Discovering human transcription factor physical interactions with genetic variants, novel DNA motifs, and repetitive elements using enhanced yeast one-hybrid assays

Shrestha, Shaleen; Sewell, Jared Allan; Santoso, Clarissa Stephanie; Forchielli, Elena; Pro, Sebastian Carrasco; Martinez, Mélissa; Bass, Juan I. Fuxman

doi:10.1101/gr.248823.119

Cited by 12 publications

(11 citation statements)

References 65 publications

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“…More importantly, PDIs identified by eY1H assays display a 30–70% validation rate in human cells and living animals, which is similar to the validation rate of ChIP-seq interactions, although likely because of different reasons (e.g., yeast versus mammalian system, DNA binding versus reporter activity) ( 10 , 12 , 13 ). We previously developed a high-throughput eY1H pipeline that interrogates the binding of approximately two-thirds (1086 of ∼1600) of all human TFs to defined regulatory regions, which we used to identify TFs that bind to human developmental enhancers, noncoding genetic variants, and repetitive DNA elements ( 10 , 14 ).…”

Section: Introductionmentioning

confidence: 99%

Comprehensive mapping of the human cytokine gene regulatory network

Santoso

Lal

et al. 2020

Nucleic Acids Research

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Proper cytokine gene expression is essential in development, homeostasis and immune responses. Studies on the transcriptional control of cytokine genes have mostly focused on highly researched transcription factors (TFs) and cytokines, resulting in an incomplete portrait of cytokine gene regulation. Here, we used enhanced yeast one-hybrid (eY1H) assays to derive a comprehensive network comprising 1380 interactions between 265 TFs and 108 cytokine gene promoters. Our eY1H-derived network greatly expands the known repertoire of TF–cytokine gene interactions and the set of TFs known to regulate cytokine genes. We found an enrichment of nuclear receptors and confirmed their role in cytokine regulation in primary macrophages. Additionally, we used the eY1H-derived network as a framework to identify pairs of TFs that can be targeted with commercially-available drugs to synergistically modulate cytokine production. Finally, we integrated the eY1H data with single cell RNA-seq and phenotypic datasets to identify novel TF–cytokine regulatory axes in immune diseases and immune cell lineage development. Overall, the eY1H data provides a rich resource to study cytokine regulation in a variety of physiological and disease contexts.

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Section: Introductionmentioning

confidence: 99%

Comprehensive mapping of the human cytokine gene regulatory network

Santoso

Lal

et al. 2020

Nucleic Acids Research

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“…We previously developed a high-throughput eY1H pipeline that interrogates the binding of approximately two-thirds (1,086 of ~1,600) of all human TFs to defined regulatory regions, which we used to identify TFs that bind to human developmental enhancers, noncoding genetic variants, and repetitive DNA elements (Fuxman Bass et al, 2015;Shrestha et al, 2019b).…”

Section: Introductionmentioning

confidence: 99%

Comprehensive mapping of the human cytokine gene regulatory network

Santoso¹,

Lal

et al. 2020

Preprint

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Proper cytokine gene expression is essential in development, homeostasis, and immune responses. Studies on the transcriptional control of cytokine genes have mostly focused on highly researched transcription factors (TFs) and cytokines, resulting in an incomplete portrait of cytokine gene regulation. Here, we use enhanced yeast one-hybrid (eY1H) assays to derive a comprehensive network comprising 1,380 interactions between 265TFs and 108 cytokine gene promoters, greatly expanding the known repertoire of TFcytokine gene interactions. We found an enrichment of nuclear receptors and confirmed their role in cytokine regulation in primary macrophages. Additionally, we used the eY1Hderived network as a framework to identify pairs of TFs that synergistically modulate cytokine gene expression, and to identify novel TF-cytokine regulatory axes in immune diseases and immune cell lineage development. Overall, the eY1H data provides a rich resource to study cytokine regulation in a variety of physiological and disease contexts. 8 High Fidelity (ThermoFisher) from a pool of human genomic DNA (Clonetech). PCR products were Gateway cloned into the pDONR P1-P4 vector and entry clones were confirmed by Sanger sequencing. Then DNA-baits were Gateway cloned upstream of two reporter genes (HIS3 and LacZ) and both reporter constructs were integrated into the yeast genome to generate chromatinized DNA-bait strains (Deplancke et al., 2006a;Deplancke et al., 2006b). Yeast DNA-bait strains were confirmed by Sanger sequencing.DNA-bait strains were mated with an array of 1,086 TF-prey yeast strains expressing human TFs fused to the yeast Gal4 activation domain (AD) (Fuxman Bass et al., 2015).Matings were performed using a Singer Rotor robotic platform that manipulates yeast strains in a 1,536-colony format. An interaction was detected when a TF-prey binds the DNA-bait and the AD moiety activates reporter gene expression, allowing the mated yeast to grow on media lacking histidine, overcome the addition of 3-amino-triazole (3AT), a competitive inhibitor of the His3p enzyme, and convert the colorless X-gal into a blue compound. Each interaction was tested in quadruplicate and interactions were considered positive if at least two of the four mated colonies tested positively (>90% of detected interactions tested positively for all four colonies).Images of mated colonies on plates lacking histidine and containing 3AT and Xgal were processed using the Mybrid web-tool to detect positive interactions (Reece-Hoyes et al., 2013). Positive interactions detected by Mybrid were then manually checked and curated. False positives detected by Mybrid, which typically occur on plates with uneven background, were removed. False negatives missed by Mybrid, which typically occur when baits exhibit high background reporter gene expression or when interactions occur next to very strong positives, were included. A total of high-quality 1,380 PDIs 9 between 265 TFs and 108 cytokine promoters were included in the final dataset (Table S3). The eY1H interactions...

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“…Advantages of the eY1H assay include that it is independent of protein abundance, it represents 66% of the known human transcription factor repertoire, and it utilizes compound cis-elements as DNA baits. eY1H assays have been used to characterize transcription factor binding to human enhancers and promoters as well as the identification of loss and gain of protein-DNA interactions for disease-associated variants (24,26,45). We used this approach to characterize transcription factor binding to HIV-1 and HIV-2 LTRs to gain insights into networks that control HIV-1 and HIV-2 transcription.…”

Section: Discussionmentioning

confidence: 99%

A functional screen identifies transcriptional networks that regulate HIV-1 and HIV-2

Pedro

Agosto

Sewell

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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The molecular networks involved in the regulation of HIV replication, transcription, and latency remain incompletely defined. To expand our understanding of these networks, we performed an unbiased high-throughput yeast one-hybrid screen, which identified 42 human transcription factors and 85 total protein–DNA interactions with HIV-1 and HIV-2 long terminal repeats. We investigated a subset of these transcription factors for transcriptional activity in cell-based models of infection. KLF2 and KLF3 repressed HIV-1 and HIV-2 transcription in CD4+ T cells, whereas PLAGL1 activated transcription of HIV-2 through direct protein–DNA interactions. Using computational modeling with interacting proteins, we leveraged the results from our screen to identify putative pathways that define intrinsic transcriptional networks. Overall, we used a high-throughput functional screen, computational modeling, and biochemical assays to identify and confirm several candidate transcription factors and biochemical processes that influence HIV-1 and HIV-2 transcription and latency.

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Discovering human transcription factor physical interactions with genetic variants, novel DNA motifs, and repetitive elements using enhanced yeast one-hybrid assays

Cited by 12 publications

References 65 publications

Comprehensive mapping of the human cytokine gene regulatory network

Comprehensive mapping of the human cytokine gene regulatory network

Comprehensive mapping of the human cytokine gene regulatory network

A functional screen identifies transcriptional networks that regulate HIV-1 and HIV-2

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