High-throughput identification of human SNPs affecting regulatory element activity

AbstractEfficient and highly organized transcription initiation and termination is fundamental to an organism’s ability to survive, proliferate, and quickly respond to its environment. Over the last decade, our simplistic outlook of bacterial transcriptional regulation and architecture has evolved to include stimulus-responsive regulation by untranslated RNA and the formation of alternate transcriptional units. In this study, we map the transcriptional landscape of the bacterial pathogen Streptococcus pneumoniae by applying a combination of high-throughput RNA-sequencing techniques. Our study reveals a complex transcriptome wherein environment-respondent alternate transcriptional units are observed within operons stemming from internal transcription start sites (TSS) and transcription terminators (TTS) suggesting that more fine-tuning of regulation occurs than previously thought. Additionally, we identify many putative cis-regulatory RNA elements and riboswitches within 5’-untranslated regions (5’-UTR) of genes. By integrating TSSs and TTSs with independently collected RNA-Seq datasets from a variety of conditions, we establish the response of these regulators to changes in growth conditions and validate several of them. Furthermore, to determine the importance of ribo-regulation by 5’-UTR elements for in vivo virulence, we show that the pyrR regulatory element is essential for survival, successful colonization and infection in mice suggesting that such RNA elements are potential drug targets. Importantly, we show that our approach of combining high-throughput sequencing with in vivo experiments can reconstruct a global understanding of regulation, but also pave the way for discovery of compounds that target (ribo-) regulators to mitigate virulence and antibiotic resistance.

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Prioritization of autoimmune disease-associated genetic variants that perturb regulatory element activity in T cells

10.1101/2021.05.30.445673 ◽

2021 ◽

Author(s):

Kousuke Mouri ◽

Michael H. Guo ◽

Carl G. de Boer ◽

Greg A. Newby ◽

Matteo Gentili ◽

...

Keyword(s):

T Cells ◽

Autoimmune Disease ◽

Association Studies ◽

Regulatory Element ◽

Genome Wide Association Studies ◽

Expression Of Genes ◽

Human T Cell ◽

Allele Specific ◽

Reporter Assays ◽

Element Activity

Genome-wide association studies have uncovered hundreds of autoimmune disease-associated loci; however, the causal genetic variant(s) within each locus are mostly unknown. Here, we perform high-throughput allele-specific reporter assays to prioritize disease-associated variants for five autoimmune diseases. By examining variants that both promote allele-specific reporter expression and are located in accessible chromatin, we identify 60 putatively causal variants that enrich for statistically fine-mapped variants by up to 57.8-fold. We introduced the risk allele of a prioritized variant (rs72928038) into a human T cell line and deleted the orthologous sequence in mice, both resulting in reduced BACH2 expression. Naive CD8 T cells from mice containing the deletion had reduced expression of genes that suppress activation and maintain stemness. Our results represent an example of an effective approach for prioritizing variants and studying their physiologically relevant effects.

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Systematic identification of human SNPs affecting regulatory element activity

10.1242/prelights.6046 ◽

2018 ◽

Author(s):

Jesus Victorino

Keyword(s):

Regulatory Element ◽

Element Activity ◽

Systematic Identification

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Locally acting transcription factors are required for p53-dependent cis-regulatory element activity

10.1101/761387 ◽

2019 ◽

Author(s):

Allison N. Catizone ◽

Gizem Karsli Uzunbas ◽

Petra Celadova ◽

Sylvia Kuang ◽

Daniel Bose ◽

...

Keyword(s):

Transcription Factors ◽

Regulatory Element ◽

Regulatory Elements ◽

Mrna Levels ◽

Sequence Context ◽

Endogenous Expression ◽

Damage Repair ◽

Local Sequence ◽

Element Activity ◽

Massively Parallel Reporter Assay

AbstractThe master tumor suppressor p53 controls transcription of a wide-ranging gene network involved in apoptosis, cell cycle arrest, DNA damage repair, and senescence. Recent studies revealed pervasive binding of p53 to cis-regulatory elements (CRE), which are non-coding segments of DNA that spatially and temporally control transcription through the combinatorial binding of local transcription factors (TFs). Although the role of p53 as a strong trans-activator of gene expression is well known, the co-regulatory factors and local sequences acting at p53-bound CREs are comparatively understudied. We designed and executed a massively parallel reporter assay (MPRA) to investigate the effect of transcription factor binding motifs and local sequence context on p53-bound CRE activity. Our data indicate that p53-bound CREs are both positively and negatively affected by alterations in local sequence context and changes to co-regulatory TF motifs. We identified a SP1/KLF family motif located in an intronic p53 CRE that is required for the endogenous expression of the p53-dependent gene CCNG1. We also identified ATF3 as a factor that co-regulates the expression of the p53-dependent gene GDF15 through binding with p53 in an upstream CRE. Loss of either p53 or ATF3 severely reduces CRE activity and alters endogenous GDF15 mRNA levels in the cell. Our data suggests that p53 has the flexibility to cooperate with a variety of transcription factors in order to regulate CRE activity. By utilizing different sets of co-factors across CREs, we hypothesize that p53 activity is guarded against loss of any one regulatory partner allowing for dynamic and redundant control of p53-mediated transcription.

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Locally acting transcription factors regulate p53-dependent cis-regulatory element activity

Nucleic Acids Research ◽

10.1093/nar/gkaa147 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4195-4213 ◽

Cited By ~ 2

Author(s):

Allison N Catizone ◽

Gizem Karsli Uzunbas ◽

Petra Celadova ◽

Sylvia Kuang ◽

Daniel Bose ◽

...

Keyword(s):

Transcription Factors ◽

Regulatory Element ◽

Regulatory Elements ◽

Sequence Context ◽

Binding Motifs ◽

Cycle Arrest ◽

Damage Repair ◽

Local Sequence ◽

Element Activity ◽

Massively Parallel Reporter Assay

Abstract The master tumor suppressor p53 controls transcription of a wide-ranging gene network involved in apoptosis, cell cycle arrest, DNA damage repair, and senescence. Recent studies revealed pervasive binding of p53 to cis-regulatory elements (CREs), which are non-coding segments of DNA that spatially and temporally control transcription through the combinatorial binding of local transcription factors. Although the role of p53 as a strong trans-activator of gene expression is well known, the co-regulatory factors and local sequences acting at p53-bound CREs are comparatively understudied. We designed and executed a massively parallel reporter assay (MPRA) to investigate the effect of transcription factor binding motifs and local sequence context on p53-bound CRE activity. Our data indicate that p53-bound CREs are both positively and negatively affected by alterations in local sequence context and changes to co-regulatory TF motifs. Our data suggest p53 has the flexibility to cooperate with a variety of transcription factors in order to regulate CRE activity. By utilizing different sets of co-factors across CREs, we hypothesize that global p53 activity is guarded against loss of any one regulatory partner, allowing for dynamic and redundant control of p53-mediated transcription.

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