Author response: Synthetic reconstruction of the hunchback promoter specifies the role of Bicoid, Zelda and Hunchback in the dynamics of its transcription

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Appendix 2 Appendix 3 Appendix 4 Appendix 5 Appendix 6 Data availability References Decision letter Author response Article and author information Metrics Abstract For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as a model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation. By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the early hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoid’s partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate. Editor's evaluation In this paper, the authors use synthetic transcriptional enhancers to probe the roles of three transcription factors, Bicoid, Hunchback and Zelda, in specifying the production of a sharp, accurately placed gene expression boundary in early fruit fly embryos. They find that Bicoid, which is expressed in an anterior-posterior gradient, is sufficient on its own to generate a boundary in the same location as a wild-type fly, but combinatorial regulation by Hunchback and Zelda is needed to ensure the boundary forms quickly enough. They further combine their experimental observations with modeling to conclude that Bicoid exists in active and inactive forms, and that an equilibrium model captures the relevant behaviors, implying energy expenditure during the binding of transcription factors to DNA or RNA polymerase is theoretically unnecessary. https://doi.org/10.7554/eLife.74509.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Morphogen gradients are used by various organisms to establish polarity along embryonic axes or within organs. In these systems, positional information stems from the morphogen concentration detected by each cell in the target tissue and mediates the determination of cell identity through the expression of specific sets of target genes. While these processes ensure the reproducibility of developmental patterns and the emergence of properly proportioned individuals, the question of whether the morphogen itself directly contributes to this robustness or whether it requires the involvement of downstream cross-regulatory networks or cell-communication remains largely debated. This question becomes even more pressing with the recent discovery that when studied at the single-cell level, transcription is frequently observed to be an extremely noisy process, hardly suggestive of such precise control. To understand how reproducible transcription patterns can robustly emerge from subtle differences of morphogen concentration, we study the Bicoid (Bcd) morphogen system which initiates pattern formation along the antero-posterior (AP) axis in the young fruit fly embryo (Driever and Nüsslein-Volhard, 1988). The Bcd gradient was shown to be steadily established at the onset of transcription, one hour after egg laying, in the form of an exponential AP gradient with a λdecay length measured in the range of 16–25% egg-length (EL) (Abu-Arish et al., 2010; Durrieu et al., 2018; Houchmandzadeh et al., 2002; Liu et al., 2013). Fluorescent correlation spectroscopy measurements (Abu-Arish et al., 2010) and single molecule tracking of GFP-tagged Bcd proteins (Mir et al., 2018) revealed that a fraction of the Bcd proteins has a fast diffusion coefficient sufficient to explain the establishment of the gradient in such a short time by the synthesis-diffusion-degradation model (Abu-Arish et al., 2010; Fradin, 2017). This was further supported with the use of a tandem fluorescent timer as a protein age sensor (Durrieu et al., 2018). Of note, the establishment of the Bcd gradient is not only rapid but also extremely precise in space with only 10% variability among embryos (Gregor et al., 2007b) and the gradient is linearly correlated to the amount of bcd mRNA maternally provided and the number of functional bcd alleles in the females (Liu et al., 2013; Petkova et al., 2014). The Bcd protein binds DNA through its homeodomain (Hanes and Brent, 1989; Treisman et al., 1989) and activates the expression of a large number of target genes carrying Bcd binding sites (BS). Among the Bcd target genes, hunchback (hb) is expressed in a large domain spanning the whole anterior half of the embryo (Driever et al., 1989). hb expression begins when the first hints of transcription are detected in the embryo, i.e. at nuclear cycle 8 (Porcher et al., 2010). About one hour later (i.e. at nuclear cycle 14), the expression domain of hb is delimited by a posterior boundary, which is both precisely positioned along the AP axis and very steep suggesting that very subtle differences in Bcd concentration in two nearby nuclei at the boundary are already precisely measured to give rise to very different transcriptional responses (Crauk and Dostatni, 2005; Gregor et al., 2007a; Houchmandzadeh et al., 2002). Detailed analysis of hb expression by RNA FISH also indicated that transcription at the hb locus is extremely dynamic in time: it is detected during the successive S-phases but not during the intervening mitoses, which punctuate this period of development. To gain insights into the dynamics of hb early expression with a higher temporal resolution, the MS2-MCP approach for fluorescent tagging of RNA (Ferraro et al., 2016) was adapted to living fruit fly embryos (Lucas et al., 2013; Garcia et al., 2013). This provided an hb-P2 MS2-reporter expressed anteriorly in a domain with a boundary of the same steepness and positioning precision as the endogenous hb (Lucas et al., 2018). Of note, despite, this highly reproducible measurement of positional information (position and steepness of the boundary) on the scale of the embryo, at the single locus level, the variability in the total mRNA production (δmRNA/mRNA) over an entire nuclear cycle for loci at the boundary was of 150 %, i.e. one locus can produce 2.5 X more mRNA than another locus (Desponds et al., 2016). This high variability (noise) was consistent with smFISH data measuring the variability of hb mRNA amounts in nuclei (Little et al., 2013). It reflects a stochastic transcription process in neighboring nuclei which nevertheless all make the precise decision to turn ON hb during the cycle. The transcription dynamics of the hb-P2 MS2-reporter indicated that its steep boundary is established at each nuclear cycle 11–13 within 180 s and therefore suggested that accurate measurements of Bcd concentration were made much more rapidly than anticipated (Lucas et al., 2018). Consistently, inactivating Bcd by optogenetics in the embryo indicated that the hb transcription exhibited a very fast sensitivity to Bcd activity (Huang et al., 2017). Modeling was used to recapitulate the observed dynamics assuming cooperative binding of Bcd proteins to the six known BS sites of the hb-P2 promoter and rate limiting concentrations of Bcd at the boundary (Tran et al., 2018a). The model was able to recapitulate the fast temporal dynamics of the boundary establishment but could not reproduce its observed steepness which, given the 20% EL decay length of the Bcd protein gradient measured with immuno-staining (Houchmandzadeh et al., 2002), corresponds to a Hill coefficient of ~7, difficult to achieve without invoking the need for additional energy expenditure (Estrada et al., 2016). As expected, the performance of the model was higher when increasing the number of Bcd BS above six with a minimum of 9 Bcd BS required to fit the experimental data with a boundary of the appropriate steepness. This indicated that either the hb-P2 promoter contained more than 6 Bcd BS or that additional mechanisms were required to account for the steepness of the boundary. While quantitative models based on equilibrium binding of transcription factors to DNA shed lights on segmentation in Drosophila (Segal et al., 2008) or on the Bcd system (Estrada et al., 2016; Tran et al., 2018a), their impact remained limited by the lack of a quantitative experimental systems for validation. Here, we combined the MS2 quantitative probing system with a synthetic approach to decipher the functioning of Bcd in the transcription process at the mechanistic level. We built Bcd-only reporters with specific numbers of Bcd BS as well as reporters with 6 Bcd BS in combination with BS for the two known maternal Bcd co-factors binding to the hb-P2 promoter, namely the Hb protein itself (Porcher et al., 2010; Simpson-Brose et al., 1994) and the Zelda (Zld) pioneer transcription factor (Hannon et al., 2017; Xu et al., 2014). We show that 6 Bcd BS are not sufficient to recapitulate the hb-P2 expression dynamics while a reporter with only 9 Bcd BS recapitulates most of its spatial features, except a slightly lower steepness of its expression boundary and a longer period to reach steady state. To account for the bursty behavior of Bcd-only reporters in excess of Bcd, we fitted our data to a model involving a first step of Bcd binding/unbinding to the BS array and a second step where the bound Bcd molecules activate transcription. Synthetic reporters combining Bcd BS with either Hb or Zld BS indicated that both Hb and Zld sites reduce the time to reach steady state and increase expression by different means: Zld sites contribute to the first step of the model by drastically lowering the Bcd concentration thresholds required for activation while Hb sites act in the second step by reducing Bcd-induced burstiness and increasing the polymerase firing rates. Importantly, in embryos maternally expressing one (1 X) vs two (2 X) bcd functional copies, the boundary shift of the Bcd-only synthetic reporter with 9 Bcd BS was small enough to set the Bcd system within the limits of an equilibrium model. Lastly, the shift observed for the hb-P2 reporter in 1 X vs 2 X bcd backgrounds was the same as for the synthetic reporters further supporting that the Bcd gradient is the main source of positional information for the early expression of hb. Results Nine Bicoid binding sites alone recapitulate most features of the hb-P2 pattern We first investigated the transcription dynamics of Bcd-only MS2 reporters carrying exclusively 6, 9, or 12 strong Bcd binding sites (BS) (Hanes and Brent, 1989; Treisman et al., 1989) upstream of an hsp70 minimal promoter (Figure 1A and Supplementary file 1), all inserted at the same genomic location (see Materials and methods and Figure 1—figure supplement 1). Videos were recorded (see Videos 1–3) and analyzed from nuclear cycle 11 (nc11) to 13 (nc13) but we focused on nc13 data, which are statistically stronger given the higher number of nuclei analyzed. Unless otherwise specified, most conclusions were also valid for nc11 and nc12. Given that the insertion of a BAC spanning the whole endogenous hb locus with all its Bcd-dependent enhancers did not affect the regulation of the wild-type gene (Lucas et al., 2018), it is unlikely that there will be competition for Bcd binding between the endogenous hb and these synthetic reporters. Figure 1 with 1 supplement see all Download asset Open asset Transcription dynamics of the hb-P2, B6, B9 and B12 reporters. (A) Arrangement of the binding sites for Bcd (yellow), Hb (purple), and Zld (blue) upstream of the TATA box (red) and the TSS (broken arrow) of each reporter. (B) The MS2 reporters express the iRFP coding sequence followed by the sequence of the 24 MS2 stem loops. In the hb-P2 reporter, the hb-P2 promoter, 5’UTR sequence of the endogenous hb and its intron are placed just upstream of the iRFP sequence. In the synthetic reporters, the minimal promoter of the hsp70 gene was used. Of note, replacing the minimal promoter of hsp70 in B6 by the hb minimal promoter leads to a reporter with lower activity (Figure 1—figure supplement 1), (F–G). (C) Kymographs of mean fraction of active loci (colormap on the right) as a function of time (Y axis in s) and nuclei position along the AP axis (X axis in %EL) at nc13. (D) Along the AP axis (%EL), mean time of first spot appearance T0 (s) with shaded standard error of the mean and calculated only for loci with observed expression. (E) Cumulative distribution function of T0 (s) in the anterior (20% ± 2.5 %EL). (F) Boundary position (%EL) of fraction of nuclei with MS2 signal along AP axis, with shaded 95% confidence interval, as a function of time. The dash vertical lines represent the time to reach the final decision boundary position ( ± 2 %EL). (G) Fraction of nuclei with any MS2 signal, averaged over n embryos, with shaded standard error of the mean, along the AP axis (%EL), at nc13. (H) Boundary position and width were extracted by fitting the patterns fraction of expressing nuclei, (G) with a sigmoid function. Bar plots with 95% confidence interval for boundary position and width as the gray region placed symmetrically around the boundary position. Average values and confidence intervals are indicated in the adjacent table. (D–H) reporter data are distinguished by color: hb-P2 (orange, n = 5 embryos), B6 (yellow, n = 5 embryos), B9 (cyan, n = 6 embryos), and B12 (green, n = 4 embryos). Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Live imaging of transcription dynamics of B6 reporter. The videos have two channels: MCP-GFP channel (green) for monitoring the dynamics of nascent mRNA production and His-RFP (red) for nuclei detection. The capture frame is from 15% to 65% of embryo length. The anterior pole is on the left side of the frame. Position along the AP axis is indicated by white vertical bars positioned every 10% EL with the tallest one corresponding to 50% EL. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Live imaging of transcription dynamics of B9 reporter. The videos have two channels: MCP-GFP channel (green) for monitoring the dynamics of nascent mRNA production and His-RFP (red) for nuclei detection. The capture frame is from 18% to 65% of embryo length. The anterior pole is on the left side of the frame. Position along the AP axis is indicated by white vertical bars positioned every 10% EL with the tallest one corresponding to 50% EL. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Live imaging of transcription dynamics of B12 reporter. The videos have two channels: MCP-GFP channel (green) for monitoring the dynamics of nascent mRNA production and His-RFP (red) for nuclei detection. The capture frame is from 20% to 70% of embryo length. The anterior pole is on the left side of the frame. Position along the AP axis is indicated by white vertical bars positioned every 10% EL with the tallest one corresponding to 50% EL. The expression of the B6 (6 Bcd BS), B9 (9 Bcd BS), and B12 (12 Bcd BS) reporters harbored similar features as expression of the hb-P2 reporter (Lucas et al., 2018), which carries the ~300 bp of the hb-P2 promoter and the hb intron (Figure 1B, Supplementary file 1, Video 4): during the cycle, transcription was first initiated in the anterior with the expression boundary moving rapidly toward the posterior to reach a stable position into nc13 (Figure 1C). For all synthetic reporters, the earliest time when transcription was detected following mitosis (averaged over nuclei at the same position, see also Materials and methods), T0 , showed a dependence on position along the AP axis (Figure 1D), as observed for hb-P2 (Lucas et al., 2018). Thus, Bcd concentration is a rate-limiting factor for the expression of all reporters. As indicated by the distributions of onset time T0 in the anterior (~20 %EL), the first transcription initiation time at high Bcd concentration were not statistically different (P-values > 0.5) for all synthetic reporters (B6, B9, or B12) and hb-P2 (Figure 1E). This contrasts to the middle of the axis where the absolute number of Bcd molecules has been evaluated to be around 700 (Gregor et al., 2007a) and where the Bcd protein is thus likely to be limiting: transcription dynamics of the various reporters was quite diverse (Figure 1F) and the time it took for the hb-P2 reporter to reach the final decision to position its boundary (converging time, Supplementary file 2) was only 225 ± 25 s while it took as much time for B6 ± 25 s) or B9 ± 25 s) and slightly for B12 ± 25 Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Live imaging of transcription dynamics of hb-P2 reporter. The videos have two channels: MCP-GFP channel (green) for monitoring the dynamics of nascent mRNA production and His-RFP (red) for nuclei detection. The capture frame is from 15% to 70% of embryo length. The anterior pole is on the left side of the frame. Position along the AP axis is indicated by white vertical bars positioned every 10% EL with the tallest one corresponding to 50% EL. For all reporters, the fraction of nuclei with MS2 signal during the cycle exhibited a pattern along the AP axis in the anterior and in the posterior (Figure We fitted these patterns with sigmoid of position along the AP axis and extracted (see Materials and quantitative values for the position and width of the expression boundary (Figure the number of Bcd BS from 6 to 9, the expression boundary toward the posterior and the width of the boundary (Figure increasing the number of Bcd sites from 9 to 12 did not the boundary position the boundary Of note, B9, and hb-P2 expression were at while the width of the hb-P2 boundary was than the width of the B9 or the B12 (Figure Thus, even 6 Bcd BS have been in the hb-P2 promoter, only 6 Bcd BS alone in a synthetic reporter is not sufficient to recapitulate the hb this number to is sufficient to recapitulate all spatial features of the hb-P2 pattern except for the steepness of the expression boundary. Of note, the Bcd-only reporters much longer than the hb-P2 reporter to reach the final decision for boundary positioning suggesting that binding of additional transcription factors in the hb-P2 promoter likely contribute to the transcription is bursty at steady state even in excess of Bicoid To study the of transcription by Bcd, we the dynamics of transcription of the hb-P2 and the Bcd-only reporters at steady state the time of the time of MS2 activity in each the of the transcription process Figure supplement at a given position along the AP axis was by , the fraction of the cycle length during which fluorescent were observed (Figure In the anterior (~20 %EL), when increasing the number of Bcd BS in synthetic reporters from 6 to 9, with = ± and = ± = ± was as high as for B9 or B12 = ± values were all than the fraction of expressing nuclei ( 1, Figure This indicated bursty transcription activity in nuclei for all reporters, as by their MS2 in this region. for all Bcd-only reporters reach a in the anterior where the Bcd concentration is in excess (Figure and Figure supplement 1). As in this region the Bcd BS on reporters are likely to be by Bcd the burstiness observed is not by the binding/unbinding of Bcd to the BS array but by downstream Meanwhile, the mean of the MS2 in the anterior region did not of between reporters (Figure suggesting that the number of bound Bcd molecules does not the firing rate within transcription Figure 2 with 3 see all Download asset Open asset Modeling transcription dynamics at steady state. (A) Fraction of loci active time at steady state of s into averaged over n embryos, as a function of nuclei position along AP axis (B) fluorescent with standard error of active MS2 loci detected in the anterior region ( 20% ± at steady state. In reporter data are distinguished by color: hb-P2 (orange, n = 5 embryos), B6 (yellow, n = 5 embryos), B9 (cyan, n = 6 embryos), and B12 (green, n = 4 embryos). (C) of Bicoid binding and to an array of binding nuclear Bcd molecules can to binding sites at rate The binding array state is by where is the number of bound The rate are the binding of Bcd to the sites of The rate are the of bound Bcd from (D) Transcription dynamics is as a bursty model with the rate and The rate on the number of bound Bcd Transcription is not with than bound Bcd during the ON state can and transcription at rate Fraction of active loci at steady state for B6 B9 B12 to the fraction of active loci at steady state from the fitting models for corresponding BS numbers for B6 for B9 (F) or for B12 In these the are the rate the promoter with bound Bcd molecules and is set to The ON at higher bound are set given the activation of transcription by bound Bcd (see Supplementary file The binding rate is by assuming that Bcd binding is diffusion limited A model to recapitulate expression dynamics from synthetic reporters To explain the observed dynamics of the expression patterns (Figure and bursty transcription in with excess Bcd (Figure and Figure supplement 1), we built a model for transcription regulation of the Bcd-only synthetic reporters (Figure In this regulation in two nuclear Bcd molecules can to and from the Bcd BS on the promoter (Figure and bound Bcd molecules can activate transcription (Figure We a Bcd gradient, i.e. the Bcd concentration at a given position is over time. This was by on the dynamics of the Bcd gradient using Bcd at during nc13 of our (Abu-Arish et al., 2010; Gregor et al., In step 1 (Figure the binding and of Bcd to an array of Bcd BS were as in et al., 2016; Tran et al., In our the binding state was by , with the number of bound Bcd molecules The binding rate on the number of BS and the Bcd rate for a single BS The rate were to account for various of and binding In step 2 (Figure we this model to account for the burstiness in transcription with Bcd binding/unbinding (Figure The promoter dynamics was as a ON and to account for the observed of transcription with a time scale between s and s et al., et al., 2016; et al., The ON rate was by the number of bound Bcd the BS than Bcd molecules transcription could not be To account for the between the burstiness of transcription and the Bcd binding and the rate did not on the Bcd BS state. the promoter is could transcription and be at rate any given time and nuclei position along the AP axis, it was to the for the promoter to be in the ON state (see Materials and methods and Appendix 1). In this each could be to the measured transcription dynamics Bcd binding rate , the pattern boundary position, Bcd rate the pattern steepness (Estrada et al., 2016; Tran et al., 2018a), the , the fraction of active loci during steady state and the firing rate the mean loci To which processes were on the number of Bcd we first the for the fit of the model with the B6 data (Figure and Appendix we each of these to either alone or in to fit the B9 (Figure and Figure supplement 2) and B12 data (Figure and Figure supplement As have more Bcd BS than B6, the fitting of the B9 and B12 data to the model also to account for higher (i.e. for > indicated that very could be for B9 and B12 by only 3 of the to , (Figure supplement 2) while the remained for Given that the expression patterns of hb-P2 and all Bcd-only reporters a in the anterior where Bcd concentration is likely in we the activation of the promoter when = 6, 9 or 12 Bcd BS were that the number of bound Bcd proteins did not affect the rate , we a of between and This is three than the of the Bcd BS numbers between B9 and In there is impact of three more sites when B9 to B12 it is the increase of in the number of This that the is not in the number of Bcd BS but that there is between bound Bcd in the for between bound Bcd molecules is in Appendix 3 and Figure supplement Hunchback reduces the burstiness of transcription the same number of Bcd BS in the B6 and hb-P2 reporters, their expression pattern and dynamics were very different 1 and and Figure supplement 1). To whether this could be by the of BS for in the hb-P2 promoter, we used our synthetic approach to decipher the impact on the various features in our model when to the reporters BS for the two partners of Bcd, and Zld also in the hb-P2 promoter (Figure As our was to to which mechanistic step of our model each of these we by BS in numbers that are much higher than in the hb-P2 Figure 3 with 1 supplement see all Download asset Open asset Transcription dynamics of the B6, and reporters. (A) Arrangement of the binding sites for Bcd (yellow), Hb (purple), and Zld (blue) upstream of the TATA box (red) and the TSS (broken arrow) of each reporter. (B) Kymographs of mean fraction of active loci (colormap on the right) as a function of time (Y axis in s) and nuclei position along the AP axis (X axis in %EL) at nc13. (C) time of first spot appearance T0 (s) along the AP axis with shaded standard error of the mean and calculated only for loci with observed

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