Fig. 9: Decreased higher-order inter-chromosome structure between two RDH gene clusters in EAF1-mutant cells. | Nature Communications

Fig. 9: Decreased higher-order inter-chromosome structure between two RDH gene clusters in EAF1-mutant cells.

From: The 3′ Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies

Fig. 9

a Wild-type HEK293T (WT) and EAF1-mutant cells (mut) were fixed with paraformaldehyde containing 0.5% Triton X-100 and subjected to DNA-FISH. Four-color microscopy images are shown. RDH gene clusters 1 (cyan) and 2 (red) were stained using Cy5- or Cy3-labeled probe. NPAT (blue) and Coilin (green) were stained using specific antibodies. Enlarged images for representative particles are shown in the upper left of each image. Scale bar, 10 μm. The frequencies of each RDH gene cluster 2 association with CB (Coilin) in wild-type (WT, n = 359 Coilin particles) and mutant cells (mut, n = 556 Coilin particles) were determined and are shown in the left panel. The frequencies of each RDH gene cluster 1 association with CB (Coilin) in wild-type (WT, n = 359 Coilin particles) and mutant cells (mut, n = 556 Coilin particles) were determined and are shown in the right panel. b Representative four-color microscopy images showing the overlap of CB, HLB, and two RDH gene clusters in wild-type HEK293T cells. RDH gene clusters, NPAT, and Coilin were stained as described above. Enlarged images for representative particles are shown in the upper left of each image. Scale bar, 5 μm. The frequency of inter-chromosome interaction between two RDH gene clusters is shown in the right panel (WT, n = 1130 RDH gene cluster 2; mut, n = 771 RDH gene cluster 2). c–f 4C-seq analysis revealed that the higher-order inter-chromosome structure between two RDH gene clusters was abolished in EAF1-mutant cells. c, e Representative 4C-seq profile using RDH gene cluster 1 (HIST1H2BK, chromosome 6) bait or RDH gene cluster 2 (HIST2H2AB, chromosome 1) bait. The detailed contact profile at RDH gene cluster 2 (chromosome 1) (c) or at RDH gene cluster 1 (chromosome 6) (e) is shown in the lower panels. d, f Comparisons of 4C-seq counts detected in each RDH gene cluster region in wild-type (WT) or EAF1-mutant (EAF1 mut) cells under each 4C-seq condition. The read counts of 4C-seq using chromosome 6 HIST1H2BK bait (d) and chromosome 1 HIST2H2AB bait (f) are shown. These plots represent the mean of n = 2 experiments. Source data are provided as a Source Data file.

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