Abstract
Pioneer transcription factors direct cell differentiation by deploying new enhancer repertoires through their unique ability to target and initiate remodelling of closed chromatin. The initial steps of their action remain undefined, although pioneers have been shown to interact with nucleosomal target DNA and with some chromatin-remodeling complexes. We now define the sequence of events that enables the pioneer Pax7 with its unique abilities. Chromatin condensation exerted by linker histone H1 is the first constraint on Pax7 recruitment, and this establishes the initial speed of chromatin remodeling. The first step of pioneer action involves recruitment of the KDM1A (LSD1) H3K9me2 demethylase for removal of this repressive mark, as well as recruitment of the MLL complex for deposition of the activating H3K4me1 mark. Further progression of pioneer action requires passage through cell division, and this involves dissociation of pioneer targets from perinuclear lamin B. Only then are the SWI–SNF remodeling complex and the coactivator p300 recruited, leading to nucleosome displacement and enhancer activation. Thus, the unique features of pioneer actions are those occurring in the lamin-associated compartment of the nucleus. This model is consistent with previous work that showed a dependence on cell division for establishment of new cell fates.
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Data availability
Sequencing data are available on the GEO as GSE87185, GSE125671, GSE225231 and GSE240090. Source data are provided with this paper.
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Acknowledgements
We are very grateful to A. Shilatifard (Northwestern University) for the MLL3 and MLL4 antibodies, to O. Neyret and S. Boisset for next-generation sequencing analyses, and to V. Magoon for her expert secretarial assistance. Data analyses were possible thanks to the support of Compute Canada. This work was supported by Foundation grant FDN-154297 to J.D. from the Canadian Institutes of Health Research. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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A.G. and J.D. conceived the study; A.G., A.B. and J.D. conceived and designed the experiments; A.G., J.D.-G., V.L.-R. and J.H. performed experiments; A.G., V.L.-R. and J.D. wrote the paper.
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Extended data
Extended Data Fig. 1 Genome-wide effect of Pax7.
a, Pax7 western blot analysis of total cell extracts from neurointermediate lobe (IL) pituitary cells (~50 % melanotropes), AtT20 cells and AtT20-ERPax7 cells showing ectopic expression of ERPax7 at physiological levels (n = 3). b, H3K4me1 deposition (ChIP-seq) and DNA accessibility (ATAC-seq) at Pax7 binding sites before (0 h) and after 48 h of Pax7 activation. Bottom plots show selection threshold for constitutively active (n = 20,775), pioneered (n = 248) and primed (n = 2,023) enhancers bound by Pax7. c, Boxplots of Pax7 recruitment, H3K4me1 deposition and ATAC signals at constitutively active and pioneered enhancers as defined in (b) as well as at random sites from no to 72 h of Pax7 activation and with stable Pax7 expression. ChIP-seq and ATAC-seq data, p-values from two-tailed t-tests. d, Frequency per bin of Pax7 recruitment (ChIP-seq) at pioneered enhancers binned according to signal strength. The randomly selected n = 14 pioneered enhancers used in the qPCR panel (Fig. 1d) are indicated by vertical dashed bars. e, t1/2 of H3K4me1 deposition at the n = 14 pioneered enhancers computed from time courses in Fig. 1e. For each site, H3K4me1 was measured either at the Pax7 binding site (centered) or shifted towards one of the lateral H3K4me1 deposition summits. ChIP-qPCR data from n ≥ 2 biological replicates.
Extended Data Fig. 2 Marker enrichment at pioneered enhancers.
Enrichment of chromatin and enhancer markers at the n = 14 pioneered enhancers (Pio, Fig. 1d) as well as constitutively active enhancers (Con) and random sites (Rnd) used as controls at no (blue) or 72 h (red) of Pax7 activation. Enrichments are expressed relative to mean enrichment at random loci (for Pax7 data) or constitutively active enhancers (all others). ChIP-qPCR data expressed as median ± min and max values of n ≥ 2 technical replicates per site. One of n ≥ 2 biological replicates is shown for each marker. Boxplots at right show the enrichment (1-2.5-5-7.5-9 deciles) at the n = 14 pioneered enhancers and the p-values (two-tailed paired t-test) compare enrichments at no vs. 72 h of Pax7 activation.
Extended Data Fig. 3 Variations in Pax7 pioneering kinetics and initial chromatin state.
a, b, Mean (± standard error of the mean) time courses of mark changes at the stronger and weaker third Pax7 recruitment subsets (n = 4 each) of the n = 14 pioneered enhancers described in Fig. 1d. Each data point was determined by ChIP-qPCR from n ≥ 2 biological replicates. The sequence of events is preserved but a delay of ~6 h in markers t1/2 is observed for the weaker compared to stronger sites. c, H3K4me1 deposition time courses at genome-wide (ChIP-seq) pioneered enhancers (n = 248, Extended Data Fig. 1b) color-coded according to Pax7 recruitment at 1 h and 48 h. d, Correlations between the indicated parameters at the n = 14 pioneered enhancers. Significant correlations are observed between the initial H1 levels, the initial Pax7 recruitment (1 h) up to 48 h and the overall speed of the pioneering process. Theses correlations are lost for long Pax7 activation (> 20 days) or for stably expressed Pax7 suggesting that pioneered enhancers ultimately reach a similar final state. No correlation is observed with the initial levels of H3K9me2 or total histone H3. ChIP-seq data and ChIP-qPCR data from n ≥ 2 biological replicates, p-values from two-tailed t-tests.
Extended Data Fig. 4 Sequential Pax7 recruitment and chromatin opening at pioneered enhancers.
Pax7 ChIP-seq and ATAC-seq profiles at the panel of n = 14 pioneered enhancers specified in Fig. 1d at the indicated times after Pax7 activation or after stable Pax7 expression.
Extended Data Fig. 5 Characterization of mimosine-arrested cells.
a, FACS profiles of AtT20 cells treated with mimosine. Cells arrested in G1 by 24 h mimosine treatment are viable and re-enter cell cycle following release from mimosine-induced cell cycle arrest. One of n = 2 biological replicates is shown. b, p300, Brg1 and Tpit western blots in nuclear extracts of normal and cells treated for 60 h with mimosine. The treatment does not affect p300 or Brg1 levels but results in a two-fold decrease of Tpit expression. n = 3 biological replicates are shown. c, FACS data from control plot in (a) exemplifying the gating strategy used to produce FACS profiles of propidium iodide treated cells.
Extended Data Fig. 6 Pioneering assessment in mimosine-arrested AtT20 cells.
Enrichment of chromatin and enhancer marks at the n = 14 pioneered enhancers (Pio, Fig. 1d) as well as at constitutively active enhancers (Con) and random sites (Rnd) used as controls at no (blue) or 48 h (red) of Pax7 activation in normal and mimosine-arrested cells. Enrichments are expressed relative to mean enrichment at random loci (for Pax7 data) or constitutively active enhancers (all others). ChIP-qPCR data expressed as median ± min and max values of n ≥ 2 technical replicates per site. One of n ≥ 2 biological replicates is shown for each marker. Boxplots at right show the enrichment (1-2.5-5-7.5-9 deciles) at the n = 14 pioneered enhancers and the p-values (two-tailed paired t-test) compare enrichments at no vs. 48 h of Pax7 activation.
Extended Data Fig. 7 Time course of KDM3A recruitment and further characterization of Pax7-primed enhancers.
a, Mean time courses of KDM3A compared to Pax7 recruitment at the n = 14 pioneered enhancers described in Fig. 1d (± standard error of the mean). Each data point was determined by ChIP-qPCR from n ≥ 2 biological replicates. b, Heatmaps (ChIP-seq) of the indicated markers at multiple subsets of Pax7-targeted sites with or without stable Pax7 expression. 4 kb window centered on Pax7 peak.
Supplementary information
Supplementary Tables 1–3
Loci and primers, genome-wide sequencing datasets and antibodies.
Source data
Source Data Fig. 2
Time course qPCR data.
Source Data Fig. 4
Cell cycle arrest qPCR data.
Source Data Fig. 5
KDM1A inhibition qPCR data.
Source Data Fig. 6
Domain-wide qPCR data.
Source Data Extended Data Fig. 1
Unprocessed western blots.
Source Data Extended Data Fig. 2
Markers enrichment qPCR data.
Source Data Extended Data Fig. 5
Unprocessed western blots.
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Gouhier, A., Dumoulin-Gagnon, J., Lapointe-Roberge, V. et al. Pioneer factor Pax7 initiates two-step cell-cycle-dependent chromatin opening. Nat Struct Mol Biol 31, 92–101 (2024). https://doi.org/10.1038/s41594-023-01152-y
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DOI: https://doi.org/10.1038/s41594-023-01152-y
