Volume 21, Issue 1, Pages 236-245 (October 2017) Quantitative Measurement and Thermodynamic Modeling of Fused Enhancers Support a Two-Tiered Mechanism for Interpreting Regulatory DNA  Md. Abul Hassan Samee, Tara Lydiard-Martin, Kelly M. Biette, Ben J. Vincent, Meghan D. Bragdon, Kelly B. Eckenrode, Zeba Wunderlich, Javier Estrada, Saurabh Sinha, Angela H. DePace  Cell Reports  Volume 21, Issue 1, Pages 236-245 (October 2017) DOI: 10.1016/j.celrep.2017.09.033 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 21, 236-245DOI: (10.1016/j.celrep.2017.09.033) Copyright © 2017 The Authors Terms and Conditions

Figure 1 eve37 and eve46 Respond Differently to the Same Repressors (A) Seven-striped expression of the even-skipped (eve) gene (top) and the genomic region containing the enhancers known to drive this pattern (bottom). Each enhancer is annotated with the stripe representing where it drives eve expression. (B) eve is regulated by the repressors Hb (green) and Kni (blue); these TFs have different spatial concentrations in the blastoderm embryo. The boundaries of eve37 and eve46 expression (black peaks) are set by differential sensitivities to Hb and Kni. (C) Information integration at multiple length scales predicts different outputs of enhancer fusions. If the enhancer-level model is operational and the fused enhancers are read as a “bag of sites,” we expect two broad stripes (top). If a two-tier model is operational and the component enhancers remain autonomous, we expect four stripes (bottom). The mechanism under a two-tier model for maintaining inter-stripe gaps between stripes 3 and 4 and stripes 6 and 7 is unknown. See also Figure S1. Cell Reports 2017 21, 236-245DOI: (10.1016/j.celrep.2017.09.033) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Expression Profiles for Fused Enhancers (A) We measured LacZ expression normalized to a hkb co-stain driven by eve37/eve46 enhancer fusions in multiple configurations. The average expression for each transgenic line is displayed as a function of A/P position with the shadow representing SEM. (B) For one fusion configuration, “Fusion C,” we placed 200-bp and 1,000-bp spacers between the enhancers and measured LacZ expression as above. (C) The length and binding site content of these synthetic constructs (orange) are comparable to other Drosophila developmental enhancers (blue). See also Figure S2. Cell Reports 2017 21, 236-245DOI: (10.1016/j.celrep.2017.09.033) Copyright © 2017 The Authors Terms and Conditions

Figure 3 The Enhancer-Level GEMSTAT Fails to Explain Readouts of Fused Enhancers (A) We applied GEMSTAT to three single eve enhancers. In all panels, experimentally measured expression profiles are shown in blue and GEMSTAT output in red. For this approach, we simultaneously fitted the model on ∼30 developmental enhancers, excluding all enhancers of eve. We then fitted the model on three enhancers of eve, starting from (P) initial parameters and letting (Q) denote the optimized parameters following a constrained strategy (see the Experimental Procedures). (B) We used GEMSTAT to fit a separate model for each of the fused enhancers, starting from (Q) as the initial estimate and using a constrained fitting strategy. (C) We also used GEMSTAT to fit a separate model for six fused enhancers using an unconstrained fitting strategy. See also Tables S1 and S2. Cell Reports 2017 21, 236-245DOI: (10.1016/j.celrep.2017.09.033) Copyright © 2017 The Authors Terms and Conditions

Figure 4 The Two-Tier GEMSTAT-GL Model Captures the Readouts of Enhancer Fusions We applied GEMSTAT-GL (for gene locus) to the six fusion constructs. Experimentally determined expression profiles (blue) and model predictions (red) are shown as a function of egg length along the A/P axis. See also Tables S1, S2, and S3. Cell Reports 2017 21, 236-245DOI: (10.1016/j.celrep.2017.09.033) Copyright © 2017 The Authors Terms and Conditions

Figure 5 GEMSTAT with Short-Range Repression (GEMSTAT-SRR) Less Accurately Predicts Salient Features of Fused Enhancer Expression We applied GEMSTAT-SRR to the six fusion constructs and compared the predictions to those made by GEMSTAT-GL. Experimentally determined expression profiles are in blue, GEMSTAT-SRR predictions are in red, and GEMSTAT-GL predictions are represented by gray dashes. See also Tables S1 and S2. Cell Reports 2017 21, 236-245DOI: (10.1016/j.celrep.2017.09.033) Copyright © 2017 The Authors Terms and Conditions

Figure 6 A “Two-Tiered” Mechanism May Define and Integrate Sub-modules of Regulatory Sequence at the Level of Single Enhancers and Entire Loci (Top) Enhancer sequences contain binding sites for different TFs that function by activating or repressing their target gene. Enhancer-level models capture each TF input independently, representing the enhancer as one “bag of sites.” Two-tier models, such as GEMSTAT-GL, also can be applied to enhancer-length sequences by first separating TF inputs into multiple regulatory segments and then integrating their weighted output to predict expression. (Bottom) Two-tier and enhancer-level models can be applied to an entire locus. Enhancer-level models consider TF binding across the locus as a large “bag of sites,” without considering individual enhancers as separate regulatory entities. We can also apply the two-tiered model to a gene locus. This approach first subdivides the regulatory sequence around a gene into smaller modules and then integrates the regulatory information from each module to predict expression. Cell Reports 2017 21, 236-245DOI: (10.1016/j.celrep.2017.09.033) Copyright © 2017 The Authors Terms and Conditions