Asxl1 truncation changes the epigenetic landscape to favor CSF3R-driven differentiation. (A) Representative flow cytometry plots of CD11b and GR1 at 48 hours after estrogen withdrawal in HoxB8 CRISPR-Cas9 modified for Asxl1 truncation and transduced with CSF3RT618I or their corresponding control plasmid producing the following 4 groups: control, Asxl1sgB2, CSF3RT618I, and double mutant or Asxl1sgB2/CSF3RT618I. Quantification of the percentage of CSF3R-mCherry–positive cells (mean ± SEM; n = 3 per group). Significance was evaluated with a 2-way ANOVA with a Tukey multiple comparison test. (B) ATAC sequencing was performed 48 hours after estrogen withdrawal with all 4 groups (n = 3 per group). GREAT analysis using differential peaks between Asxl1sgB2/CSF3RT618I and control groups reveal enrichment of immune system activation and differentiation in the double-mutant mice. (C) HOMER motif enrichment results from the ATAC sequencing differential peaks between CSF3RT618I and control. The orange trend line in the scatterplot is used to compare natural log-transformed motif P values between the sample groups. Residual values in the bar plot represent the difference between the trend line and transformed motif P values. (D) CUT&Tag was performed 48 hours after estrogen withdrawal with all 4 groups (n = 3 per group) with H3K4me1 antibody. Profile plot of mean H3K4me1 activity at regions spanning its consensus peak centers ± 3 kb. (E) Heat map displaying row-scaled (region-based z-score scaled), normalized read counts for each sample condition (averaged across replicates) in regions where H3K4me1 is uniquely active. The top 6 significant enriched motifs from HOMER are revealed for region clusters 1, 5, and 6. (F) HOMER motif enrichment results from the ATAC sequencing differential peaks between Asxl1sgB2 and control. The orange trend line in the scatterplot is used to compare natural log-transformed motif P values between the sample groups. Residual values in the bar plot represent the difference between the trend line and transformed motif P values. (G) CUT&Tag was performed 48 hours after estrogen withdrawal with all 4 groups (n = 3 per group) with H2AK119ub antibody. Profile plot of mean H2AK119ub activity at regions spanning its consensus peak centers ± 3 kb. (H) Heat map displaying row-scaled (region-based z-score scaled) normalized read counts for each sample condition (averaged across replicates) in regions where H2AK119ub is uniquely active. Top 6 significant enriched motifs from HOMER are revealed for region cluster 1. CTCF and BORIS are not specific to cluster 1 as they are top 1 and 2 in all but 1 cluster. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.

Asxl1 truncation changes the epigenetic landscape to favor CSF3R-driven differentiation. (A) Representative flow cytometry plots of CD11b and GR1 at 48 hours after estrogen withdrawal in HoxB8 CRISPR-Cas9 modified for Asxl1 truncation and transduced with CSF3RT618I or their corresponding control plasmid producing the following 4 groups: control, Asxl1sgB2, CSF3RT618I, and double mutant or Asxl1sgB2/CSF3RT618I. Quantification of the percentage of CSF3R-mCherry–positive cells (mean ± SEM; n = 3 per group). Significance was evaluated with a 2-way ANOVA with a Tukey multiple comparison test. (B) ATAC sequencing was performed 48 hours after estrogen withdrawal with all 4 groups (n = 3 per group). GREAT analysis using differential peaks between Asxl1sgB2/CSF3RT618I and control groups reveal enrichment of immune system activation and differentiation in the double-mutant mice. (C) HOMER motif enrichment results from the ATAC sequencing differential peaks between CSF3RT618I and control. The orange trend line in the scatterplot is used to compare natural log-transformed motif P values between the sample groups. Residual values in the bar plot represent the difference between the trend line and transformed motif P values. (D) CUT&Tag was performed 48 hours after estrogen withdrawal with all 4 groups (n = 3 per group) with H3K4me1 antibody. Profile plot of mean H3K4me1 activity at regions spanning its consensus peak centers ± 3 kb. (E) Heat map displaying row-scaled (region-based z-score scaled), normalized read counts for each sample condition (averaged across replicates) in regions where H3K4me1 is uniquely active. The top 6 significant enriched motifs from HOMER are revealed for region clusters 1, 5, and 6. (F) HOMER motif enrichment results from the ATAC sequencing differential peaks between Asxl1sgB2 and control. The orange trend line in the scatterplot is used to compare natural log-transformed motif P values between the sample groups. Residual values in the bar plot represent the difference between the trend line and transformed motif P values. (G) CUT&Tag was performed 48 hours after estrogen withdrawal with all 4 groups (n = 3 per group) with H2AK119ub antibody. Profile plot of mean H2AK119ub activity at regions spanning its consensus peak centers ± 3 kb. (H) Heat map displaying row-scaled (region-based z-score scaled) normalized read counts for each sample condition (averaged across replicates) in regions where H2AK119ub is uniquely active. Top 6 significant enriched motifs from HOMER are revealed for region cluster 1. CTCF and BORIS are not specific to cluster 1 as they are top 1 and 2 in all but 1 cluster. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.

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