2019 Apr 26
Quantitative Analysis of the ThrbCRM1-centered Gene Regulatory Network
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Quantitative Analysis of the ThrbCRM1-centered Gene Regulatory Network
Enhancer activity is determined by both the activity and occupancy of transcription factors as well as the specific sequences they bind. Experimental investigation of this dynamic requires the ability to manipulate components of the system, ideally in as close to an
in vivo context as possible. Here we use electroporation of plasmid reporters to define critical parameters of a specific cis-regulatory element, ThrbCRM1, during retinal development. ThrbCRM1 is associated with cone photoreceptor genesis and activated in a subset of developing retinal cells that co-express the Otx2 and Onecut1 (OC1) transcription factors. Variation of reporter plasmid concentration was used to generate dose response curves and revealed an effect of binding site availability on the number and strength of cells with reporter activity. Critical sequence elements of the ThrbCRM1 element were defined using both mutagenesis and misexpression of the Otx2 and OC1 transcription factors in the developing retina. Additionally, these experiments suggest that the ThrbCRM1 element is co-regulated by Otx2 and OC1 even under conditions of sub-optimal binding of OC1.
Cis-regulatory elements; Cone photoreceptors; Electroporation; Flow cytometry; Gene regulatory networks; Quantitative analysis; Reporter assays; Retinal development.
© 2019. Published by The Company of Biologists Ltd.
Conflict of interest statement
Competing interestsThe authors declare no competing or financial interests.
Schematic of the ThrbCRM1 reporter and sequence elements. (A) Sequence of one copy of the ThrbCRM1 element and the corresponding binding sites of Otx2 and Onecut1 (OC1). ‘*’ indicates conservation as defined in Emerson et al. (2013). (B) Schematic representation of the ThrbCRM1 construct with 4X or 2X copies of the 40 base pair ThrbCRM1 element shown as the grey box. Predicted Otx2 binding sites are indicated in yellow and the predicted OC1 binding site is indicated in blue.
Effects of ThrbCRM1::GFP plasmid concentration and ThrbCRM1 element copy number on reporter activity. (A,B) Representative flow cytometry plots of dissociated cells from chick retinas receiving 160 ng/µl of either the ThrbCRM1(4X) (A) or ThrbCRM1(2X) (B) EGFP reporter plasmid. Bins representing levels of EGFP fluorescence intensity are shown as vertical boxes and denoted on the right side of the plot. (C,D) Graphs of the percentage of total percentage of EGFP-positive cells along the y-axis relative to the concentration of the 4X (C) or 2X (D) ThrbCRM1::EGFP reporter plasmid on the x-axis. Arrow denotes 120 ng/µl of reporter plasmid (E,F) A graph of the percentage of EGFP-positive cells in each Bin out of the total number of EGFP-positive cells along the y-axis and the concentration of the reporter plasmid shown along the x-axis. Bin 1 through Bin 5 represent increasing amount of EGFP fluorescence (Bin 1, least amount of EGFP fluorescence; Bin 1, dark blue; Bin 2, red; Bin 3, light green; Bin 4, purple; Bin 5, aqua) Bin key in panel F also applies to panel E. (G,H) Graphs of the percentage of total EGFP-positive cells along the y-axis relative to the concentration of the 4X (G) or 2X (H) ThrbCRM1::EGFP reporter plasmid. Samples from G and H were generated in a single experiment, but plotted in separate graphs. Error bars represent 95% confidence intervals. Asterisk identifies a statistically significant P-value<0.05 using the Mann–Whitney U-test. n.s. denotes no significance.
Effects of misexpression of Otx2 and/or OC1 on ThrbCRM1::EGFP activity. (A-D) Representative flow cytometry plots of dissociated cells from chicken retinas receiving Ubiq::TdT reporter plasmid, 4XThrbCRM1::EGFP reporter plasmid and either TE (no DNA) (A), CAG::Otx2 (B), CAG::OC1 (C) or CAG::OC1 and CAG::Otx2 (D). (E) A plot of the percentage of ThrbCRM1::EGFP-positive cells in response to electroporation of the CAG plasmid shown along the x-axis. Average values are based on four retinas. (F) A graph of the percentage of ThrbCRM1::EGFP-positive cells in each Bin (Bins 1–5 as shown in A with Bin 1 having the least amount of EGFP fluorescence) for each of the four conditions. Plotted values represent the averages of four retinas and error bars represent 95% confidence intervals. A one-way ANOVA with a post-hoc Dunnett's statistical test was used to compare each of the misexpression groups to that of the TE group. * P<0.05, ** P<0.01 and *** P<0.001.
Effects of transcription factor misexpression plasmid concentration on reporter activity in mouse and chick retinas. (A) A graph of the percentage of electroporated cells in chicken E5 retinas that are EGFP-positive after introduction of 160 ng/µl ThrbCRM1::EGFP reporter plasmid and varying concentrations of the CAG::Otx2 and CAG::OC1 misexpression plasmids. The x-axis displays concentrations in ng/µl of each of the misexpression plasmids. (B) A graph of the percentage of EGFP-positive cells in each Bin (Bins 1–5). Bin 1 through Bin 5 represent increasing amount of EGFP fluorescence (Bin 1, least amount of EGFP fluorescence; Bin 5, the most amount of EGFP fluorescence). (C) A graph of the amount of cells positive for EGFP in a mouse P0 retina electroporated with 200 ng/µl of ThrbCRM1::EGFP reporter plasmid and varying concentrations of the CAG::OC1 misexpression plasmids. The x-axis displays concentrations in ng/µl of CAG::OC1 plasmid. (D) A graph of the amount of cells in each Bin (Bins 1–5) of the data plotted in C.
Mutational analysis of the ThrbCRM1 element. (A) Schematic and sequence representation of the ThrbCRM1 element. The putative OTX2 binding sites are highlighted in yellow and the OC1 binding site is highlighted in blue. The letters in red represent the mutation of the corresponding nucleotide. (B–G’) Confocal z-stack images of chicken retinas electroporated with CAG::Nucβ-gal and the ThrbCRM1::EGFP plasmid shown above and immunofluorescent detection of EGFP (green), Nucβ-gal (red) and DAPI (blue). (B–G) Merged images (B′–G′) EGFP signal only (H) Results of a flow cytometry evaluation of EGFP fluorescence displayed as a graph of the percentage of cells positive for the ThrbCRM1-driven EGFP reporter (x-axis). *** P<0.001 as determined by a one-way ANOVA with a post-hoc Dunnett's test. The electroporated Stagia3 reporter is shown to the left of each bar. Scale bar: 20 µm.
Activity of mutant ThrbCRM1::EGFP reporter in response to misexpression of Otx2 and/or OC1. (A,B) Graphs of the percentage of electroporated cells positive for EGFP after introduction into E5 chick retinas of 160 ng/µl of ThrbCRM::EGFP reporter plasmid with either no other plasmid, 100 ng/µl of Otx2 misexpression plasmid, or 100 ng/µl of OC1 misexpression plasmid. (A) ThrbCRM1[Otx2Mut1/2] reporter (B) ThrbCRM1[OCMut] reporter. (C) A graph of the percentage of EGFP-positive cells (y-axis) in the electroporated population after electroporation of E5 chick retinas with the ThrbCRM1 mutant reporter plasmid and the concentration of the Otx2 and OC1 misexpression plasmids (ng/µl) shown along the x-axis for each condition. Error bars represent 95% confidence intervals. Statistical significance is denoted by * P<0.05 or ** P<0.01 as determined by a one-way ANOVA with a post-hoc Dunnett's test.
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