Figure 5.
FBXO11 targets BAHD1 at bivalent erythroid genes. (A) Heat maps showing H3K27me3 and H3K4me3 histone marks near the transcription start site (TSS, ±10 kb) of genes with decreased, increased, or unchanged expression after FBXO11 KO in HUDEP-2 cells grown under expansion conditions. Results are shown for WT and F11KO (clone F11KO #2) HUDEP-2 cells with analysis from 2 biological replicates. (B) The histone marks H3K4me3 and H3K27me3 within 10 kb of the TSS of FBXO11-regulated genes in human peripheral blood (PB) CD34+ cell–derived proerythroblasts at differentiation day 7 and in CD36+CD71+CD235a+ human umbilical cord blood CD34+ cell–derived erythroblasts from the Blueprint consortium (http://www.blueprint-epigenome.eu/). (C) Metagene representation of histone marks for genes with decreased expression after FBXO11 KO, as shown in panel A. Results are from an analysis of 2 biological replicates. (D) Violin plots showing normalized densities of H3K27me3 and H3K4me3 marks in genes with decreased or increased expression after FBXO11 KO in WT or F11KO HUDEP-2 cells grown in culture under expansion conditions. The data summarize the results of 2 biological replicate experiments. (E) Genomic localization of BAHD1-V5 ChIP-seq peaks. Promoter, 2 kb on either side of the TSS; TES, 2 kb on either side of the transcription end site (TES); 5′ distal region, 50 kb upstream of the TSS to 2 kb downstream of the TSS; 3′ distal region, 2 kb downstream of the TES to 50 kb downstream of the TES; intergenic, >50 kb upstream of the TSS and >50 kb downstream of the TES. Results are shown from analysis of 3 biological replicates. (F) Overlap between BAHD1-V5 binding (ChIP-seq signals) and H3K27me3 marks in HUDEP-2 cells. The P value was determined by Fisher exact test. (G) Analysis of genes that are deregulated by ectopic expression of BAHD1-V5 in HUDEP-2 cells. Pie charts show the mRNA transcripts that are decreased (upper left) or increased (upper right) in BAHD1-V5–expressing vs WT cells, color-coded according to the BAHD1-V5 binding of the corresponding genes. The P values refer to the significance of BAHD1-V5 occupancy on genes with decreased or increased expression caused by BAHD1-V5 (as determined by Fisher exact test). BAHD1-V5 binding in genes with decreased (lower left) or increased (lower right) expression in F11KO vs WT HUDEP-2 cells. The P values refer to the significance of BAHD1-V5 occupancy in each group (Fisher exact test). (H) Promoter bivalency in genes with decreased (left) or increased (right) expression in F11KO vs WT HUDEP-2 cells. The P values refer to the significance of promoter bivalency in each group (Fisher exact test). (I) ChIP-seq analysis of WT and F11KO (clone F11KO #2) HUDEP-2 cells for GATA1 occupancy near the TSS (±5 kb) of genes with decreased or increased expression after FBXO11 KO. Heat maps of GATA1 signals are shown on the left. Violin plots on the right show normalized densities of GATA1 signals (data from 2 biological replicate experiments). P values were determined by the Wilcoxon test. (J) Motif-enrichment analysis of ATAC-seq data from WT and FBXO11-KO (F11KO #2) HUDEP-2 cells. The volcano plot shows the transcription factor binding motifs that are enriched near ATAC-seq peaks (reflecting open chromatin) and that are lost or gained in F11KO cells compared with WT cells. (K) Overlap between BAHD1-V5 binding sites and GATA1 occupancy in WT (FBXO11+/+) HUDEP-2 cells. P value was determined by Fisher exact test. (L) Violin plots showing normalized densities of H3K27ace and H3K9ace marks in genes with decreased expression after FBXO11 KO in WT vs F11KO HUDEP-2 cells. The P value was determined by the Wilcoxon test.

FBXO11 targets BAHD1 at bivalent erythroid genes. (A) Heat maps showing H3K27me3 and H3K4me3 histone marks near the transcription start site (TSS, ±10 kb) of genes with decreased, increased, or unchanged expression after FBXO11 KO in HUDEP-2 cells grown under expansion conditions. Results are shown for WT and F11KO (clone F11KO #2) HUDEP-2 cells with analysis from 2 biological replicates. (B) The histone marks H3K4me3 and H3K27me3 within 10 kb of the TSS of FBXO11-regulated genes in human peripheral blood (PB) CD34+ cell–derived proerythroblasts at differentiation day 7 and in CD36+CD71+CD235a+ human umbilical cord blood CD34+ cell–derived erythroblasts from the Blueprint consortium (http://www.blueprint-epigenome.eu/). (C) Metagene representation of histone marks for genes with decreased expression after FBXO11 KO, as shown in panel A. Results are from an analysis of 2 biological replicates. (D) Violin plots showing normalized densities of H3K27me3 and H3K4me3 marks in genes with decreased or increased expression after FBXO11 KO in WT or F11KO HUDEP-2 cells grown in culture under expansion conditions. The data summarize the results of 2 biological replicate experiments. (E) Genomic localization of BAHD1-V5 ChIP-seq peaks. Promoter, 2 kb on either side of the TSS; TES, 2 kb on either side of the transcription end site (TES); 5′ distal region, 50 kb upstream of the TSS to 2 kb downstream of the TSS; 3′ distal region, 2 kb downstream of the TES to 50 kb downstream of the TES; intergenic, >50 kb upstream of the TSS and >50 kb downstream of the TES. Results are shown from analysis of 3 biological replicates. (F) Overlap between BAHD1-V5 binding (ChIP-seq signals) and H3K27me3 marks in HUDEP-2 cells. The P value was determined by Fisher exact test. (G) Analysis of genes that are deregulated by ectopic expression of BAHD1-V5 in HUDEP-2 cells. Pie charts show the mRNA transcripts that are decreased (upper left) or increased (upper right) in BAHD1-V5–expressing vs WT cells, color-coded according to the BAHD1-V5 binding of the corresponding genes. The P values refer to the significance of BAHD1-V5 occupancy on genes with decreased or increased expression caused by BAHD1-V5 (as determined by Fisher exact test). BAHD1-V5 binding in genes with decreased (lower left) or increased (lower right) expression in F11KO vs WT HUDEP-2 cells. The P values refer to the significance of BAHD1-V5 occupancy in each group (Fisher exact test). (H) Promoter bivalency in genes with decreased (left) or increased (right) expression in F11KO vs WT HUDEP-2 cells. The P values refer to the significance of promoter bivalency in each group (Fisher exact test). (I) ChIP-seq analysis of WT and F11KO (clone F11KO #2) HUDEP-2 cells for GATA1 occupancy near the TSS (±5 kb) of genes with decreased or increased expression after FBXO11 KO. Heat maps of GATA1 signals are shown on the left. Violin plots on the right show normalized densities of GATA1 signals (data from 2 biological replicate experiments). P values were determined by the Wilcoxon test. (J) Motif-enrichment analysis of ATAC-seq data from WT and FBXO11-KO (F11KO #2) HUDEP-2 cells. The volcano plot shows the transcription factor binding motifs that are enriched near ATAC-seq peaks (reflecting open chromatin) and that are lost or gained in F11KO cells compared with WT cells. (K) Overlap between BAHD1-V5 binding sites and GATA1 occupancy in WT (FBXO11+/+) HUDEP-2 cells. P value was determined by Fisher exact test. (L) Violin plots showing normalized densities of H3K27ace and H3K9ace marks in genes with decreased expression after FBXO11 KO in WT vs F11KO HUDEP-2 cells. The P value was determined by the Wilcoxon test.

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