A map of human PRDM9 binding provides evidence for novel behaviors of PRDM9 and other zinc-finger proteins in meiosis
- University of Oxford, United Kingdom
- École Polytechnique Fédérale de Lausanne, Switzerland
Figures
Figure 1 with 4 supplements
Comparison of seven distinct motifs bound by human PRDM9 (B allele).
(a) Seven motif logos produced by our algorithm (applied to the top 5,000 PRDM9 binding peaks ranked by enrichment, after filtering out repeat-masked sequences) were aligned to each other and to an …
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Figure 1—source data 1
List of all ChIP-seq samples.
- https://doi.org/10.7554/eLife.28383.008
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Figure 1—source data 2
PWMs for all motifs, in MEME format.
- https://doi.org/10.7554/eLife.28383.009
Figure 1—figure supplement 1
DMC1, H3K4me3, and H3K36me3 signals surrounding human PRDM9 peaks.
(a) A comparison our autosomal PRDM9 peaks, called at various p-value thresholds ranging from 10−8 to 10−3 (minimum peak separation 250 bp), to a set of published DSB hotspots corresponding to the …
Figure 1—figure supplement 2
Comparison of PRDM9 and H3K4me3/DMC1 enrichment values.
H3K4me3 ChIP-seq data from transfected HEK293T cells (this study) and H3K4me3/DMC1 data from testes (Pratto et al., 2014) were force-called in a 1 kb window centered on each PRDM9 binding peak …
Figure 1—figure supplement 3
All motifs found in human PRDM9 peaks.
All 17 motif logos returned by our motif-finding algorithm are listed, along with histograms indicating their positions within the central 300 bp of our human PRDM9 peaks, as a measure of how …
Figure 1—figure supplement 4
Motif 7 represents a binding mode favored by the B allele.
(a) Peak enrichment quartiles (filtered to remove promoters) were separated by motif type (Motifs 2, 3, and 5 were combined due to low abundance), and mean force-called H3K4me3 enrichment was …
Figure 2 with 4 supplements
Human PRDM9 can bind promoters, though recombination is suppressed.
(a) The chance-corrected proportion of protein-coding genes that have a PRDM9 peak center occurring within 500 bp of the TSS, stratified by different PRDM9 enrichment value thresholds (shades of …
Figure 2—figure supplement 1
Chimp w11a PRDM9 binds a T-rich motif away from human binding sites.
(a) Comparison of the number of human (B allele; blue) and chimp (w11a allele; semitransparent green) PRDM9 ChIP-seq peaks in 1 Mb bins across human Chr1. Consistent with their different predicted …
Figure 2—figure supplement 2
Human PRDM9 can bind promoters, though DSBs do not occur.
(a) FIMO was used to identify the top 1 million matches for Motif 1 in hg19 (Bailey et al., 2015. For 0.1 percentile bins of increasing FIMO score, the proportion of motif matches occurring within …
Figure 2—figure supplement 3
ATAC-seq profiles showing nucleosome phasing around PRDM9 binding sites.
(a) ATAC-seq profile plots surrounding a set of the ~15,000 strongest human PRDM9 ChIP-seq peaks (filtered to require a motif match and to not overlap an annotated DNase hypersensitive site) in …
Figure 2—figure supplement 4
PRDM9s ZF domain is necessary and sufficient for nuclear localization.
Representative results of ImmunoFluorescence detection of V5 tags in HEK293T cells transfected with full-length human PRDM9 (first column), the ZF domain alone (‘ZF-only’, second column), or …
Figure 3 with 1 supplement
Spermatogenesis-specific genes VCX and CTCFL are activated by human PRDM9 in HEK293T cells.
(a) left: Bar plots showing the log2 fold change relative to untransfected HEK293T cells in computed FPKM values (fragments per kilobase of transcript per million mapped RNA-seq reads) for HEK293T …
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Figure 3—source data 1
qPCR primers, Ct values, and calculations.
- https://doi.org/10.7554/eLife.28383.017
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Figure 3—source data 2
PRDM9-bound genes with differential expression.
- https://doi.org/10.7554/eLife.28383.018
Figure 3—figure supplement 1
Raw coverage values surrounding the VCX promoter.
A browser screenshot (Zhou et al., 2011) from ChrX containing the VCX gene with custom tracks indicating ChIP-seq and RNA-seq raw coverage data. Human PRDM9, but not chimp PRDM9, (green) binds a …
Figure 4 with 2 supplements
Influences on recombination in cis downstream of PRDM9 binding.
(a) Analysis of THE1B repeats shows the positions along the THE1B consensus (bottom, gray) of motifs influencing PRDM9 binding (top row), motifs influencing recombination hotspot occurrence at bound …
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Figure 4—source data 1
Detailed information on all THE1B motifs.
- https://doi.org/10.7554/eLife.28383.022
Figure 4—figure supplement 1
Features associated with recombination outcomes given PRDM9 binding.
(a) DMC1-based recombination rates around the centers of THE1B repeats containing different approximate matches to the PRDM9 binding motif CCTCCC[CT]AGCCA[CT] (colors) and the motif ATCCATG (lines …
Figure 4—figure supplement 2
Large-scale recombination rate affects testis DMC1 but not H3K4me3.
Profiles of mean DMC1 and H3K4me3 read coverage from human male testes (with a PRDM9 A/B genotype; Pratto et al., 2014) around all THE1B repeats, stratified into quantiles based on the …
Figure 5 with 3 supplements
PRDM9 multimer formation is mediated by the ZF domain in an allele-biased manner.
(a) Overview of the different C-terminally tagged PRDM9 constructs used. Both an HA and a V5 version of each construct were generated for co-IP experiments. (b) Barplot showing the relative …
Figure 5—figure supplement 1
Confirmation of PRDM9 multimer formation.
Left: Western blots illustrating controls and experimental results. Samples were split and run on two blots separately, one imaged using an anti-HA antibody (upper) and one using an anti-V5 antibody …
Figure 5—figure supplement 2
Multimerization is mediated primarily by ZF-ZF binding.
Western blots illustrating co-IP results for various combinations of full-length Human, noZF, and ZFonly constructs. (a) The third and fourth blots show only a very faint co-IP signal despite strong …
Figure 5—figure supplement 3
Benzonase treatment does not affect co-IP results.
(a) Replication of ZFonly-ZFonly co-IP western experiment with and without benzonase treatment. Lanes 1 and 7 are protein ladder. Lane 4 is empty. Lanes 2 and 5 are input lysate (50 μg protein …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.28383.027
