FIREWACh: high-throughput functional detection of transcriptional regulatory modules in mammalian cells

Murtha, Matthew; Tokcaer-Keskin, Zeynep; Tang, Zuojian; Strino, Francesco; Chen, Xi; Wang, Yatong; Xi, Xiangmei; Basilico, Claudio; Brown, Stuart M.; Bonneau, Richard; Kluger, Yuval; Dailey, Lisa A.

doi:10.1038/nmeth.2885

Cited by 93 publications

(108 citation statements)

References 41 publications

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“…Our capture-and-clone approach provides a strategy for assaying candidate CREs with lengths of a desired range. Moreover, the capture approach can be used in conjunction with any existing MPRA-like approach, including those that already rely on DNA fragmentation (Dickel et al 2014;Murtha et al 2014). For example, STARR-seq (Arnold et al 2013) has been used to assess long DNA fragments obtained by whole-genome shotgun cloning of the Drosophila genome.…”

Section: Discussionmentioning

confidence: 99%

Massively parallel cis-regulatory analysis in the mammalian central nervous system

et al. 2015

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Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell-derived organoids.

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Section: Discussionmentioning

confidence: 99%

Massively parallel cis-regulatory analysis in the mammalian central nervous system

et al. 2015

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“…We adopted a similar approach to compare H2BK20ac-only and H3K27ac-only enhancers. For this analysis, we exploited the availability of data from FIREWACh, an MPRA that was used to test ∼80,000 genomic fragments for enhancer activity in mESCs (Murtha et al 2014). At the default P-value threshold for peak detection, DFilter detected a negligible number of H3K27ac-only enhancer peaks.…”

Section: Genome-wide Pattern Of H2bk20ac At Different Enhancer Classesmentioning

confidence: 99%

“…In this case, the ChIP-seq peak-calling threshold was lowered to 1 × 10 −4 so as to detect the mE-Enh6 class, which was marked by H3K27ac in the absence of H2BK20ac (∼800 mEenh6 regions). Bar graph shows the success rate of tested regions from each enhancer class in the massively parallel FIREWACh enhancer assay (Murtha et al 2014). Nonacetylated regions tested in the enhancer assay (non-ac) are shown as a control.…”

Section: Genome-wide Pattern Of H2bk20ac At Different Enhancer Classesmentioning

confidence: 99%

“…FIREWACh assay (Murtha et al 2014) according to their histone modification-based enhancer class and calculated the enhancer success rate for each class. Notably, sequences from the mE-Enh4 class, which were only marked by H2BK20ac, showed almost twice the enhancer activity of H3K27ac-only sequences (mE-Enh6).…”

Section: Genome-wide Pattern Of H2bk20ac At Different Enhancer Classesmentioning

confidence: 99%

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Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters

et al. 2016

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Although over 35 different histone acetylation marks have been described, the overwhelming majority of regulatory genomics studies focus exclusively on H3K27ac and H3K9ac. In order to identify novel epigenomic traits of regulatory elements, we constructed a benchmark set of validated enhancers by performing 140 enhancer assays in human T cells. We tested 40 chromatin signatures on this unbiased enhancer set and identified H2BK20ac, a little-studied histone modification, as the most predictive mark of active enhancers. Notably, we detected a novel class of functionally distinct enhancers enriched in H2BK20ac but lacking H3K27ac, which was present in all examined cell lines and also in embryonic forebrain tissue. H2BK20ac was also unique in highlighting cell-type-specific promoters. In contrast, other acetylation marks were present in all active promoters, regardless of cell-type specificity. In stimulated microglial cells, H2BK20ac was more correlated with cell-state-specific expression changes than H3K27ac, with TGF-beta signaling decoupling the two acetylation marks at a subset of regulatory elements. In summary, our study reveals a previously unknown connection between histone acetylation and cell-type-specific gene regulation and indicates that H2BK20ac profiling can be used to uncover new dimensions of gene regulation.

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“…However, these do not allow direct quantification of enhancer activity. Besides, previous studies have focused their efforts on the isolation of enhancers using high-throughput gene reporter assays coupled to cell sorting 2,3 or tags sequencing 4 , providing limited quantitative information. Recently, a technique named Starr-seq enabling genome-wide quantification of enhancer activity in Drosophila cell lines has been developed [5][6][7] .…”

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confidence: 99%

High-throughput and quantitative assessment of enhancer activity in mammals by CapStarr-seq

et al. 2015

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Cell-type specific regulation of gene expression requires the activation of promoters by distal genomic elements defined as enhancers. The identification and the characterization of enhancers are challenging in mammals due to their genome complexity. Here we develop CapStarr-Seq, a novel high-throughput strategy to quantitatively assess enhancer activity in mammals. This approach couples capture of regions of interest to previously developed Starrseq technique. Extensive assessment of CapStarr-seq demonstrates accurate quantification of enhancer activity. Furthermore, we find that enhancer strength is associated with binding complexity of tissue-specific transcription factors and super-enhancers, while additive enhancer activity isolates key genes involved in cell identity and function. The CapStarr-Seq thus provides a fast and cost-effective approach to assess the activity of potential enhancers for a given cell type and will be helpful in decrypting transcription regulation mechanisms.

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FIREWACh: high-throughput functional detection of transcriptional regulatory modules in mammalian cells

Cited by 93 publications

References 41 publications

Massively parallel cis-regulatory analysis in the mammalian central nervous system

Massively parallel cis-regulatory analysis in the mammalian central nervous system

Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters

High-throughput and quantitative assessment of enhancer activity in mammals by CapStarr-seq

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