Figure 7.
Chromatin accessibility in ELDR-overexpressing AML cell lines. (A) ATAC-seq was performed in ELDR-overexpressing THP-1 cell lines ELDR-C1 and ELDR-C3 vs EV control cell line (EV1) and NT cells. UpSet plot revealing the intersection of annotated regions in DARs from the ATAC-seq analysis indicated comparisons colored by genomic localization. The overlap of ELDR-overexpressing lines ELDR-C1 and ELDR-C3 vs control EV1 cells each contains 94 DARs (black arrow). Overlap of ELDR-overexpressing lines ELDR-C1 and ELDR-C3 vs control EV1 cells and vs NT cells each contain 44 DARs (asterisk). (B) Volcano plot of the 94 DARs identified in the UpSet plot under panel (A) indicating the DARs that are lost (closed) or gained (opened). The 39 α-satellite–containing regions are more closed in ELDR-C1 and ELDR-C3 vs control EV1 cells. (C) Venn diagram illustrating the overlap between proteins identified by ChIRP-MS to interact with ELDR and proteins that bind the centromere specific histone variant CENP-A.35,36 (D) Alignment of RNA-seq (from supplemental Figure 6C), ATAC-seq, and indicated ChIP-seq data at the α-satellite repeat containing centromere and neighboring regions of chromosome 5 to illustrate the localization of DARs and the binding of MLL-AF9, MLL, AF9, RUNX, and transcribed genes using the integrated genome viewer. Data for MLL-AF9 target genes by the MLL-AF9-Flag fusion protein were from GSE103947, data for MLL and AF9 were from GSE79899,37 and the data set for RUNX1 was from GSE217171. (E) Representative IMARIS-based 3D reconstruction of nuclei stained for centromeres for MLL-AF9 and smFISH for ELDR. (F) Magnification of the areas labeled “i-v” in panel (E) revealing colocalizations of ELDR and MLL-AF9 within 240 nm (blue and purple spots), centromere and MLL-AF9 within 500 nm (green and pink spots), and centromere and ELDR within 500 nm (green and pale blue spots). (G) Quantification of the colocalization of ELDR with MLL-AF9 per cell. n = 78 cells per condition. (H) Percentage of MLL-AF9 spots colocalized with ELDR or not over all MLL-AF9 spots. (I) Quantification of spots indicating colocalization of ELDR and centromere per cell. mEldr (murine Eldr) and IgG control (78 cells), ELDR and IgG control (78 cells), ELDR and centromere (74 cells). (J) Quantification of the percentage of cells among all analyzed cells with localization of ELDR/MLL-AF9 complexes in the vicinity of the centromere (radius 0.5 μm). (K) Quantification of ELDR-MLL-AF9 complexes in the vicinity of a centromere per cell with controls. n = 78 cells per condition. (L) Expression levels of the RNA of the HOR D5Z2 at the centromere of chromosome 5 in ELDR-overexpressing lines ELDR-C1 and ELDR-C3 vs EV1 assessed by RT-qPCR. Data are presented as mean +SD from 3 independent experiments. ∗∗∗P < .001; ∗∗∗∗P < .0001. P values were calculated using parametric unpaired t test (panel L).

Chromatin accessibility in ELDR-overexpressing AML cell lines. (A) ATAC-seq was performed in ELDR-overexpressing THP-1 cell lines ELDR-C1 and ELDR-C3 vs EV control cell line (EV1) and NT cells. UpSet plot revealing the intersection of annotated regions in DARs from the ATAC-seq analysis indicated comparisons colored by genomic localization. The overlap of ELDR-overexpressing lines ELDR-C1 and ELDR-C3 vs control EV1 cells each contains 94 DARs (black arrow). Overlap of ELDR-overexpressing lines ELDR-C1 and ELDR-C3 vs control EV1 cells and vs NT cells each contain 44 DARs (asterisk). (B) Volcano plot of the 94 DARs identified in the UpSet plot under panel (A) indicating the DARs that are lost (closed) or gained (opened). The 39 α-satellite–containing regions are more closed in ELDR-C1 and ELDR-C3 vs control EV1 cells. (C) Venn diagram illustrating the overlap between proteins identified by ChIRP-MS to interact with ELDR and proteins that bind the centromere specific histone variant CENP-A.35,36 (D) Alignment of RNA-seq (from supplemental Figure 6C), ATAC-seq, and indicated ChIP-seq data at the α-satellite repeat containing centromere and neighboring regions of chromosome 5 to illustrate the localization of DARs and the binding of MLL-AF9, MLL, AF9, RUNX, and transcribed genes using the integrated genome viewer. Data for MLL-AF9 target genes by the MLL-AF9-Flag fusion protein were from GSE103947, data for MLL and AF9 were from GSE79899,37 and the data set for RUNX1 was from GSE217171. (E) Representative IMARIS-based 3D reconstruction of nuclei stained for centromeres for MLL-AF9 and smFISH for ELDR. (F) Magnification of the areas labeled “i-v” in panel (E) revealing colocalizations of ELDR and MLL-AF9 within 240 nm (blue and purple spots), centromere and MLL-AF9 within 500 nm (green and pink spots), and centromere and ELDR within 500 nm (green and pale blue spots). (G) Quantification of the colocalization of ELDR with MLL-AF9 per cell. n = 78 cells per condition. (H) Percentage of MLL-AF9 spots colocalized with ELDR or not over all MLL-AF9 spots. (I) Quantification of spots indicating colocalization of ELDR and centromere per cell. mEldr (murine Eldr) and IgG control (78 cells), ELDR and IgG control (78 cells), ELDR and centromere (74 cells). (J) Quantification of the percentage of cells among all analyzed cells with localization of ELDR/MLL-AF9 complexes in the vicinity of the centromere (radius 0.5 μm). (K) Quantification of ELDR-MLL-AF9 complexes in the vicinity of a centromere per cell with controls. n = 78 cells per condition. (L) Expression levels of the RNA of the HOR D5Z2 at the centromere of chromosome 5 in ELDR-overexpressing lines ELDR-C1 and ELDR-C3 vs EV1 assessed by RT-qPCR. Data are presented as mean +SD from 3 independent experiments. ∗∗∗P < .001; ∗∗∗∗P < .0001. P values were calculated using parametric unpaired t test (panel L).

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