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Differential cofactor dependencies define distinct types of human enhancers

This article has been updated

Abstract

All multicellular organisms rely on differential gene transcription regulated by genomic enhancers, which function through cofactors that are recruited by transcription factors1,2. Emerging evidence suggests that not all cofactors are required at all enhancers3,4,5, yet whether these observations reflect more general principles or distinct types of enhancers remained unknown. Here we categorized human enhancers by their cofactor dependencies and show that these categories provide a framework to understand the sequence and chromatin diversity of enhancers and their roles in different gene-regulatory programmes. We quantified enhancer activities along the entire human genome using STARR-seq6 in HCT116 cells, following the rapid degradation of eight cofactors. This analysis identified different types of enhancers with distinct cofactor requirements, sequences and chromatin properties. Some enhancers were insensitive to the depletion of the core Mediator subunit MED14 or the bromodomain protein BRD4 and regulated distinct transcriptional programmes. In particular, canonical Mediator7 seemed dispensable for P53-responsive enhancers, and MED14-depleted cells induced endogenous P53 target genes. Similarly, BRD4 was not required for the transcription of genes that bear CCAAT boxes and a TATA box (including histone genes and LTR12 retrotransposons) or for the induction of heat-shock genes. This categorization of enhancers through cofactor dependencies reveals distinct enhancer types that can bypass broadly utilized cofactors, which illustrates how alternative ways to activate transcription separate gene expression programmes and provide a conceptual framework to understand enhancer function and regulatory specificity.

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Fig. 1: Rapid COF degradation coupled to STARR-seq reveals COF-specific effects on enhancer activity.
Fig. 2: Differential COF requirements define distinct enhancer types with distinguishing sequence and chromatin features.
Fig. 3: P53 target genes and enhancers are insensitive to MED14 depletion.
Fig. 4: Combination of the TATA box and the CCAAT box renders the transcription of LTR12 retrotransposons and histone genes independent of BRD4.

Data availability

All raw deep-sequencing data (STARR-seq, PRO-seq, ChIP-seq and STAP-seq) and associated processed data generated in this study have been deposited in the NCBI GEO database under accession number GSE156741. Previously published datasets re-analysed in this study are available in the GEO repository under the following accession numbers: GSE100432 (genome-wide STARR-seq input library), GSE97889 (ATAC-seq), GSE71510 (H3K4me1, H3K4me3, H3K27ac, SMARCC1 and SMARCA4 ChIP-seq), GSE51176 (P300 and MLL4 ChIP-seq), GSE57628 (BRD4 ChIP-seq), GSE38258 (CDK8 ChIP-seq) and GSE86164 (P53 ChIP-seq). Peak files for the following ChIP-seq datasets are available from ENCODE (https://www.encodeproject.org/): DNase-seq (ENCFF001SQU, ENCFF001WIJ, ENCFF001WIK, ENCFF175RBN, ENCFF228YKV, ENCFF851NWR, ENCFF927AHJ, ENCFF945KJN and ENCFF360XGA), H3K36me3 (ENCFF467KXG, ENCFF742ZBG and ENCFF922EIA), H3K27me3 (ENCFF237TTT, ENCFF991HKN and ENCFF029ZPV), H3K9me2 (ENCFF586SOS, ENCFF808XMV and ENCFF346SOF), H3K9me3 (ENCFF751VFZ, ENCFF577FKU and ENCFF909UTX), JUND (ENCFF001UDY, ENCFF001UDZ, ENCFF950JTT and ENCFF088WYS) and FOSL1 (ENCFF001UDW and ENCFF001UDX). The vertebrate transcription factor motif collection is available from the JASPAR database (http://jaspar.genereg.net/download/CORE/ JASPAR2020_CORE_vertebrates_non-redundant_ pfms_jaspar.zip). The SwissProt Human database is available at

https://www.uniprot.org/proteomes/UP000005640. No restrictions on data availability apply.

Code availability

All custom codes used for data processing and computational analyses are available from the authors upon request.

Change history

  • 07 July 2022

    In the version of this article initially published, a typesetting error truncated the first sentence of the Acknowledgements, which has now been restored.

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Acknowledgements

We thank F. Muerdter, M. Muhar, J. Zuber and U. Schoeberl (all Research Institute of Molecular Pathology (IMP), Vienna BioCenter) for advice and help establishing the AID system and PRO-seq, respectively; R. Imre (IMP and Institute of Molecular Biotechnology, Vienna BioCenter) for help with analysing MS data; D. Hnisz (MPI Berlin) for help with Mediator ChIP-seq; R. C. Casellas, J. Kalchschmidt and S. K. Jung (NIH NIAMS) for sharing the mouse Mediator KO cell lines and data; D. Taatjes (University of Colorado), C. Bernecky (IST) and R. Young (MIT) for discussions; B. Sabari (UTSW) for feedback and help; and C. Buecker and H. Thomas (MPL), C. Plaschka (IMP) and A. Andersen (Life Science Editors) for comments on the manuscript; the staff at the IMP/IMBA in-house FACS facility, in particular G. Schmauss, and staff at the Molecular Biology Service. Deep sequencing was performed at the Vienna BioCenter Core Facilities. V.H. is supported by the Human Frontier Science Program (grant number LT000324/2016-L) and A.B. by the Swedish Research Council Postdoctoral Fellowship (VR 2017-00372). Research in the Stark group is supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement number 647320) and by the Austrian Science Fund (FWF, F4303-B09). Basic research at the IMP is supported by Boehringer Ingelheim and the Austrian Research Promotion Agency (FFG).

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Authors and Affiliations

Authors

Contributions

C.N. and A.S. conceived the project. C.N., K.B., O.H. and L.S. generated the CRISPR–Cas9-edited cell lines. M.P., M.R. and K.B. cultured cells and performed transfections. C.N., K.K. and C.D.A. performed STARR-seq. C.N., C.D.A., L.S., O.H. and M.P. performed PRO-seq, and C.D.A. performed STAP-seq. A.B. and C.H.L. performed MED1 ChIP-seq, and A.B. and J.E.H. performed and analysed MED1 IF with RNA FISH. K.S. performed the MS experiments (under the supervision of K.M.). C.N., L.S., C.D.A., M.P. and O.H. performed qPCR experiments. V.H. and G.L. performed the computational analyses. C.N., V.H. and A.S. interpreted the data and wrote the manuscript. A.S. supervised the project.

Corresponding author

Correspondence to Alexander Stark.

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Extended data figures and tables

Extended Data Fig. 1 Validation of cofactor degradation and effect on cell growth.

a, Western blots of denoted cofactors (COF) in the cell line where the respective COF is tagged by AID, without and with auxin (IAA) treatment for 1h. done once; validated by mass spectrometry; gel source data: Supplementary Figure 1. b, Schematic of the Mediator complex structure with head, middle and tail domains shown in different colors. Core structural subunit MED14 targeted in this study is shown in green. Subunits that cannot be detected anymore in mass-spectrometry upon MED14 depletion are semi-transparent. c, Protein abundance change as measured by shot-gun mass-spectrometry upon MED14 depletion by IAA treatment. All detected Mediator subunits are marked and colored according to different Mediator modules/domains shown in b. Subunits marked in italic were not detected anymore (i.e. were below detection limit) in all replicates of IAA treatment. N = 3 independent replicates. d, Protein abundance of denoted COFs as measured by targeted mass-spectrometry approach in the cell line where the respective COF is tagged by AID, without and with IAA treatment for 3h. N = 3 independent replicates; mean ± s.d. shown. e, Growth curves over a course of 3 days comparing untreated (solid line) and IAA-treated (dashed line) cells, for each COF-AID cell line. N = 2 independent replicates. f, Growth curves over a course of 5 days comparing untreated (solid line) and IAA-treated (dashed line) cells for MLL4- and CDK8-AID cell line. N = 3 independent replicates. P value of two-sided Student’s t-test at final day 5 timepoint is shown.

Extended Data Fig. 2 Effect of cofactor tagging and targeted cofactor degradation on enhancer activity.

a, Pearson’s correlations for pair-wise comparisons of replicates for each cofactor (COF) with and without IAA treatment calculated across a reference set of 6249 enhancers. For majority of COFs there are 4 independent replicates in each condition, except for our positive and negative controls, CDK9 and the Parental cell line, that have 2 and 3 replicates per condition, respectively. Inset on the right shows correlations between BRD4 samples pre-treated with IAA before STARR-seq library transfection, i.e. with an extended period of protein degradation. b, Hierarchical clustering of untreated and IAA-treated Parental and different COF-AID cell lines based on enhancer activity for a reference set of 6249 enhancers. All untreated cell lines (except p300/CBP which shows high level of COF pre-degradation in absence of IAA) cluster together with the Parental cell line, as well as IAA-treated MLL4- and CDK8-AID cell lines. c, Differential analysis of STARR-seq enhancer activity between each individual COF-AID cell line and Parental cell line without any treatment to assess the effect of COF-tagging on enhancer activity. Number of significantly up- or down-regulated enhancers is denoted (FDR ≤ 0.05). d, Differential analysis of STARR-seq enhancer activity for each COF-AID cell line with and without IAA treatment to assess the effect of COF degradation on enhancer activity. Number of significantly up- or down-regulated enhancers is denoted (FDR ≤ 0.05). e, Log2 fold-change in enhancer activity for enhancers pre-affected by P300 and CBP tagging (left; N = 301) and the rest of non-affected enhancers (right; N = 5948) in Parental and P300/CBP-AID cells upon IAA treatment. Boxes: median and interquartile range; whiskers: 5th and 95th percentiles. P values: two-sided Wilcoxon rank-sum test. f, Significance of change in enhancer activity (P values from differential analysis corrected for multiple testing/FDR) for a reference set of 6249 enhancers sorted individually by fold-change in each COF-AID cell line, from unaffected (or upregulated) enhancers on the left to most downregulated enhancers on the right.

Extended Data Fig. 3 Features of the four different groups of enhancers.

a, Significance of change in enhancer activity (P values from differential analysis corrected for multiple testing/FDR) upon individual cofactor degradation for four groups of enhancers defined by PAM (partitioning-around-medoids) clustering. Significant P values (FDR≤0.05) for down- and up-regulated enhancers are shown in shades of blue and red, respectively. Non-significant P values are shown in yellow. N = 1392, 1660, 1519, 1678 for Groups 1-4, respectively. b, Percent of variance explained by clustering of 6249 enhancers with partitioning around medoids (PAM) algorithm into different number of clusters. Four clusters explain ~85% of the variance. c, Hierarchical clustering of enhancers based on change in enhancer activity upon individual cofactor degradation. Boxplots summarize the log2 fold-change values per COF for each of the 5 clusters defined by cutting the dendrogram as denoted with a dashed line. Enhancer group assignment (from PAM clustering shown in Fig. 2a) is denoted by the coloured stripe below the dendrogram. N = 1156, 1391, 1531, 1052, 1119 for Groups 1-5, respectively. Boxes: median and interquartile range; whiskers: 5th and 95th percentiles. d, Agreement between clusters defined by hierarchical clustering and enhancer groups defined by PAM. For each hierarchical cluster (row) percent of enhancers falling into each PAM enhancer group is shown. e, Two-dimensional visualization of the data after dimensionality reduction with UMAP algorithm. Points represent individual enhancers coloured by their group membership (from PAM clustering). f, Percent of enhancers accessible/open according to DNase-seq in HCT116 cells or in other cell types in the four groups of enhancers defined in Fig. 2a. g, Percent of enhancers accessible/open according to DNase-seq in different number of cell lines ranging from enhancers closed in all cell lines (0 - yellow) to enhancers open in many/all (125 - red) cell lines assayed by DNase-seq in ENCODE. h, Mutual enrichment of transcription factor motifs for the four groups of enhancers. For each motif from the JASPAR vertebrate core collection of 579 non-redundant TF motifs (http://jaspar.genereg.net/download/CORE/JASPAR2020_ CORE_vertebrates_non-redundant_pfms_jaspar.zip) the enrichment/depletion in each group is assessed against the remaining three groups using two-sided Fisher’s exact test and only motifs with P value ≤ 0.001 and odds-ratio ≥ 2 are shown. The motifs are hierarchically clustered based on pair-wise Pearson’s correlation between motif position-weight matrices (PWMs) to group together similar motifs. A selection of representative motifs from these groups of similar motifs is shown in Fig. 2e. i, Enrichment analysis of 579 non-redundant TF motifs from the JASPAR vertebrate core collection (http://jaspar.genereg.net/download/CORE/JASPAR2020_CORE_ vertebrates_non-redundant_pfms_jaspar.zip) between unaffected and down-regulated enhancers upon MED14 depletion. Significantly enriched and depleted motifs (two-sided Fisher’s exact test; P value ≤0.01) are shown in red and blue, respectively. j, Differential analysis of enhancer activity upon MED14 depletion with enhancers containing a P53 motif marked in yellow. k, Differential analysis of enhancer activity upon MED14 (left) or BRD4 (right) depletion with enhancers overlapping a P53 ChIP-seq peak marked in yellow.

Extended Data Fig. 4 Induction of P53 target genes and enhancers is insensitive to MED14 depletion, but sensitive to BRD4 depletion.

a, Differential analysis of PRO-seq signal at genes between Nutlin-3a-treated and untreated WT HCT116 (left), MED14- (middle) or BRD4-AID (right) cells. Number of significantly upregulated genes in each cell line is denoted in yellow (FDR≤0.05 and fold-change≥2). N = 2 independent replicates for each condition. b, Venn diagram showing overlap of significantly upregulated genes in the 3 cell lines shown in panel a, defining in total 151 P53 target genes induced after 3h of Nutlin-3a treatment. c, Venn diagram showing overlap of 151 P53 target genes induced after 3h of Nutlin-3a (this study) and 175 P53 target genes defined previously after 1h of Nutlin-3a treatment76, defining a set of 243 direct P53 target genes used in panels d and h, and in Fig. 3e. d, Comparison of induction of direct P53 target genes (defined in panel c) in different cell lines and conditions. Top row compares induction in MED14- (left) or BRD4-AID (right) cells when the respective factor is present (-IAA) or depleted (+IAA). P53 targets are induced to the same extent upon MED14 depletion, but their induction is impeded upon BRD4 depletion. Bottom row compares induction between the two cell lines in the condition without IAA (left) or with IAA (right). Without IAA both MED14- and BRD4-AID cells induce P53 target genes to the same extent, however with IAA the induction in the BRD4-AID cells is impeded compared to the MED14-AID cells. e, Loci of the P53 target genes FAS (left) and RPS27L (right) with intronic P53-bound enhancers. Enhancer activity in different COF-AID cell lines with and without IAA treatment is shown (normalized STARR-seq signal for merged replicates), together with nascent transcription (normalized PRO-seq signal for merged replicates) upon induction of P53 signalling with Nutlin-3a in MED14- and BRD4-AID cells with and without IAA treatment. Transcription of both genes is induced upon Nutlin-3a treatment in both conditions with MED14 present (-IAA) or degraded (+IAA), but is strongly reduced with BRD4 degraded due to a pause-release defect that persists upon Nutlin-3a treatment. Activity of their associated P53-bound enhancers is unchanged upon MED14 depletion but is abolished upon BRD4 depletion. f, Locus with a FOS-bound MED14-depletion sensitive (left) and a P53-bound MED14-depletion insensitive (right) enhancer. Activity in different COF-AID cell lines with and without IAA treatment is shown (normalized STARR-seq signal for merged replicates), together with nascent transcription (normalized PRO-seq signal for merged replicates) upon induction of P53 signalling with Nutlin-3a in MED14- and BRD4-AID cells with and without IAA treatment. Activity of the FOS-bound enhancer is strongly reduced by both MED14 and BRD4 depletion, whereas the activity of the P53-bound enhancer is unchanged upon MED14 depletion but is abolished upon BRD4 depletion. Endogenous bidirectional transcription of the P53-bound enhancer is induced upon Nutlin-3a treatment in both conditions with MED14 present (-IAA) or degraded (+IAA), but is reduced with BRD4 degraded due to a pause-release defect that persists upon Nutlin-3a treatment. g, Differential analysis of PRO-seq signal at distal P53 or FOS bound sites (enhancers) upon Nutlin-3a treatment in IAA-treated BRD4-AID cell line. h, Log2 fold-change of PRO-seq signal for direct P53 target genes (left; genes defined in panel c) and distal P53 bound sites around direct P53 target genes (enhancers; right) in BRD4-AID cell line upon Nutlin-3a induction in background with BRD4 present (-IAA) or depleted (+IAA). N = 151, 20964, 244, 359 for P53 targets, other genes, P53- and FOS-bound enhancers, respectively. Boxes: median and interquartile range; whiskers: 5th and 95th percentiles; P values: two-sided Wilcoxon rank-sum test. i-k, Endogenous induction of known P53 target genes with Nutlin-3a as measured by qPCR in BRD4- (i), CDK9- (j) or TAF1-AID (k) cells without or with IAA treatment, i.e. with the respective factor present or degraded. N = 3 independent replicates; fold-change for each replicate calculated independently by dividing the treatment value with the corresponding control value; mean ± s.d. shown; P values: two-sided Student’s t-test. l, Growth curves over a course of 3 days comparing untreated (solid line) and IAA-treated (dashed line) TAF1-AID cells. N = 2 independent replicates. Inset shows Western blot for TAF1 in cells without and with IAA treatment for 1h.

Extended Data Fig. 5 P53 target genes and enhancers are not bound by MED1.

a, Locus of the MYC gene with an upstream cluster of endogenously active MED1-bound enhancers. ChIP-seq signal and called MED1 peaks in MED14-AID cells treated with IAA or/and Nutlin-3a and in WT HCT116 cells treated with Nutlin-3a are shown. b, Number of MED1 peaks called in each condition in MED14-AID and WT cells (MACS2, FDR ≤ 0.05). c, Average plot of MED1 ChIP-seq enrichment over input for a common set of MED1 peaks called in MED14-AID (638 peaks; left) and in WT HCT116 cells (1545 peaks; right). d, Example of an endogenously active MED14-dependent enhancer bound by MED1 (left) and a P53-bound MED14-independent enhancer not bound by MED1 (right). MED14-dependent enhancer is bound by MED1 in both WT and MED14-AID cells and this binding is abolished upon IAA treatment, i.e. upon MED14 depletion. P53-bound enhancer shows no MED1 binding in any condition, not even upon P53 induction with Nutlin-3a in either WT or MED14-AID cells.e, MED1 ChIP-seq enrichment over input for 2 groups of STARR-seq enhancers: 1) MED14-independent, P53-bound enhancers (N = 586) and 2) endogenously open and H3K27ac-marked MED14-dependent enhancers (N = 315), upon Nutlin-3a treatment in control and MED14-depleted MED14-AID cells (left) or in WT cells (right). While MED14-dependent enhancers show some MED1 binding in both WT and MED14-AID cells, which is abolished upon MED14 depletion (i.e. IAA treatment), P53-bound enhancers show no binding in any condition, including after Nutlin-3a treatment when these enhancers are activated. Boxes: median and interquartile range; whiskers: 5th and 95th percentiles. P values: two-sided Wilcoxon rank-sum test. f, MED1 IF with concurrent RNA FISH against P53 target gene RRM2B (top row) and Mediator-regulated positive control gene MYC (bottom row) in Nutlin-3a-treated WT HCT116 cells. Examples of individual cells with merged view of the FISH and MED1 IF signal at the FISH spot are shown on the left. Hoechst staining was used to determine the nuclear periphery, highlighted with a dashed white line. Mean RNA FISH and mean MED1 IF signal in 1x1μm window centred at FISH spots, or at random spots is shown on the right. Number of spots analysed is indicated in the lower right corner (n). g, Distribution of distance between each random spot and the nearest MED1 IF spot for random spots picked in different FISH experiments. Boxes: median and interquartile range; whiskers: 5th and 95th percentiles. P value: Kruskal-Wallis rank sum test.

Extended Data Fig. 6 P53 target gene induction is independent of multiple Mediator subunits in human and mouse cells.

a-c, Endogenous expression of known P53 target genes as measured by qPCR in IAA or/and Nutlin-3a-treated MED15- (a, tail module), MED19- (b, middle module) or MED1-AID (c, middle module) cells. Western blot of the denoted Mediator subunit in the respective COF-AID cell line, without and with IAA treatment for 3h is shown on top. gel s ource data: Supplementary Figure 1. d, Endogenous expression of known P53 target genes as measured by qPCR upon Nutlin-3a treatment before and after MED17 (head module) knock-down via RNAi in WT HCT116 cells. e, Endogenous expression of P53 target genes as measured by qPCR in DMSO or Nutlin-3a-treated mouse CH12 cells, either wild-type (WT) or knock-out (KO) cell lines for different Mediator subunits (cell lines from ref. 18). Experiment was performed in two batches (shown in two rows), each time using a re-thawed WT cell line as a control. Tailless = quintuple knock-out for MED15, MED16, MED23, MED24 and MED25 subunits. In a-g, N = 3 independent replicates; fold-change for each replicate calculated independently by dividing the treatment value with the corresponding control value; mean ± s.d. shown; P values: two-sided Student’s t-test.

Extended Data Fig. 7 LTR12 family repeats act as BRD4 independent enhancers/promoters that contain a combination of TATA-box and multiple CCAAT-box motifs.

a, Enrichment of retrotransposons in enhancers up- vs. down-regulated upon BRD4 depletion. b, Differential analysis of enhancer activity upon BRD4 depletion with LTR12 family repeat-overlapping enhancers marked in yellow. c, Fold-change of endogenous LTR12 expression as measured by qPCR in IAA-treated vs. untreated BRD4-AID K562 (left) and A549 (right) cells. In both cell lines BRD4 depletion leads to upregulation of LTR12C and D. d, Multiple alignment of LTR12 family repeats with detected enhancer activity in STARR-seq. Occurrences of CCAAT-box and TATA-box motifs, and the endogenous transcription initiation previously mapped by CAGE are marked below the alignment. e, Enrichment analysis of 579 non-redundant TF motifs from the JASPAR vertebrate core collection (http://jaspar.genereg.net/download/CORE/JASPAR2020_ CORE_vertebrates_non-redundant_pfms_jaspar.zip) between upregulated and down-regulated enhancers upon BRD4 depletion in HCT116 cells. Significantly enriched and depleted motifs (two-sided Fisher’s exact test; P value ≤0.05) are shown in red and blue, respectively. Logo of the most highly enriched CCAAT-box motif bound by NFYA/B is shown on the right. f, Endogenous expression of NFYA and NFYB as measured by qPCR without or with NFYA & NFYB siRNA treatment in BRD4-AID HCT116 cells. g, Western blots of NFYA (left) and NFYB (right) with and without treatment with the respective siRNA. gel source data: Supplementary Figure 1. h, Endogenous expression of LTR12C and D as measured by qPCR in IAA or/and NFYA & NFYB siRNA treated BRD4-AID HCT116 cells. i, Endogenous expression of NFYA and NFYB as measured by qPCR without or with NFYA & NFYB siRNA treatment in BRD4-AID A549 cells. j, Endogenous expression of LTR12C and D as measured by qPCR in IAA or/and NFYA & NFYB siRNA treated BRD4-AID A549 cells. k, Growth curves over a course of 4 days comparing untreated (solid line) and IAA-treated (dashed line) BRD4-AID and Parental A549 cells. N = 3 independent replicates. In c, f, h, i and j, mean ± s.d. shown; P values: two-sided Student’s t-test. N = 3 (c, i and j) or N = 6 (f and h) independent replicates; fold-change for each replicate calculated independently by dividing the treatment value with the corresponding control value.

Extended Data Fig. 8 Histone genes have a promoter with TATA-box and CCAAT-box motifs and do not require BRD4 for productive transcription.

a, Gene ontology term enrichment for genes with promoters containing both TATA-box and CCAAT-box motifs. Top 5 terms for cellular compartment (top), molecular function (middle) and biological process (bottom) categories are shown. Bars show fold-enrichment and are colored according to the P value of the one-sided hypergeometric test. b, Occurrence of TATA- and CCAAT-boxes in histone genes promoters relative to TSSs. c, Loci of the histone genes HIST1H2BJ and HIST1H2AG (left) and ribosomal protein gene RPS9 (right) with enhancer activity (normalized STARR-seq signal for merged replicates) and nascent transcription (normalized PRO-seq signal for merged replicates) in BRD4- and MED14-AID cells with and without IAA treatment. While RPS9 shows typical pause release defect with loss of RNA polymerase II signal throughout the gene body and increase at the promoter, the two histone genes do not lose signal in the gene body and still have high levels of actively elongating RNA polymerase II. d, Log2 fold-change of endogenous nascent transcription for histone genes from previously published datasets. Left: SLAM-seq in different cell lines upon rapid BRD4 degradation via AID system or BRD4 inhibition by JQ1 (from ref. 33); Right: NET-seq in MOLT4 cell line upon BRD4 inhibition by JQ1 or dBET6 (from ref. 32). e, STARR-seq signal enrichment over input in BRD4-AID cell line separated by strand for enhancers overlapping TATA-box promoters (N = 190), distal enhancers not overlapping promoters (N = 4917) and random inactive regions (negative control; N = 5151). Sense strand corresponds to orientation of the gene for enhancers overlapping promoters and is randomly assigned for distal enhancers and random regions. In d and e, boxes: median and interquartile range; whiskers: 5th and 95th percentiles. f, Examples of STARR-seq enhancers overlapping TATA-box promoters with evidence of endogenous initiation (CAGE): promoter of the MMP13 gene (left) and an instance of LTR12 repeat element (right). STARR-seq signal in BRD4-AID cell line and input library coverage is shown for + and – strands separately. Fragments from both strands are enriched over input, i.e. these promoter-overlapping fragments work as enhancers in both orientations.

Extended Data Fig. 9 Combination of a TATA-box core promoter and CCAAT-box-containing proximal enhancer is required and sufficient to drive high levels of BRD4 independent transcription.

a, Design of a sequence library to assess the requirement and sufficiency of the TATA-box and CCAAT-box motifs in the core and proximal promoter region, respectively, for the BRD4-independent transcriptional activity with massive parallel reporter assay. For the loss of function approach (left) 10 different BRD4-independent promoters (from LTR12 repeats and histone genes) were selected and variants with either TATA- and/or CCAAT-box motifs mutated were designed. For the gain of function approach (right) the TATA- and/or CCAAT-box motifs from the 10 selected promoters were inserted into 18 randomly picked neutral sequences. Each sequence variant is present in the library 5 times, coupled to a different 10bp long barcode at the 3’ end. b, Schematic of the massive parallel reporter assay (STAP-seq) to measure transcriptional activity at a single base-pair resolution in BRD4-AID cells without or with IAA treatment. 5’ ends of transcripts arising from each sequence present in the library are captured, amplified and sequenced, and the sequenced tags are uniquely mapped to the sequence variant of origin via the 10bp identification barcode. Correlation between transcriptional activity across all sequences in the library measured in two independent replicates for IAA-treated (right) and untreated (left) cells is shown at the bottom. c, Transcriptional activity at single base-pair resolution measured by STAP-seq for wild-type (WT) and different mutant versions of the LTR12 promoter instance. Transcription from each sequence variant was assessed 5 times in the library (coupled to 5 different barcodes) and the mean normalized STAP-seq signal across different barcodes is shown for the 2 independent replicates. STAP-seq signal in IAA-treated (red) vs. untreated (blue) BRD4-AID cells is shown as semi-transparent overlay. d, Transcriptional activity at single base-pair resolution measured by STAP-seq for a random neutral sequence upon insertion of TATA- and CCAAT-box motifs from an LTR12C, an LTR12D instance or from the HIST1H2AJ promoter. e-f, Endogenous expression of known heat-shock responsive genes as measured by qPCR in IAA or/and heat-shock treated BRD4-AID HCT116 (left), K549 (middle) and A549 (right) cells (e), and CDK9-AID HCT116 cells (f). In all three BRD4-AID cell lines heat-shock genes are equally strongly induced with BRD4 present or depleted but fail to get induced with CDK9 depleted. g, Endogenous expression of AFF1, AFF4 and known heat-shock responsive genes as measured by qPCR without or with AFF1 & AFF4 siRNA treatment in HCT116 cells. The induction of heat-shock genes is decreased after AFF1 & AFF4 knock-down. In e-g, N = 3 independent replicates; fold-change for each replicate calculated independently by dividing the treatment value with the corresponding control value; mean ± s.d. shown; P values: two-sided Student’s t-test. h, i, Changes in gene expression (log2 fold-change in PRO-seq signal) upon BRD4 (h) or MED14 (i) depletion for two groups of genes: (1) genes that have an enhancer insensitive to respective COF depletion (Group 4 enhancer for BRD4 or Group 3 enhancer for MED14) and (2) genes that have an enhancer downregulated upon respective COF depletion within 50 kb of their TSS. Number of genes in each group (N) is denoted in parentheses. Boxes: median and interquartile range; whiskers: 5th and 95th percentiles; P values: one-sided Wilcoxon rank-sum test. Barplots show percentage of genes in each group that are unaffected (not significantly downregulated) by COF depletion in PRO-seq. P values: one-sided Fisher’s exact test.

Extended Data Fig. 10 STARR-seq for additional AID-tagged cofactors shows no effect on enhancer activity.

a, Growth curves over a course of four days comparing untreated (solid line) and IAA-treated (dashed line) cells, for BRD7- (left), BRD9- (middle) and MLL1-AID (right) cell line. N = 2 independent replicates. Insets show Western blot for the respective cofactor in cells without and with IAA treatment for 3h. Upon IAA treatment none of the cofactors were detectable either in Western blot or in mass spectrometry. b, Examples of four enhancers detected by STARR-seq in the BAC library. For each enhancer the activity in BRD7-, BRD9- and MLL1-AID cell lines in the BAC-STARR-seq screen with and without IAA treatment is shown (normalized STARR-seq signal for merged replicates), alongside with endogenous chromatin accessibility and histone modifications in wild-type HCT116 cells. For comparison, enhancer activity in different COF-AID cell lines from the genome-wide STARR-seq screen is shown. None of the enhancers are affected by the loss of neither BRD7, BRD9 nor MLL1, while they are sensitive to depletion of other COFs (e.g. BRD4, MED14 or CDK9). c, Differential analysis of STARR-seq enhancer activity for 114 enhancers detected in the BAC library in each COF-AID cell line with and without IAA treatment to assess the effect of COF degradation on enhancer activity. Number of significantly up- or down-regulated enhancers is denoted (FDR ≤ 0.05). Depletion of none of the three COFs has an effect on enhancer activity, suggesting that they are not required for enhancer activity in the unperturbed HCT116 cells.

Supplementary information

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Supplementary Fig. 1 | Source images of western blots presented in Fig. 1c, Extended Data Figs 1a, 5l, 7a–d, 8f and 11a. a–n, Source images of western blots shown in Fig. 1c (a–g), Extended Data Fig. 1a (a–g), Extended Data Fig. 5l (h), Extended Data Fig. 7a–d (i–l) and Extended Data Fig. 11a (m, n), detecting BRD2 (a), BRD4 (b), p300 (c), CBP (d), MED14 (e), CDK7 (f), CDK9 (f), CDK8 (g), TAF1 (h), MED15 (i), MED19 (j), MED1 (k), MED17 (l), BRD7 (m), MLL1 (m) or BRD9 (n) in the cell line for which the respective cofactor is tagged by an AID tag, comparing control (–IAA) and auxin (+IAA) treatment for 1h (a–k, m, n), or in the parental HCT116 cell line comparing control and MED17 siRNA treatment for 24 h (l). o-p, Source images of western blots shown in Extended Data Fig. 8f detecting NFYA (o) or NFYB (p) in BRD4-AID cells comparing control and combined NFYA and NFYB siRNA treatment for 24 h. In each panel, the top image always shows the immunoblot with antibody against the V5 Tag or the endogenous protein (denoted in the top right corner). The bottom image always shows tubulin, which was blotted from same gel and serves as a loading control. Regions cropped for presentation in final figures are boxed in red.

Reporting Summary

Peer Review File

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Supplementary Table 1 | List of materials. List of sequences of gRNAs to establish HCT116 parental cell line; list of sequences of gRNAs to target individual COFs within the parental cell line; table of MS peptide sequences utilized to measure abundance of individual COFs; list of primary and secondary antibodies used to measure COF-AID degradation, to assess siRNA knockdown efficiency and to perform ChIP and IF experiments; list of utilized qPCR primers; list of custom designed intronic RNA FISH probes.

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Supplementary Table 2 | COF-AID STARR-seq mapping statistics. Summary of total sequenced reads, mapped reads and spike-in reads for genome-wide and BAC STARR-seq screens; individual spike-in counts and calculated normalization factor used to scale each COF-AID STARR-seq screen; list of selected STARR-seq spike-in sequences (mouse enhancers and human enhancers with D. melanogaster flanking sequence) used for normalizing STARR-seq counts; number of called peaks with MACS2 for each COF/condition from merged replicates.

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Supplementary Table 3 | COF-AID STARR-seq counts. Raw counts for a reference set of 6,249 enhancers in all STARR-seq experiments; table of log2(FC) values between treatment and control in each COF-AID STARR-seq experiment for a reference set of enhancers; table of adjusted P values (FDR) from the differential analysis between treatment and control in each COF-AID STARR-seq experiment for a reference set of enhancers..

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Supplementary Table 4 | Re-analysed published datasets. Table of used, previously published STARR-seq input libraries (genome-wide and BAC); list of all previously published datasets analysed in this study, with respective references and GEO or ENCODE database accessions.

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Supplementary Table 5 | Reads statistics for NGS experiments. Summary of total sequenced and mapped reads for PRO-seq experiments in MED14-AID, BRD4-AID and WT cells; MED1 ChIP-seq in MED14-AID and WT cells; and STAP-seq in BRD4-AID cells. Where applicable, statistics of spike-in reads and derived normalization factors are provided.

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Supplementary Table 6 | PRO-seq counts. Raw PRO-seq counts in promoter and gene body regions of 21,116 analysed genes for MED14-AID, BRD4-AID and WT HCT116 cell lines with different treatments (IAA and/or Nutlin-3a).

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Supplementary Table 7 | BRD4-AID CCAAT- & TATA-box promoter library counts. Raw STAP-seq counts per position for 2,000 promoter candidate sequences each 250-bp long, including WT CCAAT-box and TATA-box-containing promoters, their mutated versions and insertions of these motifs into random neutral sequences.

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Neumayr, C., Haberle, V., Serebreni, L. et al. Differential cofactor dependencies define distinct types of human enhancers. Nature 606, 406–413 (2022). https://doi.org/10.1038/s41586-022-04779-x

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