Systematic analysis of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs
- University of California, San Diego, United States
- University of Arizona, United States
- Nippon Medical School, Japan
- University of California San Diego, United States
Abstract
Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq. We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPβ binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPβ at these locations tolerated changes in spacing ranging from 5-bp increase to >30-bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation.
Data availability
All sequencing data generated during this study have been deposited in GEO under accession code GSE178080. For reviewer access, please go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE178080 and enter token inyjgyqcbrsnrwz into the box.
Article and author information
Author details
Christopher K Glass
Funding
National Institutes of Health (DK091183)
- Christopher K Glass
National Institutes of Health (HL147835)
- Christopher K Glass
Leducq Transatlantic Network (16CVD01)
- Christopher K Glass
National Institutes of Health (T32DK007044)
- Thomas A Prohaska
American Heart Association (postdoctoral grant)
- Marten A Hoeksema
Netherlands Organization for Scientific Research (Rubicon grant)
- Marten A Hoeksema
Amsterdam Cardiovascular Sciences Institute (postdoctoral grant)
- Marten A Hoeksema
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Bone marrow cells were isolated from femurs and tibias of Cas9-expressing transgenic mice (Jackson Laboratory, No.028555) housed at the University of California San Diego animal facility on a 12-hour/12-hour light/dark cycle with free access to normal chow food and water. All of the mice were handled according to approved institutional animal care and use committee (IACUC) protocols (S01015) of the University of California San Diego to minimize pain and suffering.
Copyright
© 2022, Shen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Systematic analysis of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs
eLife 11:e70878.
https://doi.org/10.7554/eLife.70878
Further reading
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Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.
