Enhancer regions show high histone H3.3 turnover that changes during differentiation
- Massachusetts General Hospital, United States
- Harvard Medical School, United States
- Instituto Gulbenkian de Ciencia, Portugal
- Massachusetts General Hospital and Harvard Medical School, United States
Figures
Figure 1 with 3 supplements
Time-ChIP assay in H3.3_SNAP ESCs.
(A) Western blot for total H3 in H3.3_SNAP ESCs after 48 hr dox induction of transgene. (B) Outline of time-ChIP protocol. (C) Quantification of DNA recovered from time-ChIP experiments. The amount …
Figure 1—figure supplement 1
Time-ChIP assay in H3.3_SNAP ESCs.
(A) Generation of H3.3_SNAP ESCs. The p2lox H3.3_SNAP plasmid was introduced into A2lox.Cre ESCs resulting in H3.3_SNAP ESCs expressing H3.3_SNAP_HA under dox inducible control from the hprt locus …
Figure 1—figure supplement 2
Post-translational modification of SNAP-tagged histones.
(A) Western blot for H3K4me3 H3.3_SNAP ESCs. Uninduced sample ('un') does not have any H3.3_SNAP, upon dox induction ('dox') H3K4me3 modified H3.3_SNAP is observed. Non-specific bands present in …
Figure 1—figure supplement 3
Spike-in control.
Amount of DNA recovered relative to 0 hr time point (red bars) and proportion of sequencing reads mapping to the mouse genome relative to 0 hr time point based on the number of reads recovered from …
Figure 2 with 1 supplement
Measuring H3.3 turnover in ESCs.
(A) Average H3.3 profiles for 0 hr, 3 hr, 6 hr and 12 hr time points over the transcription start sites (TSSs) of expressed and silent genes in ESCs. (B) H3.3 in 1kb bins profiles for 0 hr, 3 hr, 6 h…
Figure 2—figure supplement 1
H3.3 enrichment in ESCs and TI correlation between replicates.
(A) H3.3 enrichment in ESCs at annotated genomic elements – promoters, 5’ end of genes, gene bodies, TTSs and enhancers. Genes are grouped by ESC expression level and ESC enhancers are from Whyte et …
Figure 3 with 2 supplements
Regions with high TI in ESCs have regulatory functions.
(A) Representation of annotated genomic features in the 1000 highest TI regions in ESCs relative to their representation in the genome as a whole. Overrepresentation was calculated by taking the …
Figure 3—figure supplement 1
H3.3 turnover at enhancers and super-enhancers calculated using a 300 bp bin size.
Average H3.3 TI over conventional ESC enhancers ('Enhancers' – purple) and super-enhancer constituent enhancers ('Super-enhancers' – pink) where TI was calculated using a 300 bp bin size. The mean …
Figure 3—figure supplement 2
H3.1 turnover at ESC enhancers and super-enhancers.
Average H3.1 TI over conventional ESC enhancers ('Enhancers' – purple), super-enhancer constituent enhancers ('Super-enhancers' – pink) and random genomic regions with a similar size distribution to …
Figure 4 with 1 supplement
H3.3 turnover at PcG targets in ESCs.
(A) TI averaged over PcG-bound genomic regions (turquoise) and all CpG islands (red). (B) TI averaged over the TSSs of active genes (red), PcG targets (turquoise) and silent genes (blue) in ESCs. (C)…
Figure 4—figure supplement 1
H3.3 enrichment at PcG targets in ESCs.
(A) H3.3 enrichment in ESCs at PcG targets (grey) compared to H3K9me3 regions (green). (B) H3.3 enrichment in ESCs at PcG targets (blue), CpG islands (CGI – red), enhancers (purple) and randomized …
Figure 5 with 2 supplements
H3.3 turnover and distribution changes during neural differentiation of ESCs.
(A) Average TI profile around TSSs of genes that lose PcG binding and increase gene expression upon differentiation to NSCs. (B) The region containing the Olig1 and Olig2 genes, which are activated …
Figure 5—figure supplement 1
Generation of H3.3_SNAP NSCs.
(A) Screening for chimeric NSC lines expressing H3.3_SNAP based on puromycin resistance. Red bars indicate cell lines that scored as highly chimeric. NSC line '3_1' was used for time-ChIP studies. (B…
Figure 5—figure supplement 2
Measuring turnover in H3.3_SNAP NSCs.
(A) Western blot showing expression of H3.3_SNAP upon dox induction in ESCs compared to NSCs. For dox induced samples ('dox'), increasing amounts of protein were loaded, for uninduced samples ('un') …
Figure 6
Changes in TI occurring during neural differentiation often occur at enhancers.
(A) Volcano plot showing differences in TI between ESCs and NSCs. Regions with a TI difference in the top 2.5% with a p-value≤0.01 were selected as changing during differentiation (red dots). (B) …
Figure 7 with 2 supplements
H3.3 turnover correlates with DNA accessibility at enhancers.
(A) Boxplot of average ESC TI and MACC at ESC enhancers, the TSSs of expressed genes, and PcG-bound regions. (B) Pearson’s correlation between DNA accessibility as measured by MNase titration (MACC) …
Figure 7—figure supplement 1
Measuring DNA accessibility using MNase titration.
(A) Boxplot of average TI over DNase hypersensitive regions in E14 ESCs (Encode accession ENCSR000CMW). TI index was plotted over called DNase peaks (E14-DS18505.peaks.fdr0.01). (B) MNase digestion …
Figure 7—figure supplement 2
Comparing turnover to DNA accessibility measured by MNase titration.
(A) Pearson’s correlation between DNA accessibility as measured by MNase titration (MACC) and TI at the TSSs of genes differentially expressed in ESCs and NSCs. For MACC four replicates are shown …
Additional files
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Supplementary file 1
Top 1000 regions with highest turnover in ESCs.
- https://doi.org/10.7554/eLife.15316.021
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Supplementary file 2
Top 1000 regions with highest turnover in NSCs.
- https://doi.org/10.7554/eLife.15316.022
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Supplementary file 3
Differences in turnover between ESCs and NSCs.
- https://doi.org/10.7554/eLife.15316.023
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Supplementary file 4
External datasets used.
- https://doi.org/10.7554/eLife.15316.024
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Supplementary file 5
Spike-in controls.
- https://doi.org/10.7554/eLife.15316.025
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Supplementary file 6
Primer sequences.
- https://doi.org/10.7554/eLife.15316.026
