Correction: Corrigendum: Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity

Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas‐Chollier, Morgane; Meijsing, Sebastiaan H.

doi:10.1038/ncomms13784

Cited by 10 publications

(6 citation statements)

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“…To study what is required to convert endogenous genes into GR targets, we first set out to add a single GBS near the TSS of four candidate genes using CRISPR/Cas9 and HDR templates (Fig 1A). To increase our chances of observing GR-dependent regulation, we picked a GBS variant (CGT, a synthetic sequence matching the consensus motif), which showed the highest GR-dependent activation in previous studies (10, 11). The candidate genes were selected based on the following criteria.…”

Section: Resultsmentioning

confidence: 99%

“…GR is known to bind directly to a broad spectrum of sequences that differ in their precise sequence composition (23). In addition to recruiting GR to defined genomic loci, the sequence of the binding site can also modulate the activity of GR downstream of binding (10, 11, 23). To test if GBS variants, other than the CGT variant we initially tested, can accommodate GR-dependent regulation of the IL1R2 gene, we generated single-cell–derived clonal lines for three additional GBS variants (Figs 4A and S2).…”

Section: Resultsmentioning

confidence: 99%

“…To test if GBS variants, other than the CGT variant we initially tested, can accommodate GR-dependent regulation of the IL1R2 gene, we generated single-cell–derived clonal lines for three additional GBS variants (Figs 4A and S2). We picked two GBS variants PAL (a synthetic sequence with perfect palindromic half sites) and GILZ (a GBS derived from a GR-bound region near the GILZ gene) that showed markedly lower activities than the CGT sequence in previous studies using reporter assays and one with comparable activity FKBP5-2 (a GBS derived from a GR-bound region near the FKBP5 gene) (10, 11). Next, we tested if these GBS variants could also convert the IL1R2 gene into a GR target and found that this was the case for each of the variants tested (Fig 4B).…”

Section: Resultsmentioning

confidence: 99%

“…The second trait we evaluated is the sequence of GR’s DNA-binding site, which can influence the magnitude of transcriptional activation by GR (10, 11, 27). By changing the sequence identity of the introduced GR-binding site at the IL1R2 gene and of a GBS at an endogenous GR-bound region near the GILZ gene, we found that distinct variants facilitate equivalent levels of GR-dependent activation.…”

Section: Discussionmentioning

confidence: 99%

“…The sequence composition of cis -regulatory elements also plays a role in fine-tuning the expression level of individual genes. For instance, GR binds as a dimer to typically imperfect half sites separated by a 3-bp spacer, and the exact sequence of the half site, the spacer, and of the nucleotides flanking the GR-binding sequence (GBS) can modulate GR’s activity towards target genes (10, 11).…”

Section: Introductionmentioning

confidence: 99%

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Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements

et al. 2019

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The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used CRISPR/Cas-mediated homology-directed repair to add a single GR-binding site directly upstream of the transcriptional start site of each of four genes. To our surprise, we found that the addition of a single GR-binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Furthermore, by introducing GR-binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR-binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements

et al. 2019

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Power-law behavior of transcription factor dynamics at the single-molecule level implies a continuum affinity model

Garcia

Fettweis

Presman

et al. 2021

Nucleic Acids Research

109

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Single-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the diffusion and binding behavior of these proteins in the nuclear environment. Dwell time distributions obtained by SMT for most TFs appear to follow bi-exponential behavior. This has been ascribed to two discrete populations of TFs—one non-specifically bound to chromatin and another specifically bound to target sites, as implied by decades of biochemical studies. However, emerging studies suggest alternate models for dwell-time distributions, indicating the existence of more than two populations of TFs (multi-exponential distribution), or even the absence of discrete states altogether (power-law distribution). Here, we present an analytical pipeline to evaluate which model best explains SMT data. We find that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution of dwell-times, blurring the temporal line between non-specific and specific binding, suggesting that productive binding may involve longer binding events than previously believed. From these observations, we propose a continuum of affinities model to explain TF dynamics, that is consistent with complex interactions of TFs with multiple nuclear domains as well as binding and searching on the chromatin template.

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Single-molecule DNA unzipping reveals asymmetric modulation of a transcription factor by its binding site sequence and context

Rudnizky

Khamis

Malik

et al. 2017

Preprint

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Most functional transcription factor (TF) binding sites deviate from their "consensus" recognition motif, although their sites and flanking sequences are often conserved across species. Here, we used singlemolecule DNA unzipping with optical tweezers to study how Egr-1, a TF harbouring 3 zinc fingers (ZF1,ZF2 and ZF3), is modulated by the sequence and context of its functional sites in the Lhb gene promoter. We find that both the core 9 base pairs bound to Egr-1 in each of the sites, and the base pairs flanking them, modulate the affinity and structure of the protein-DNA complex. The effect of the flanking sequences is asymmetric, with a stronger effect for the sequence flanking ZF3. Characterization of the dissociation time of Egr-1 revealed that a local, mechanical perturbation of the interactions of ZF3 destabilizes the complex more effectively than a perturbation of the ZF1 interactions. Our results reveal a novel role for ZF3 in the interaction of Egr-1 with other proteins and the DNA, providing insight on the regulation of Lhb and other genes by Egr-1. Moreover, our findings reveal the potential of small changes in DNA sequence to alter transcriptional regulation, and may shed light on the organization of regulatory elements at promoters.

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Correction: Corrigendum: Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity

Cited by 10 publications

References 0 publications

Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements

Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements

Power-law behavior of transcription factor dynamics at the single-molecule level implies a continuum affinity model

Single-molecule DNA unzipping reveals asymmetric modulation of a transcription factor by its binding site sequence and context

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