Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

Guzmán, Carlos; Duttke, Sascha H.; Zhu, Yixin; Saldanha, Camila De Arruda; Downes, Nicholas; Benner, Chris; Heinz, Sven

doi:10.1093/nar/gkad562

Cited by 2 publications

(1 citation statement)

References 76 publications

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“…Though not explicitly considered in the work presented here, choice of using an episomal or integrated MPRA vector has also been examined previously (Inoue et al 2017; Klein et al 2020; Guzman et al 2023). Here, we used an integrated construct to compare activity at a specific locus in the LHCN-M2 human skeletal muscle cell line compared to an electroporated episomal construct in the 832/13 rat beta cell line ( Figure 4D-E ), due to technical limitations that make electroporation or transfection of LHCN-M2 cells unfeasible.…”

Section: Discussionmentioning

confidence: 99%

Using a modular massively parallel reporter assay to discover context-specific regulatory grammars in type 2 diabetes

Tovar,

Kyono,

Nishino

et al. 2023

Preprint

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Recent genome-wide association studies have established that most complex disease-associated loci are found in noncoding regions where defining their function is nontrivial. In this study, we leverage a modular massively parallel reporter assay (MPRA) to uncover sequence features linked to context-specific regulatory activity. We screened enhancer activity across a panel of 198-bp fragments spanning over 10k type 2 diabetes- and metabolic trait-associated variants in the 832/13 rat insulinoma cell line, a relevant model of pancreatic beta cells. We explored these fragments’ context sensitivity by comparing their activities when placed up-or downstream of a reporter gene, and in combination with either a synthetic housekeeping promoter (SCP1) or a more biologically relevant promoter corresponding to the human insulin gene (INS). We identified clear effects of MPRA construct design on measured fragment enhancer activity. Specifically, a subset of fragments (n = 702/11,656) displayed positional bias, evenly distributed across up- and downstream preference. A separate set of fragments exhibited promoter bias (n = 698/11,656), mostly towards the cell-specificINSpromoter (73.4%). To identify sequence features associated with promoter preference, we used Lasso regression with 562 genomic annotations and discovered that fragments withINSpromoter-biased activity are enriched for HNF1 motifs. HNF1 family transcription factors are key regulators of glucose metabolism disrupted in maturity onset diabetes of the young (MODY), suggesting genetic convergence between rare coding variants that cause MODY and common T2D-associated regulatory variants. We designed a follow-up MPRA containing HNF1 motif-enriched fragments and observed several instances where deletion or mutation of HNF1 motifs disrupted theINSpromoter-biased enhancer activity, specifically in the beta cell model but not in a skeletal muscle cell line, another diabetes-relevant cell type. Together, our study suggests that cell-specific regulatory activity is partially influenced by enhancer-promoter compatibility and indicates that careful attention should be paid when designing MPRA libraries to capture context-specific regulatory processes at disease-associated genetic signals.

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Section: Discussionmentioning

confidence: 99%

Using a modular massively parallel reporter assay to discover context-specific regulatory grammars in type 2 diabetes

Tovar,

Kyono,

Nishino

et al. 2023

Preprint

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Position-dependent function of human sequence-specific transcription factors

Duttke,

Guzman,

Chang

et al. 2024

Nature

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Patterns of transcriptional activity are encoded in our genome through regulatory elements such as promoters or enhancers that, paradoxically, contain similar assortments of sequence-specific transcription factor (TF) binding sites1–3. Knowledge of how these sequence motifs encode multiple, often overlapping, gene expression programs is central to understanding gene regulation and how mutations in non-coding DNA manifest in disease4,5. Here, by studying gene regulation from the perspective of individual transcription start sites (TSSs), using natural genetic variation, perturbation of endogenous TF protein levels and massively parallel analysis of natural and synthetic regulatory elements, we show that the effect of TF binding on transcription initiation is position dependent. Analysing TF-binding-site occurrences relative to the TSS, we identified several motifs with highly preferential positioning. We show that these patterns are a combination of a TF’s distinct functional profiles—many TFs, including canonical activators such as NRF1, NFY and Sp1, activate or repress transcription initiation depending on their precise position relative to the TSS. As such, TFs and their spacing collectively guide the site and frequency of transcription initiation. More broadly, these findings reveal how similar assortments of TF binding sites can generate distinct gene regulatory outcomes depending on their spatial configuration and how DNA sequence polymorphisms may contribute to transcription variation and disease and underscore a critical role for TSS data in decoding the regulatory information of our genome.

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Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation

Cited by 2 publications

References 76 publications

Using a modular massively parallel reporter assay to discover context-specific regulatory grammars in type 2 diabetes

Using a modular massively parallel reporter assay to discover context-specific regulatory grammars in type 2 diabetes

Position-dependent function of human sequence-specific transcription factors

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