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. 2021 Nov 24;49(21):12178–12195. doi: 10.1093/nar/gkab1100

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — RA-induced changes in enhancer activity at RARα-occupied sites correlate with specific sequence and chromatin features. (A) Genome browser view of an exemplary genomic region encompassing RA-inducible and non-inducible RARα binding sites. Normalized ATAC-, FAIRE-STARR and RNA-seq signals for LIF or RA treated cells and RefSeq genes for this region in either black (protein coding genes) or red (non-coding genes). (B) Distribution of changes in STARR activity (log₂ fold change STARR score RA/LIF) and mean STARR activity (log score, for both treatments) of RARα-occupied regions that are covered in our FAIRE-STARR input library (as shown in Supplementary Figure S6A). Only regions with a minimum mean STARR activity ≥2.5 were included for further analysis. The 10% most induced, 10% most repressed and an equal number of regions that do not respond to RA treatment (non-resp.) were used for motif enrichment and TF binding analyses. (C) Enriched TF motif clusters (JASPAR 2018 clustered motif matrices) at induced, repressed, and non-responsive RARα-occupied sites. TF motif clusters with a maximum E-value of 1e−5 for at least one group and a log fold change ≥2 of induced or repressed over non-responsive regions are shown. Z-score normalization of E-values per row was performed. (D) Different spacings (0–8 nucleotides) and orientations (direct (DR), inverted (IR), and everted repeat (ER)) of the RARα::RXRα consensus motif (MA0159.1, upper panel, arrows highlight repeat orientation) were constructed in silico and used for motif enrichment analysis using AME. Only motifs which showed significant enrichment (E-value ≤ 1e−3) for at least one RARα binding site group are shown. Z-score normalization of E-values per row was performed. (E) Enhancer activity measured by STARR-RT-qPCR for selected spacing variants of the RARα::RXRα consensus motif (MA0159.1) and neighboring TF motifs as indicated (scr = scrambled motif) after 4 h of LIF or RA treatment. Bar plots depict the mean GFP expression + SE for three biological replicates. (F) Mean enrichment of RARα and H3K27ac as well as chromatin accessibility (ATAC) at induced, repressed, and non-responsive RARα-occupied sites.