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• W3037892274 abstract "•Systematic analysis of TFBS architecture by using the c-AMP response element (CRE)•Assay CRE affinity, number, placement, spacing, and surrounding sequence content•Similar expression trends between an episomal and single-copy, genomic MPRA In eukaryotes, transcription factors (TFs) orchestrate gene expression by binding to TF-binding sites (TFBSs) and localizing transcriptional co-regulators and RNA polymerase II to cis-regulatory elements. However, we lack a basic understanding of the relationship between TFBS composition and their quantitative transcriptional responses. Here, we measured expression driven by 17,406 synthetic cis-regulatory elements with varied compositions of a model TFBS, the c-AMP response element (CRE) by using massively parallel reporter assays (MPRAs). We find CRE number, affinity, and promoter proximity largely determines expression. In addition, we observe expression modulation based on the spacing between CREs and CRE distance to the promoter, where expression follows a helical periodicity. Finally, we compare library expression between an episomal MPRA and a genomically integrated MPRA, where a single cis-regulatory element is assayed per cell at a defined locus. These assays largely recapitulate each other, although weaker, non-canonical CREs exhibit greater activity in a genomic context. In eukaryotes, transcription factors (TFs) orchestrate gene expression by binding to TF-binding sites (TFBSs) and localizing transcriptional co-regulators and RNA polymerase II to cis-regulatory elements. However, we lack a basic understanding of the relationship between TFBS composition and their quantitative transcriptional responses. Here, we measured expression driven by 17,406 synthetic cis-regulatory elements with varied compositions of a model TFBS, the c-AMP response element (CRE) by using massively parallel reporter assays (MPRAs). We find CRE number, affinity, and promoter proximity largely determines expression. In addition, we observe expression modulation based on the spacing between CREs and CRE distance to the promoter, where expression follows a helical periodicity. Finally, we compare library expression between an episomal MPRA and a genomically integrated MPRA, where a single cis-regulatory element is assayed per cell at a defined locus. These assays largely recapitulate each other, although weaker, non-canonical CREs exhibit greater activity in a genomic context. The ability of organisms to precisely control gene expression levels and responses is crucial for almost all biological processes. Expression levels are controlled by cis-regulatory elements such as promoters and enhancers, trans-acting factors such as transcription factors (TFs), and cell epigenetic and environmental states. A variety of large-scale projects seek to determine gene-expression levels across various cell lines and cell types (Lizio et al., 2015Lizio M. Harshbarger J. Shimoji H. Severin J. Kasukawa T. Sahin S. Abugessaisa I. Fukuda S. Hori F. Ishikawa-Kato S. et al.Gateways to the FANTOM5 promoter level mammalian expression atlas.Genome Biol. 2015; 16: 22Crossref PubMed Scopus (418) Google Scholar, Lizio et al., 2017Lizio M. Harshbarger J. Abugessaisa I. Noguchi S. Kondo A. Severin J. Mungall C. Arenillas D. Mathelier A. Medvedeva Y.A. et al.Update of the FANTOM web resource: high resolution transcriptome of diverse cell types in mammals.Nucleic Acids Res. 2017; 45: D737-D743Crossref PubMed Scopus (68) Google Scholar), identifying functional elements that could control expression (ENCODE Project Consortium, 2012ENCODE Project ConsortiumAn integrated encyclopedia of DNA elements in the human genome.Nature. 2012; 489: 57-74Crossref PubMed Scopus (10539) Google Scholar), the genome-wide characterization of the epigenetic states of DNA (Roadmap Epigenomics Consortium et al., 2015Kundaje A. Meuleman W. Ernst J. Bilenky M. Yen A. Heravi-Moussavi A. Kheradpour P. Zhang Z. Wang J. et al.Roadmap Epigenomics ConsortiumIntegrative analysis of 111 reference human epigenomes.Nature. 2015; 518: 317-330Crossref PubMed Scopus (3374) Google Scholar), and the binding specificities of TFs (Jolma et al., 2013Jolma A. Yan J. Whitington T. Toivonen J. Nitta K.R. Rastas P. Morgunova E. Enge M. Taipale M. Wei G. et al.DNA-binding specificities of human transcription factors.Cell. 2013; 152: 327-339Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar, Jolma et al., 2015Jolma A. Yin Y. Nitta K.R. Dave K. Popov A. Taipale M. Enge M. Kivioja T. Morgunova E. Taipale J. DNA-dependent formation of transcription factor pairs alters their binding specificity.Nature. 2015; 527: 384-388Crossref PubMed Scopus (276) Google Scholar; Yin et al., 2017Yin Y. Morgunova E. Jolma A. Kaasinen E. Sahu B. Khund-Sayeed S. Das P.K. Kivioja T. Dave K. Zhong F. et al.Impact of cytosine methylation on DNA binding specificities of human transcription factors.Science. 2017; 356Crossref Scopus (483) Google Scholar; Zhu et al., 2018Zhu F. Farnung L. Kaasinen E. Sahu B. Yin Y. Wei B. Dodonova S.O. Nitta K.R. Morgunova E. Taipale M. et al.The interaction landscape between transcription factors and the nucleosome.Nature. 2018; 562: 76-81Crossref PubMed Scopus (119) Google Scholar). Collectively, these efforts generally give us a parts list of putatively functional elements, yet it is still not well understood how these parts define quantitative levels of expression. The combination of sequence motifs that recruit TFs, or TF-binding sites (TFBS), functionalize cis-regulatory elements via unique arrangements that help determine quantitative regulatory responses (Lambert et al., 2018Lambert S.A. Jolma A. Campitelli L.F. Das P.K. Yin Y. Albu M. Chen X. Taipale J. Hughes T.R. Weirauch M.T. The human transcription factors.Cell. 2018; 172: 650-665Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar; Spitz and Furlong, 2012Spitz F. Furlong E.E.M. Transcription factors: from enhancer binding to developmental control.Nat. Rev. Genet. 2012; 13: 613-626Crossref PubMed Scopus (1100) Google Scholar). Thus, the consequences of subtle changes to TFBS compositions can be drastic. For example, clusters of weak-affinity Gal4 sites in yeast promoters increase expression synergistically, whereas strong-affinity sites contribute additively to expression (Giniger and Ptashne, 1988Giniger E. Ptashne M. Cooperative DNA binding of the yeast transcriptional activator GAL4.Proc. Natl. Acad. Sci. USA. 1988; 85: 382-386Crossref PubMed Scopus (134) Google Scholar). TF occupancy of similar motifs can vary across the genome, following differences in GC content of the surrounding sequence (Dror et al., 2015Dror I. Golan T. Levy C. Rohs R. Mandel-Gutfreund Y. A widespread role of the motif environment in transcription factor binding across diverse protein families.Genome Res. 2015; 25: 1268-1280Crossref PubMed Scopus (75) Google Scholar). Additionally, the placement of TFBSs can be highly conserved in close proximity to core transcriptional machinery, such as surrounding transcription start sites (TSSs) of genes (Tabach et al., 2007Tabach Y. Brosh R. Buganim Y. Reiner A. Zuk O. Yitzhaky A. Koudritsky M. Rotter V. Domany E. Wide-scale analysis of human functional transcription factor binding reveals a strong bias towards the transcription start site.PLoS One. 2007; 2: e807Crossref PubMed Scopus (46) Google Scholar), and such placement can be critical for transcriptional activity (Kim and Maniatis, 1997Kim T.K. Maniatis T. The mechanism of transcriptional synergy of an in vitro assembled interferon-beta enhanceosome.Mol. Cell. 1997; 1: 119-129Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar; Kim et al., 1998Kim T.K. Kim T.H. Maniatis T. Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-beta enhanceosome in vitro.Proc. Natl. Acad. Sci. USA. 1998; 95: 12191-12196Crossref PubMed Scopus (93) Google Scholar). Lastly, the positional arrangement of TFBS combinations within cis-regulatory elements can modulate TF-binding strength (Jolma et al., 2013Jolma A. Yan J. Whitington T. Toivonen J. Nitta K.R. Rastas P. Morgunova E. Enge M. Taipale M. Wei G. et al.DNA-binding specificities of human transcription factors.Cell. 2013; 152: 327-339Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar, Jolma et al., 2015Jolma A. Yin Y. Nitta K.R. Dave K. Popov A. Taipale M. Enge M. Kivioja T. Morgunova E. Taipale J. DNA-dependent formation of transcription factor pairs alters their binding specificity.Nature. 2015; 527: 384-388Crossref PubMed Scopus (276) Google Scholar), and TF activity can vary across the composition of TFBS combinations (Stampfel et al., 2015Stampfel G. Kazmar T. Frank O. Wienerroither S. Reiter F. Stark A. Transcriptional regulators form diverse groups with context-dependent regulatory functions.Nature. 2015; 528: 147-151Crossref PubMed Scopus (97) Google Scholar). Deciphering the logic imbued in cis-regulatory elements is difficult, as the limited set of natural variants and cell types are typically insufficient to control variables, such as sequence composition, TFBS composition and arrangements, and activity of trans-acting factors. Proving that particular sequences have causative effects on gene expression requires carefully controlled and high-throughput reverse-genetic studies. The emergence of the massively parallel reporter assay (MPRA) allows for the testing of such reverse-genetic transcriptional assays, and has become a powerful tool for the large-scale functional validation of cis-regulatory elements across genomic and organismal contexts (White, 2015White M.A. Understanding how cis-regulatory function is encoded in DNA sequence using massively parallel reporter assays and designed sequences.Genomics. 2015; 106: 165-170Crossref PubMed Scopus (39) Google Scholar). These assays utilize the scale of synthetic DNA libraries and next-generation (next-gen) sequencing to determine the expression of thousands of individual cis-regulatory elements in pooled expression measurements, enabling high-throughput functional characterizations of cis-regulatory logic. MPRAs have been used to quantify the transcriptional strengths of cis-regulatory elements and identify the motifs integral to element activity (Ernst et al., 2016Ernst J. Melnikov A. Zhang X. Wang L. Rogov P. Mikkelsen T.S. Kellis M. Genome-scale high-resolution mapping of activating and repressive nucleotides in regulatory regions.Nat. Biotechnol. 2016; 34: 1180-1190Crossref PubMed Scopus (70) Google Scholar; Kheradpour et al., 2013Kheradpour P. Ernst J. Melnikov A. Rogov P. Wang L. Zhang X. Alston J. Mikkelsen T.S. Kellis M. Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay.Genome Res. 2013; 23: 800-811Crossref PubMed Scopus (171) Google Scholar). Furthermore, several groups are using these systems to dissect how TFBSs drive quantitative regulatory responses in bacteria (Belliveau et al., 2018Belliveau N.M. Barnes S.L. Ireland W.T. Jones D.L. Sweredoski M.J. Moradian A. Hess S. Kinney J.B. Phillips R. Systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria.Proc. Natl. Acad. Sci. USA. 2018; 115: E4796-E4805Crossref PubMed Scopus (43) Google Scholar; Kinney et al., 2010Kinney J.B. Murugan A. Callan Jr., C.G. Cox E.C. Using deep sequencing to characterize the biophysical mechanism of a transcriptional regulatory sequence.Proc. Natl. Acad. Sci. USA. 2010; 107: 9158-9163Crossref PubMed Scopus (176) Google Scholar), yeast (van Dijk et al., 2017van Dijk D. Sharon E. Lotan-Pompan M. Weinberger A. Segal E. Carey L.B. Large-scale mapping of gene regulatory logic reveals context-dependent repression by transcriptional activators.Genome Res. 2017; 27: 87-94Crossref PubMed Scopus (10) Google Scholar; Levo et al., 2017Levo M. Avnit-Sagi T. Lotan-Pompan M. Kalma Y. Weinberger A. Yakhini Z. Segal E. Systematic investigation of transcription factor activity in the context of chromatin using massively parallel binding and expression assays.Mol. Cell. 2017; 65: 604-617.e6Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar; Sharon et al., 2012Sharon E. Kalma Y. Sharp A. Raveh-Sadka T. Levo M. Zeevi D. Keren L. Yakhini Z. Weinberger A. Segal E. Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters.Nat. Biotechnol. 2012; 30: 521-530Crossref PubMed Scopus (291) Google Scholar), human cell lines (Fiore and Cohen, 2016Fiore C. Cohen B.A. Interactions between pluripotency factors specify cis -regulation in embryonic stem cells.Genome Res. 2016; 26: 778-786Crossref PubMed Scopus (23) Google Scholar; Grossman et al., 2017Grossman S.R. Zhang X. Wang L. Engreitz J. Melnikov A. Rogov P. Tewhey R. Isakova A. Deplancke B. Bernstein B.E. et al.Systematic dissection of genomic features determining transcription factor binding and enhancer function.Proc. Natl. Acad. Sci. USA. 2017; 114: E1291-E1300Crossref PubMed Scopus (82) Google Scholar; Weingarten-Gabbay et al., 2019Weingarten-Gabbay S. Nir R. Lubliner S. Sharon E. Kalma Y. Weinberger A. Segal E. Systematic interrogation of human promoters.Genome Res. 2019; 29: 171-183Crossref PubMed Scopus (40) Google Scholar), and animals (Kwasnieski et al., 2012Kwasnieski J.C. Mogno I. Myers C.A. Corbo J.C. Cohen B.A. Complex effects of nucleotide variants in a mammalian cis-regulatory element.Proc. Natl. Acad. Sci. USA. 2012; 109: 19498-19503Crossref PubMed Scopus (152) Google Scholar; Smith et al., 2013Smith R.P. Taher L. Patwardhan R.P. Kim M.J. Inoue F. Shendure J. Ovcharenko I. Ahituv N. Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model.Nat. Genet. 2013; 45: 1021-1028Crossref PubMed Scopus (139) Google Scholar; White et al., 2016White M.A. Kwasnieski J.C. Myers C.A. Shen S.Q. Corbo J.C. Cohen B.A. A simple grammar defines activating and repressing cis-regulatory elements in photoreceptors.Cell Rep. 2016; 17: 1247-1254Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Collectively, these studies have begun to dissect TFBS logic by exploring how the regulatory grammar of different site combinations, numbers, and placements affect transcriptional activity. In this work, we use MPRAs to demonstrate how a range of factors guiding cis-regulatory architecture shape the activity of a single TFBS, the c-AMP response element (CRE). The CRE binding (CREB) protein binds CRE and drives expression downstream of adenylyl cyclase activation (Gonzalez and Montminy, 1989Gonzalez G.A. Montminy M.R. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133.Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2037) Google Scholar; Montminy et al., 1986Montminy M.R. Sevarino K.A. Wagner J.A. Mandel G. Goodman R.H. Identification of a cyclic-AMP-responsive element within the rat somatostatin gene.Proc. Natl. Acad. Sci. USA. 1986; 83: 6682-6686Crossref PubMed Scopus (1051) Google Scholar) across most cell types (Mayr and Montminy, 2001Mayr B. Montminy M. Transcriptional regulation by the phosphorylation-dependent factor CREB.Nat. Rev. Mol. Cell Biol. 2001; 2: 599-609Crossref PubMed Scopus (2019) Google Scholar). CRE is ideally suited for exploring associations between TFBS architecture and regulation because of its ability to drive expression without other TFBSs in cis-regulatory elements (Melnikov et al., 2012Melnikov A. Murugan A. Zhang X. Tesileanu T. Wang L. Rogov P. Feizi S. Gnirke A. Callan C.G. Kinney J.B. et al.Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay.Nat. Biotechnol. 2012; 30: 271-277Crossref PubMed Scopus (378) Google Scholar) and its ease of inducibility in a cell (Gonzalez and Montminy, 1989Gonzalez G.A. Montminy M.R. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133.Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2037) Google Scholar; Montminy et al., 1986Montminy M.R. Sevarino K.A. Wagner J.A. Mandel G. Goodman R.H. Identification of a cyclic-AMP-responsive element within the rat somatostatin gene.Proc. Natl. Acad. Sci. USA. 1986; 83: 6682-6686Crossref PubMed Scopus (1051) Google Scholar), allowing finer control over active concentrations of the CREB protein. The most conserved, and likely to be functional, CREs generally localize within 200 base pairs (bp) of a TSS in the human genome (Mayr and Montminy, 2001Mayr B. Montminy M. Transcriptional regulation by the phosphorylation-dependent factor CREB.Nat. Rev. Mol. Cell Biol. 2001; 2: 599-609Crossref PubMed Scopus (2019) Google Scholar; Zhang et al., 2005Zhang X. Odom D.T. Koo S.H. Conkright M.D. Canettieri G. Best J. Chen H. Jenner R. Herbolsheimer E. Jacobsen E. et al.Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues.Proc. Natl. Acad. Sci. USA. 2005; 102: 4459-4464Crossref PubMed Scopus (752) Google Scholar). The function of a commercial CRE reporter has been characterized with a MPRA by using a scanning mutagenesis approach. They found that mutations to CREs in closer proximity to both the promoter, and the promoter-proximal regions immediately flanking these CREs had a greater effect on induced, CRE-dependent gene expression than mutations to and adjacent to CREs further from the promoter (Melnikov et al., 2012Melnikov A. Murugan A. Zhang X. Tesileanu T. Wang L. Rogov P. Feizi S. Gnirke A. Callan C.G. Kinney J.B. et al.Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay.Nat. Biotechnol. 2012; 30: 271-277Crossref PubMed Scopus (378) Google Scholar). Here, we explore the relationship between CRE’s distance to promoter elements and its activity in greater detail. Additionally, we characterize the quantitative relationships between CRE protein activity and CRE affinity, number, the spacing between multiple CREs, and the surrounding sequence content. Although many MPRAs are performed episomally because of their ease and quickness (Fiore and Cohen, 2016Fiore C. Cohen B.A. Interactions between pluripotency factors specify cis -regulation in embryonic stem cells.Genome Res. 2016; 26: 778-786Crossref PubMed Scopus (23) Google Scholar; Grossman et al., 2017Grossman S.R. Zhang X. Wang L. Engreitz J. Melnikov A. Rogov P. Tewhey R. Isakova A. Deplancke B. Bernstein B.E. et al.Systematic dissection of genomic features determining transcription factor binding and enhancer function.Proc. Natl. Acad. Sci. USA. 2017; 114: E1291-E1300Crossref PubMed Scopus (82) Google Scholar; Kheradpour et al., 2013Kheradpour P. Ernst J. Melnikov A. Rogov P. Wang L. Zhang X. Alston J. Mikkelsen T.S. Kellis M. Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay.Genome Res. 2013; 23: 800-811Crossref PubMed Scopus (171) Google Scholar; Melnikov et al., 2012Melnikov A. Murugan A. Zhang X. Tesileanu T. Wang L. Rogov P. Feizi S. Gnirke A. Callan C.G. Kinney J.B. et al.Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay.Nat. Biotechnol. 2012; 30: 271-277Crossref PubMed Scopus (378) Google Scholar), episomal cis-regulatory element expression does not always correlate with their genomic counterparts (Inoue et al., 2017Inoue F. Kircher M. Martin B. Cooper G.M. Witten D.M. McManus M.T. Ahituv N. Shendure J. A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity.Genome Res. 2017; 27: 38-52Crossref PubMed Scopus (119) Google Scholar; Klein et al., 2019Klein J. Agarwal V. Inoue F. Keith A. Martin B. Kircher M. Ahituv N. Shendure J. A systematic evaluation of the design, orientation, and sequence context dependencies of massively parallel reporter assays.bioRxiv. 2019; https://doi.org/10.1101/576405v1Crossref Google Scholar). Thus, in this work, we test our library both transiently, by using episomes, and in a newly developed, singly integrated genomic MPRA. We observe strong correlations between our episomal and genomic MPRAs, but also identify key differences, most notably that variants containing weak CREs drive higher expression in the genomic context. We designed libraries with one or more CRE(s) by replacing sequence within three putatively inactive 150-bp background sequences to assay a range of features contributing to TFBS architecture (Figure 1A). These backgrounds were adapted from sequences with little reported activity from the Vista enhancer database (Visel et al., 2007Visel A. Minovitsky S. Dubchak I. Pennacchio L.A. VISTA Enhancer Browser--a database of tissue-specific human enhancers.Nucleic Acids Res. 2007; 35: D88-D92Crossref PubMed Scopus (650) Google Scholar) or a commercial reporter modified by removing previously identified CREs (Fan and Wood, 2007Fan F. Wood K.V. Bioluminescent assays for high-throughput screening.Assay Drug Dev. Technol. 2007; 5: 127-136Crossref PubMed Scopus (328) Google Scholar). We generated regulatory variants by replacing 12-bp regions of the backgrounds with either the consensus CRE (AT TGACGTCA GC), in which the central 8-bp region binds a CREB dimer (two monomer binding sites), or a weaker CRE (AT TGAAGTCA GC), where one of the central dinucleotides bound by both monomers was mutated and has been previously shown to reduce activity (Mayr and Montminy, 2001Mayr B. Montminy M. Transcriptional regulation by the phosphorylation-dependent factor CREB.Nat. Rev. Mol. Cell Biol. 2001; 2: 599-609Crossref PubMed Scopus (2019) Google Scholar; Melnikov et al., 2012Melnikov A. Murugan A. Zhang X. Tesileanu T. Wang L. Rogov P. Feizi S. Gnirke A. Callan C.G. Kinney J.B. et al.Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay.Nat. Biotechnol. 2012; 30: 271-277Crossref PubMed Scopus (378) Google Scholar). For the majority of analysis, we used two CRE libraries. The first library, the CRE spacing and distance library, assays CREB activity as a function of both the spacing between CREs and CRE distance to the minimal promoter by moving two consensus CREs across the 150-bp backgrounds at six defined spacings (0, 5, 10, 15, 20, and 70 bp) between the two sites. In the CRE number and affinity library, we explore the effect of both CRE number and affinity upon expression by designating 6 equally spaced locations across all backgrounds in which each location is replaced with either the weak CRE, consensus CRE, or no CRE. We used Agilent oligonucleotide library synthesis to construct our designed libraries, added random 20 nt barcodes to the 3′ end, and mapped these barcode-variant associations. For MPRA analyses, we only considered barcodes corresponding to perfect matches to our designs, identified at this stage via next-gen sequencing. We cloned these libraries into a reporter construct we engineered to maximize signal to noise when integrated into the genome (Figure S1C) and then cloned a minimal promoter and luciferase gene between variant and barcode, placing the barcode in the 3′ UTR of the luciferase gene. The assays were conducted at varying induction conditions, either episomally by transient transfection (Episomal MPRA), or singly integrated into the intergenic H11 safe-harbor locus (Zhu et al., 2014Zhu F. Gamboa M. Farruggio A.P. Hippenmeyer S. Tasic B. Schüle B. Chen-Tsai Y. Calos M.P. DICE, an efficient system for iterative genomic editing in human pluripotent stem cells.Nucleic Acids Res. 2014; 42: e34Crossref PubMed Scopus (63) Google Scholar) by using BxBI-mediated recombination (Genomic MPRA) (Duportet et al., 2014Duportet X. Wroblewska L. Guye P. Li Y. Eyquem J. Rieders J. Rimchala T. Batt G. Weiss R. A platform for rapid prototyping of synthetic gene networks in mammalian cells.Nucleic Acids Res. 2014; 42: 13440-13451Crossref PubMed Scopus (70) Google Scholar; Jones et al., 2019Jones E.M. Lubock N.B. Venkatakrishnan A.J. Wang J. Tseng A.M. Paggi J.M. Latorraca N.R. Cancilla D. Satyadi M. Davis J. et al.Structural and functional characterization of G protein-coupled receptors with deep mutational scanning.bioRxiv. 2019; https://doi.org/10.1101/623108Crossref Scopus (0) Google Scholar; Matreyek et al., 2017Matreyek K.A. Stephany J.J. Fowler D.M. A platform for functional assessment of large variant libraries in mammalian cells.Nucleic Acids Res. 2017; 45: e102Crossref PubMed Scopus (42) Google Scholar; Xu et al., 2013Xu Z. Thomas L. Davies B. Chalmers R. Smith M. Brown W. Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome.BMC Biotechnol. 2013; 13: 87Crossref PubMed Scopus (42) Google Scholar) (Figures 1B, S1A, and S1B). The episomal MPRAs were run in biological duplicate across 8 different concentrations of forskolin (Figures 1D and S1E), which stimulates phosphorylation and activation of the CREB protein by activating adenylyl cyclase (Gonzalez and Montminy, 1989Gonzalez G.A. Montminy M.R. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133.Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2037) Google Scholar). The genomic MPRA was run in biological duplicate at full induction (Figure S1D). After forskolin stimulations, we isolated barcoded transcripts from cells and used next-gen sequencing to determine barcode prevalence per RNA sample and plasmid (Episomal MPRA) or genomic (Genomic MPRA) DNA sample. Given that each variant was mapped to multiple barcodes, we first determined the expression of each barcode via the ratio of normalized reads in the RNA over the DNA sample. We then determined variant expression from the median expression of all barcodes mapped to each variant. Both episomal and genomic MPRAs indicated high reproducibility between separately stimulated replicates (Figure 1E, episomal Pearson’s r = 0.99, genomic r = 0.91, and Figure S1E). In addition, we found strong correlation between episomal and genomic MPRAs across all variants tested (Figure 1F, r = 0.92). Although variant expression differed between backgrounds (Figure 1F), such trends were consistent between MPRA contexts. Thus, local sequence features play a larger role in determining expression trends across CRE architecture than the assay context. As barcode sequences can influence MPRA expression measurements (Melnikov et al., 2012Melnikov A. Murugan A. Zhang X. Tesileanu T. Wang L. Rogov P. Feizi S. Gnirke A. Callan C.G. Kinney J.B. et al.Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay.Nat. Biotechnol. 2012; 30: 271-277Crossref PubMed Scopus (378) Google Scholar), we performed a second round of barcoding on a subset of variants and repeated the episomal MPRA. Variant expression strongly correlated across episomal MPRAs performed two years apart, with different variant-barcode associations (Figure 1G), and overall numbers of barcodes used to determine variant expression (Figure 1H). We first focused our analysis on CRE positioning within a cis-regulatory element and its role in expression. In exploring this, we hoped to understand a single CRE’s expression variability before adding the complexity of multiple sites, as with the CRE number and affinity library. Thus, we initially explored the relationship between CRE positioning in relation to a downstream promoter (referred to as CRE distance) and variant expression by using a single consensus CRE in a separate library. Yet, we found one consensus CRE drove minimal expression in the episomal MPRA after CREB activation (Figure 2A). Thus, to explore these effects, we next examined the expression driven by two CREs in the CRE spacing and distance library. This library varied the distance of two consensus CREs to the minimal promoter and altered the number of nucleotides between these two sites (referred to as CRE spacing). We tested spacings of 0, 5, 10, 15, 20, and 70 bp between the two CREs, and then tested distance by moving CREs with each of these set spacings across the backgrounds one base at a time, spanning the 150-bp backgrounds. With two CREs, we observe a ∼10-bp expression periodicity that is more apparent at higher concentrations of forskolin, and at distances closer to the promoter (Figure 2A). This 10-bp periodicity was consistent across most CRE spacings and backgrounds, displayed similar patterns between the genomic and episomal assays, but differed between backgrounds (Figures 2B and S2B). Such periodicity has been observed before with translocations of TFBSs upstream of a promoter in a variety of model systems (Kim and Maniatis, 1997Kim T.K. Maniatis T. The mechanism of transcriptional synergy of an in vitro assembled interferon-beta enhanceosome.Mol. Cell. 1997; 1: 119-129Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar; Kim et al., 2013Kim S. Broströmer E. Xing D. Jin J. Chong S. Ge H. Wang S. Gu C. Yang L. Gao Y.Q. et al.Probing allostery through DNA.Science. 2013; 339: 816-819Crossref PubMed Scopus (164) Google Scholar; Sharon et al., 2012Sharon E. Kalma Y. Sharp A. Raveh-Sadka T. Levo M. Zeevi D. Keren L. Yakhini Z. Weinberger A. Segal E. Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters.Nat. Biotechnol. 2012; 30: 521-530Crossref PubMed Scopus (291) Google Scholar; Takahashi et al., 1986Takahashi K. Vigneron M. Matthes H. Wildeman A. Zenke M. Chambon P. Requirement of stereospecific alignments for initiation from the simian virus 40 early promoter.Nature. 1986; 319: 121-126Crossref PubMed Scopus (180" @default.
• W3037892274 created "2020-07-02" @default.
• W3037892274 creator A5010815697 @default.
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• W3037892274 date "2020-07-01" @default.
• W3037892274 modified "2023-10-13" @default.
• W3037892274 title "Dissection of c-AMP Response Element Architecture by Using Genomic and Episomal Massively Parallel Reporter Assays" @default.
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