholar). CRISPR-based methods have emerged as a powerful tool for studying the functions of chromatin regulation, because the site specificity of Cas9 binding, together with its ability to be fused to enzymatic domains, permits the isolated experimental manipulation of histone and DNA modifications at specific sites across the genome (Thakore et al., 2016Thakore P.I. Black J.B. Hilton I.B. Gersbach C.A. Editing the epigenome: technologies for programmable transcription and epigenetic modulation.Nat. Methods. 2016; 13: 127-137Crossref PubMed Scopus (274) Google Scholar). Here, to discover how neuronal activity-induced enhancer histone acetylation regulates the transcription of neuronal activity-inducible genes, we first applied quantitative single-molecule fluorescence in situ hybridization (smFISH) to establish transcriptional burst kinetics of the neuronal activity-inducible Fos and Npas4 genes in primary neurons in culture and in vivo and then used CRISPR-based dead Cas9 (dCas9) epigenome editing to locally mimic or block activity-induced histone acetylation at well-established enhancers of these genes. Our data show how enhancer histone acetylation modulates the burst dynamics of activity-inducible genes and demonstrate that the regulation of Fos bursting is transmitted to regulate Fos protein levels and functional properties of neurons. Membrane depolarization mediated by the elevation of extracellular potassium chloride (KCl) levels is a robust stimulus for the induction of neuronal activity-regulated genes, acting via well-established intracellular calcium signal transduction mechanisms and known to induce histone acetylation of activity-regulated enhancers and promoters (Bito et al., 1996Bito H. Deisseroth K. Tsien R.W. CREB phosphorylation and dephosphorylation: a Ca(2+)- and stimulus duration-dependent switch for hippocampal gene expression.Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (978) Google Scholar, Halder et al., 2016Halder R. Hennion M. Vidal R.O. Shomroni O. Rahman R.-U. Rajput A. Centeno T.P. van Bebber F. Capece V. Garcia Vizcaino J.C. et al.DNA methylation changes in plasticity genes accompany the formation and maintenance of memory.Nat. Neurosci. 2016; 19: 102-110Crossref PubMed Scopus (213) Google Scholar, Lyons and West, 2011Lyons M.R. West A.E. Mechanisms of specificity in neuronal activity-regulated gene transcription.Prog. Neurobiol. 2011; 94: 259-295Crossref PubMed Scopus (143) Google Scholar, Malik et al., 2014Malik A.N. Vierbuchen T. Hemberg M. Rubin A.A. Ling E. Couch C.H. Stroud H. Spiegel I. Farh K.K. Harmin D.A. Greenberg M.E. Genome-wide identification and characterization of functional neuronal activity-dependent enhancers.Nat. Neurosci. 2014; 17: 1330-1339Crossref PubMed Scopus (165) Google Scholar). The signaling steps that comprise this process, as well as Fos mRNA induction and subsequent degradation, occur on the time course of seconds to minutes (Bito et al., 1996Bito H. Deisseroth K. Tsien R.W. CREB phosphorylation and dephosphorylation: a Ca(2+)- and stimulus duration-dependent switch for hippocampal gene expression.Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (978) Google Scholar, Dolmetsch et al., 2001Dolmetsch R.E. Pajvani U. Fife K. Spotts J.M. Greenberg M.E. Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway.Science. 2001; 294: 333-339Crossref PubMed Scopus (741) Google Scholar, Zhai et al., 2013Zhai S. Ark E.D. Parra-Bueno P. Yasuda R. Long-distance integration of nuclear ERK signaling triggered by activation of a few dendritic spines.Science. 2013; 342: 1107-1111Crossref PubMed Scopus (86) Google Scholar). However, most prior studies of membrane depolarization-inducible H3K27ac have used persistent or hours-long stimulation paradigms (Kim et al., 2010Kim T.K. Hemberg M. Gray J.M. Costa A.M. Bear D.M. Wu J. Harmin D.A. Laptewicz M. Barbara-Haley K. Kuersten S. et al.Widespread transcription at neuronal activity-regulated enhancers.Nature. 2010; 465: 182-187Crossref PubMed Scopus (1709) Google Scholar, Malik et al., 2014Malik A.N. Vierbuchen T. Hemberg M. Rubin A.A. Ling E. Couch C.H. Stroud H. Spiegel I. Farh K.K. Harmin D.A. Greenberg M.E. Genome-wide identification and characterization of functional neuronal activity-dependent enhancers.Nat. Neurosci. 2014; 17: 1330-1339Crossref PubMed Scopus (165) Google Scholar, Tyssowski et al., 2018Tyssowski K.M. DeStefino N.R. Cho J.-H. Dunn C.J. Poston R.G. Carty C.E. Jones R.D. Chang S.M. Romeo P. Wurzelmann M.K. et al.Different neuronal activity patterns induce different gene expression programs.Neuron. 2018; 98: 530-546Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Thus, here we chose to use a minimal stimulation paradigm to ask whether histone acetylation is dynamically regulated on the same timescale as gene transcription. We stimulated dissociated embryonic mouse cortical neurons in culture with a 5 min pulse of membrane depolarization by elevating extracellular KCl to 55 mM (Lyons et al., 2016Lyons M.R. Chen L.F. Deng J.V. Finn C. Pfenning A.R. Sabhlok A. Wilson K.M. West A.E. The transcription factor calcium-response factor limits NMDA receptor-dependent transcription in the developing brain.J. Neurochem. 2016; 137: 164-176Crossref PubMed Scopus (20) Google Scholar). We then measured levels of Fos mRNA by qPCR and H3K27ac by chromatin immunoprecipitation (ChIP) at regulatory elements of the Fos gene (Figure 1A) as a function of time after the stimulus was removed. 5 min of membrane depolarization was sufficient to drive stimulus-dependent increases in both Fos mRNA and H3K27ac at the Fos promoter and distal enhancers (Figure 1B). Induction of Fos mRNA, as well as H3K27ac, in response to this stimulus was both rapid and transient, significantly increasing within 10 min following stimulus induction and falling back to basal levels within an hour after cessation of the stimulus (Figure 1B). These data establish a system in which we can study the dynamics of neuronal activity-inducible gene transcription and H3K27a. Furthermore, these data establish the coincident regulation of H3K27ac at Fos regulatory elements during the time course of active Fos transcription. Transcriptional bursts occur whenever a promoter transitions from an inactive to an active state. However, because the biochemical events that mediate these bursts are stochastic, bursts fluctuate randomly over time such that only a subset of cells in a population will be bursting at any given moment (Symmons and Raj, 2016Symmons O. Raj A. What’s luck got to do with it: single cells, multiple fates, and biological nondeterminism.Mol. Cell. 2016; 62: 788-802Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). It is possible to use mathematical models of stochastic gene expression to infer dynamic properties of transcriptional bursting from the measured distribution of RNA expression in cells sampled from a population (Gómez-Schiavon et al., 2017Gómez-Schiavon M. Chen L.-F. West A.E. Buchler N.E. BayFish: Bayesian inference of transcription dynamics from population snapshots of single-molecule RNA FISH in single cells.Genome Biol. 2017; 18: 164Crossref PubMed Scopus (23) Google Scholar, Vera et al., 2016Vera M. Biswas J. Senecal A. Singer R.H. Park H.Y. Single-cell and single-molecule analysis of gene expression regulation.Annu. Rev. Genet. 2016; 50: 267-291Crossref PubMed Scopus (77) Google Scholar). To determine the burst dynamics of activity-dependent genes in neurons, we first quantified Fos mRNA at the single-neuron level in cultured embryonic mouse hippocampal neurons by smFISH (Raj et al., 2008Raj A. van den Bogaard P. Rifkin S.A. van Oudenaarden A. Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes.Nat. Methods. 2008; 5: 877-879Crossref PubMed Scopus (1321) Google Scholar). We fixed cells for smFISH both under basal conditions (in the presence of tetrodotoxin [TTX]) and at various times following a brief period (as in Figure 1B) of membrane depolarization (Figure 2A). The distributions of fluorescence intensities of cytoplasmic mRNA spots fit a Gaussian distribution, consistent with each spot representing a single mRNA molecule (Figures S1A and S1B). The time course and magnitude of the average number of mRNAs detected per neuron by smFISH across all cells in the population precisely paralleled the changes we observed for the same stimulus and time course using qPCR, validating the measure (Figure 2B). Because multiple nascent RNAs from stimulus-induced genes accumulate near the transcription site (TS) in the nucleus before splicing and export (Bhatt et al., 2012Bhatt D.M. Pandya-Jones A. Tong A.J. Barozzi I. Lissner M.M. Natoli G. Black D.L. Smale S.T. Transcript dynamics of proinflammatory genes revealed by sequence analysis of subcellular RNA fractions.Cell. 2012; 150: 279-290Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, Senecal et al., 2014Senecal A. Munsky B. Proux F. Ly N. Braye F.E. Zimmer C. Mueller F. Darzacq X. Transcription factors modulate c-Fos transcriptional bursts.Cell Rep. 2014; 8: 75-83Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), smFISH also reveals the total number of active TSs (0, 1, or 2) for each gene within each neuron that are actively being transcribed at any given time point, providing a key measure of promoter state at each allele in a given cell (Figures 2A and 2C). Colocalization of smFISH signal for Fos introns with the nuclear Fos exon signal confirmed that these nuclear clusters are composed of nascent RNA (Figure 2C). We performed parallel measurements for the neuronal activity-regulated gene Npas4 in an independent set of neurons (Figures 2A and S1C) (Gómez-Schiavon et al., 2017Gómez-Schiavon M. Chen L.-F. West A.E. Buchler N.E. BayFish: Bayesian inference of transcription dynamics from population snapshots of single-molecule RNA FISH in single cells.Genome Biol. 2017; 18: 164Crossref PubMed Scopus (23) Google Scholar). In the presence of TTX, both Fos and Npas4 mRNA levels were low but detectable in most neurons, with a distribution of cytoplasmic mRNA levels and either 0 or 1 active TSs detected per cell (Figures 2D and S1D). Following membrane depolarization, the mean levels of mRNAs were elevated over time (Figures 2D and S1D). However, there was increased variability in the distributions of mRNA between cells, and although the proportion of neurons showing both active alleles (TS = 2) for Fos or Npas4 increased following membrane depolarization and reached a peak 10 min following cessation of the stimulus (Figures 2D and S1D), we observed a substantial fraction of neurons that had only 0 or 1 active alleles. When we compared the levels of Fos protein in single neurons induced by the same pulse of membrane depolarization, we found wide variation in Fos protein levels when comparing single cells in the population. This strongly suggests that the transcriptional variation in Fos mRNA induction propagates and contributes to differences in the levels of Fos protein between neurons (Figure 2E). The cell-to-cell and allele-to-allele variability in RNA expression that we observed in neurons is consistent with the hypothesis that these genes are transcribed in stochastic bursts in neurons both under basal conditions and following transcriptional induction by membrane depolarization. To quantify these dynamic transcriptional properties and to understand how they change upon neuronal activation, we used a computational pipeline (BayFish) (Gómez-Schiavon et al., 2017Gómez-Schiavon M. Chen L.-F. West A.E. Buchler N.E. BayFish: Bayesian inference of transcription dynamics from population snapshots of single-molecule RNA FISH in single cells.Genome Biol. 2017; 18: 164Crossref PubMed Scopus (23) Google Scholar) to infer kinetic parameters of transcription and promoter-state transitions from the measured distributions of mRNA and active alleles (Figure 2D) at time points before stimulation (basal) and after stimulation (5 min KCl + 0 min, 10 min, or 20 min). Parameter inference using BayFish derives from an underlying mathematical model of gene transcription and promoter states. We considered the simplest model of transcriptional bursting (known as a two-state promoter model), in which the promoter of each allele can be either active (ON) or inactive (OFF) (Figure 2F). Our two-state model has a minimum of five kinetic parameters to be inferred: the rate at which each promoter turns on (kON), the rate at which each promoter turns off (kOFF), the RNA synthesis rate for each ON promoter (μ) and for each OFF promoter (μ0), and a delay (τ) between transcription initiation and production of mature, cytoplasmic mRNAs. The rate of Fos RNA degradation (δ) was measured previously (Shyu et al., 1989Shyu A.B. Greenberg M.E. Belasco J.G. The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways.Genes Dev. 1989; 3: 60-72Crossref PubMed Scopus (452) Google Scholar) and kept constant in our model. However, upon membrane depolarization, one or more of the kinetic parameters could change to cause increased levels of transcription. Previously, we showed for Npas4 that increasing the promoter activation rate (from kUON to stimulated kSON) upon membrane depolarization had the best and most parsimonious fit to the smFISH data when compared to other models of induction (Gómez-Schiavon et al., 2017Gómez-Schiavon M. Chen L.-F. West A.E. Buchler N.E. BayFish: Bayesian inference of transcription dynamics from population snapshots of single-molecule RNA FISH in single cells.Genome Biol. 2017; 18: 164Crossref PubMed Scopus (23) Google Scholar). Thus, we applied the same induction model to both Fos and Npas4 smFISH data to infer kinetic parameters using BayFish (Figures S1E and S1F; Table S1). Inferred rates of promoter-state transitions were comparable to or slower than the timescale of transcript elongation and maturation (τ of ∼3 min). The average length of time each promoter spent in the ON state was ∼11 min, whereas the average duration of the OFF state changed from ∼100 to ∼4 min upon membrane depolarization, thus increasing the fraction of time spent in the ON state (Figure 2G). These data indicate that the measured cell-to-cell variations in the levels of Fos and Npas4 mRNA likely arise from transcriptional bursting due to slow promoter-state transitions and that induction increases the probability Fos or Npas4 promoter transitions to the ON state, thus increasing burst frequency, in agreement with previous work (Gómez-Schiavon et al., 2017Gómez-Schiavon M. Chen L.-F. West A.E. Buchler N.E. BayFish: Bayesian inference of transcription dynamics from population snapshots of single-molecule RNA FISH in single cells.Genome Biol. 2017; 18: 164Crossref PubMed Scopus (23) Google Scholar, Senecal et al., 2014Senecal A. Munsky B. Proux F. Ly N. Braye F.E. Zimmer C. Mueller F. Darzacq X. Transcription factors modulate c-Fos transcriptional bursts.Cell Rep. 2014; 8: 75-83Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). The variability in Fos and Npas4 mRNA expression between neurons could arise from differences in the capacity of individual neurons to receive or propagate calcium-dependent signaling events to the nucleus (extrinsic variability), or it could arise from gene-local chromatin features that influence the probabilistic activation of gene promoters (intrinsic variability). Several pieces of data pointed toward an important role for gene-intrinsic determinants of transcriptional activation in our neurons. First, our hippocampal cultures are relatively homogeneous populations of excitatory neurons (Figure S2A). Second, all neurons responded to membrane depolarization with a robust increase in intracellular calcium concentration (Figure S2B). Finally, when we varied the extracellular calcium concentration in the medium, we found that although the accumulation of Fos RNA at the TSs depends on a minimum calcium concentration, the number of active Fos TSs did not increase further when extracellular calcium rose above this threshold (Figure S2C). At all concentrations of calcium tested, we still found a substantial number of neurons with only 1 TS active, although both TSs in a single cell have exposure to the same upstream calcium signaling events. To provide a direct comparison of intrinsic versus extrinsic variability at the single-cell level, we reasoned that if there is substantial cell-to-cell variation in the activation of calcium-dependent transcriptional signaling pathways in our population, we should see concordant activation of multiple activity-dependent genes in any single neuron. Alternatively, if the variability we observe for any single gene mostly arises from probabilistic activation intrinsic to the gene promoter, then we should see uncoordinated induction of multiple activity-dependent genes in a single neuron. To examine transcriptional concordance in single neurons, we simultaneously quantified mRNA for Fos and Npas4 in single hippocampal neurons by two-color smFISH following membrane depolarization in culture (Figure 3A). Even though Fos and Npas4 have similar kinetics and magnitude of induction at the population level (Figures 2B and S1C), we found a relatively weak correlation between the depolarization-induced levels of these two mRNAs in single neurons (Figure 3B). Our data also reveal variability in the transcriptional activation of each of the two Fos and Npas4 alleles within a single neuron (Figure 3C). Although many neurons in our population transitioned from having both alleles of Fos and Npas4 off (0,0) to having both alleles on (2,2) following membrane depolarization, at each time point, we also found substantial numbers of neurons in which only a single allele of one gene was active in various combinations with 0, 1, or 2 active alleles of the other gene. To assess whether intrinsic determination of promoter activation also occurs in adult neurons in response to physiologically relevant environmental stimuli in vivo, we performed dual-color Fos and Npas4 smFISH on sections from visual cortex of dark-adapted mice before and after light exposure (Figure 3D). Because Npas4 is a neural-selective gene, whereas Fos is also inducible in non-neuronal cells, we first identified Npas4-positive neurons in the visual cortex and then quantified the number of active Npas4 and Fos alleles in the nuclei of these neurons (Figures 3E and 3F). Few cells had detectable Npas4 mRNA in the visual cortex of dark-adapted mice, and all of these had no detectable active TSs for Fos (Figure 3G). Consistent with previous studies (Hrvatin et al., 2018Hrvatin S. Hochbaum D.R. Nagy M.A. Cicconet M. Robertson K. Cheadle L. Zilionis R. Ratner A. Borges-Monroy R. Klein A.M. et al.Single-cell analysis of experience-dependent transcriptomic states in the mouse visual cortex.Nat. Neurosci. 2018; 21: 120-129Crossref PubMed Scopus (228) Google Scholar), light exposure led to a robust induction of Npas4 and Fos mRNA in visual cortex, as m
