Figures and data in Large-scale analysis of the integration of enhancer-enhancer signals by promoters

Figures
Tables
Additional files

5 figures, 1 table and 5 additional files

Figures

Figure 1 with 1 supplement

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Schematic of three-way combinatorial approach.

(A) Three-way combinatorial massively parallel reporter assay (MPRA) design to test enhancer-enhancer-promoter combinations. Eight barcoded reporter assay libraries, one per promoter, were constructed. Pairs of DNA elements (enhancers and scrambled control sequences) were inserted after barcoded reporter. The enhancers and controls can be placed in both orientations in either the enhancer position 1 (E1) or enhancer position 2 (E2). (B) The design of the library yields eight matrices that contain control-control combinations (CC), enhancer-control combinations (EC and CE), and enhancer-enhancer combinations (EE). (C and D) Two example loci, *Sox2* LCR (locus control region) (C) and *Otx2* (D) from where we selected enhancers to test in the reporter libraries. Enhancers (orange) were defined around DNAse I hypersensitivty sites from mouse embryonic stem cells (mESC). Promoters (blue) were chosen according to transcription start site (TSS) annotation and mESC DNAse I hypersensitivty sites. DNAse I data is from Joshi et al., 2015.

Figure 1—figure supplement 1

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Schematic of cloning strategy to generate enhancer-enhancer-promoter libraries.

First the eight different promoters were cloned into a reporter vector. Each of the eight vectors was barcoded and linearised, random enhancer-enhancer, enhancer-control, and control-control combinations were cloned downstream of the barcoded reporter.

Figure 2 with 3 supplements

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Effects of single enhancers across promoters.

(A) Enhancer-promoter median boost index matrix of single enhancers. For each single enhancer a median boost index (see figure and Methods, log₂(Activity_Combination/Activity_control-control_baseline)) was calculated across all enhancer-control combinations for that particular enhancer in any position and orientation, the baseline is the median activity of the promoter across all control-control combinations. For each control-control combination a boost index was calculated as the activity of the control-control combination over the median activity of all controls. A median control-control boost index was calculated based on this. Colour coding of the matrix corresponds to the median boost indexes, white spaces are missing data. (B) Distribution of boost indices for all enhancer+control combinations for the *Sox2* promoter. Leftmost column corresponds to the boost index distribution for all control-control combinations for the *Sox2* promoter. Each dot represents one enhancer+control combination. Horizontal lines correspond to the median of each distribution (same median as represented in the matrix in A). (C) Distribution of boost indices for all enhancer+control combinations for the enhancer Nanog_E074 across all promoters. Each dot represents one enhancer+control combination. Horizontal lines correspond to the median of each distribution. (D) Distribution of median boost indices for each single enhancer across promoters. Each dot represents the median of an enhancer across all enhancer+control combinations for that enhancers (same median as represented in the matrix in A). Colouring in B and D represents the significance at a 1% false discovery rate (FDR) for a Wilcoxon test comparing boost index distributions between enhancer+control combinations and the controls for each enhancer.

Figure 2—figure supplement 1

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Reproducibility of experimental data matrix of replicate correlations for all eight enhancer-enhancer-promoter (EEP) libraries.

Lower left panels represent the two-dimensional density plots of replicate-replicate activity correlations. Middle panels represent the one-dimensional density plot of each replicate. Upper right panels are the Pearson’s correlation coefficients of the mirror lower left panels.

Figure 2—figure supplement 2

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Position and orientation bias.

(A) Relationship between single enhancer boost indices across all enhancer-control (EC) combinations in position 1 versus in position 2 (see Figure 1). (B) Relationship between single enhancer boost indices across all EC combinations plus versus minus orientation, in position 1 (left) and in position 2 (right). For both panels each dot is one enhancer with one promoter. R is Pearson’s correlation.

Figure 2—figure supplement 3

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Selectivity of single enhancers.

(A) Relationship between average boost index of each single enhancer across all promoters and the F-statistic of a Welch F-test for that enhancer across all promoters. Colours indicate significance at a 5% false discovery rate (FDR). (B) Distributions of single enhancer boost indices for each enhancer across promoters. Each dot is one enhancer with one promoter and the boost index is the average boost index across all enhancer-control combinations. Colours of each point represents promoter identity. Vertical bars indicate the median boost index, and their grey or black shading indicates significance according to the Welch F-test at a 5% FDR.

Figure 3 with 1 supplement

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Effects of enhancer-enhancer (EE) combinations.

(A) Fragment-fragment combinatorial boost index matrix for the *Lefty1* promoter. Each square represents one control-control (CC), enhancer-control (EC or CE), or EE combination. Colour coding corresponds to the average boost index for each combination across all orientations measured over the median control-control baseline. (B) Boost index distributions across all eight promoters for each combination type, control-control (CC), enhancer-control (EC regardless of position) or EE combinations. p-Values correspond to the result of a Wilcoxon test. (C) Relationship between observed boost index for each EE combination and the observed boost index of the strongest single enhancer of the pair for *Sox2* and *Lefty1* promoters. Blue lines represent the LOESS fit of the data. (D) Observed and expected additive activities for the Sox2_E178+Sox2_E182 combination with the *Sox2* promoter and the individual activities of each of the elements. Each column represents the observed activities for the control-contol combinations, the enhancer-control combinations, and the EE combinations. The horizontal bars represent the median of each distribution. The horizontal black line represents the expected additive activity of the EE combination as calculated by the formula in the panel in the linear space. The horizontal red lines represent the propagated standard deviations of the expected additive activity of the EE combinations as calculated by the formula in the panel. (**E and F**) Relationship between observed and expected activities (additive in E, multiplicative in F) for all EE combinations for the *Sox2* promoter. The blue lines represent the linear fit of the data. Grey diagonal line is the x=y identity line. In all panels R represents Pearson’s correlation coefficient. Expected activities are calculated in the linear space and then plotted in the log₂ space.

Figure 3—figure supplement 1

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Additivity versus multiplicativity for all promoters.

Relationship between observed and expected activities (additive in A, multiplicative in B) for all enhancer-enhancer combinations for the all promoters. The blue lines represent the linear fit of the data. In all panels R and R2 are based on Pearson’s correlation.

Figure 4

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Supra- and sub-additive behaviours of enhancer combinations.

(A) Distributions of log₂ observed activities over expected additive activities ratios of enhancer-enhancer combinations across promoters. Coloured in turquoise are supra- and sub-additive combinations for which the observed activity is more than one standard deviation away from the expected activity. Horizontal bars represent the median of each distribution. Numbers on the top part are the percentage of supra-additive combinations for each promoter. Numbers on the lower part are the percentage of sub-additive combinations for each promoter. (B) Relationship between the percentages of supra- and sub-additive enhancer-enhancer combinations and promoter control-control baselines. Blue lines are the linear fit of the data. R is Pearson’s correlation. (C) Average supra- or sub-additive behaviour of each single enhancer across enhancer-enhancer combinations for each promoter. Each dot represents the median log₂ observed over expected for all enhancer-enhancer combinations of a single enhancer and a particular promoter. Grey bars represent the median of each distribution. (D) For two example enhancers, distribution of log₂ observed over expected ratios for combinations of that enhancer with any other enhancer and promoter. Horizontal black bars represent the median of the distribution. (E) Distribution of log₂ observed over expected ratios for enhancer-enhancer pairs from the same enhancer cluster (within clusters) or from different enhancer clusters (between clusters).The p-value results from comparing both distributions using a Wilcoxon test. Horizontal grey bars represent the median of each distribution. In all panels Obs/exp refers to observed activity over expected additive activity.

Figure 5 with 2 supplements

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Non-linear responses of promoters to enhancer-enhancer combinations.

(A) Relationship between observed boost indices and average boost index across promoters for all shared enhancer-enhancer combinations. Blue lines represent the linear fit of the data. (B) Relationship between the slopes extracted from the linear fits in A and the baseline promoter activities derived from the control-control combinations. The formulae depict the relationship between the average boost indices and the observed boost indices of each promoter through the extracted slopes. For both panels R is Pearson’s correlation coefficient.

Figure 5—figure supplement 1

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Promoter-promoter boost index correlations for all shared enhancer-enhancer combinations.

Lower left panels represent the two-dimensional density plots of promoter-promoter boost index correlations. Middle panels represent the one-dimensional density plot of each promoter. Upper right panels are the Pearson’s correlation of the mirror lower left panels. Blue lines are the linear fit of the data.

Figure 5—figure supplement 2

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Non-linear responses of promoters to enhancer-control (single enhancers) combinations.

(A) Relationship between observed boost indices of single enhancers and average boost index across promoters for all shared single enhancer-control combinations. Blue lines represent the linear fit of the data. (B) Relationship between the slopes extracted from the linear fits in A and the baseline promoter activities derived from the control-control combinations. For both panels R is Pearson’s correlation coefficient.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Cell line (Mus musculus)	E14tg2a mouse embryonic stem cell (mESC)	ATCC	CRL-1821
Gene (M. musculus)	Klf2	GenBank	16598	Promoters and enhancers; coordinates in Supplementary file 1
Gene (M. musculus)	Sox2	GenBank	20674	Promoters and enhancers; coordinates in Supplementary file 1
Gene (M. musculus)	Otx2	GenBank	18424	Promoters and enhancers; coordinates in Supplementary file 1
Gene (M. musculus)	Lefty1	GenBank	13590	Promoters and enhancers; coordinates in Supplementary file 1
Gene (M. musculus)	Fgf5	GenBank	14176	Promoters and enhancers; coordinates in Supplementary file 1
Gene (M. musculus)	Tbx3	GenBank	21386	Promoters and enhancers; coordinates in Supplementary file 1
Gene (M. musculus)	Nanog	GenBank	71950	Promoters and enhancers
Gene (M. musculus)	Ap1m1	GenBank	11767	Promoters and enhancers
Recombinant DNA reagent	Downstream Assay vector (JvAp102)	Martinez-Ara et al., 2022	JvAp102	Downstream reporter assay plasmid; ‘see Methods
Sequence-based reagent	Enhancer and promoter oligos	This paper	Primers	Supplementary file 4
Sequence-based reagent	Barcoding primers and sequencing primers	Martinez-Ara et al., 2022	Barcoding reagents	Supplementary file 4
Sequence-based reagent	Synthetic DNA negative controls	This paper	Negative controls	Supplementary file 2
Peptide, recombinant protein	Gibson Assembly Master Mix	NEB	Cat# E2611S
Peptide, recombinant protein	I-CeuI	NEB	#R0699S
Peptide, recombinant protein	I-SceI	NEB	#R0694S
Commercial assay or kit	Mouse Embryonic Stem Cell Nucleofector Kit	Lonza	#VPH-1001
Chemical compound, drug	TRIsure	Bioline	(#BIO-38032)
Commercial assay or kit	GeneJET RNA extraction kit	Thermo Fisher	#K0732
Peptide, recombinant protein	DNase I	Roche	#04716728001
Peptide, recombinant protein	Maxima Reverse transcriptase	Thermo Fisher	#EP0743
Peptide, recombinant protein	MyTaq Red mix	Bioline	#BIO-25043
Strain, strain background (Escherichia coli)	e. cloni 10G supreme	Lucigen	#60081-1	Electrocompetent cells
Peptide, recombinant protein	Takara ligation kit version 2.1	Takara	#6022
Peptide, recombinant protein	Klenow HC 3′ ->5′ exo	NEB	#M0212L
Commercial assay or kit	ISOLATE II PCR and Gel Kit	Bioline	BIO-52059
Peptide, recombinant protein	Fast-link ligase	Lucigen	LK0750H
Peptide, recombinant protein	End-It DNA End-Repair Kit	Epicentre	#ER0720
Commercial assay or kit	dsDNA High sensitivity Qubit kit	Invitrogen	#Q33231
Commercial assay or kit	CleanPCR magnetic beads	CleanNA	#CPCR-0050
Peptide, recombinant protein	XcmI	NEB	#R0533S
Peptide, recombinant protein	T4 DNA ligase	Roche	#10799009001
Peptide, recombinant protein	AvrII	Thermo Fisher	#ER1561
Peptide, recombinant protein	NheI	NEB	#R0131S
Strain, strain background (Escherichia coli)	5-alpha Competent E. coli	NEB	#C2987	Competent cells
Peptide, recombinant protein	LIF	Sigma-Aldrich	#ESG1107	2i+LIF media
Chemical compound, drug	Monothioglycerol	Sigma-Aldrich	#M6145-25ML	2i+LIF media
Chemical compound, drug	CHIR-99021	MedChemExpress	#HY-10182	2i+LIF media
Chemical compound, drug	PD0325901	MedChemExpress	#HY-10254	2i+LIF media
Other	BSA	Gibco	#15260-037	2i+LIF media
Other	DMEM-F12medium	Gibco	#11320-033	2i+LIF media
Other	Neurobasal medium	Gibco	#21103-049	2i+LIF media
Chemical compound, drug	N27	Gibco	#17504-044	2i+LIF media
Chemical compound, drug	B2	Gibco	#17502-048	2i+LIF media
Commercial assay or kit	MycoAlert Mycoplasma Detection Kit	Lonza	#LT07-318
Software, algorithm	BatchPrimer3 version 1.0	You et al., 2008	version 1.0	https://wheat.pw.usda.gov/demos/BatchPrimer3/
Software , algorithm	Starcode	Zorita et al., 2015	version 1.1	https://github.com/gui11aume/starcode
Software, algorithm	Python	Rossum and Drake, 2009	version 3.6	https://www.python.org/downloads/release/python-362/
Software, algorithm	Bowtie2	Langmead and Salzberg, 2012	version 2.3.4	http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
Software, algorithm	R	R Development Core Team, 2021	version 4.0.5	https://www.r-project.org/
Software, algorithm	ggplot2	Wickham, 2016	ggplot2	https://ggplot2.tidyverse.org/
Software, algorithm	Snakemake	Köster and Rahmann, 2012	version 4.4.0	https://anaconda.org/bioconda/snakemake/files?version=4.4.0