Asxl1 deletion disrupts MYC and RNA polymerase II function in granulocyte progenitors

Abstract

Mutations in the gene Additional Sex-Combs Like 1 (ASXL1) are recurrent in myeloid malignancies as well as the pre-malignant condition clonal hematopoiesis, where they are universally associated with poor prognosis. However, the role of ASXL1 in myeloid lineage maturation is incompletely described. To define the role of ASXL1 in myelopoiesis, we employed single cell RNA sequencing and a murine model of hematopoietic-specific Asxl1 deletion. In granulocyte progenitors, Asxl1 deletion leads to hyperactivation of MYC and a quantitative decrease in neutrophil production. This loss of granulocyte production was not accompanied by significant changes in the landscape of covalent histone modifications. However, Asxl1 deletion results in a decrease in RNAPII promoter-proximal pausing in granulocyte progenitors, indicative of a global increase in productive transcription. These results suggest that ASXL1 inhibits productive transcription in granulocyte progenitors, identifying a new role for this epigenetic regulator in myeloid development.

Figure 1.. Single Cell RNA Reveals a Granulocyte Maturation Defect in Asxl1^Δ/Δ mice at the point of cell cycle exit.

A. Bone marrow was isolated from Asxl1^WT and Asxl1^Δ/Δ mice 4 weeks and 6 months after induction of recombination with Poly I:C (n=3–4/group). Lineage positive cells were depleted using immunomagnetic purification and labeled with the indicated CITE-seq antibodies. Each individual mouse was labeled with a unique anti CD45 HTO antibody allowing for mouse-level bioinformatic deconvolution. Single cell transcriptional profiling was then performed using the Chromium platform (10X Genomics). UMAP projection demonstrating transcriptionally defined clusters identified using published datasets and data integration. Marker genes defining transcriptional cell clusters. B. UMAP projection demonstrating clusters defined by CITE-Seq. C. UMAP projections split by genotype (Asxl1^WT and Asxl1^Δ/Δ) and timepoint (4 weeks and 6 months) post induction of recombination. Circle surrounding terminal neutrophil populations D. Cell trajectories for all lineages and expanded view of granulocytic lineage. E. Linearized granulocyte trajectory showing transcriptional and CITE-seq clusters along with expression of key marker genes, cell cycle phase (phase), and CytoTRACE score. Arrows indicate point of divergence between Asxl1^WT and Asxl1^Δ/Δ.

Figure 2.. Asxl1 Deletion Leads to Hyperactivation of a Myc Transcriptional Signature in Granulocyte Progenitors.

A. Schematic of CausalPath analysis which utilizes literature derived gene regulatory networks and gene correlations from the query dataset to predict causal relationships. B. Number of network edges as a measure transcriptional network complexity between Asxl1^WT and Asxl1^Δ/Δ mice. C. Differential MYC network activity in Asxl1^Δ/Δ mice relative to wild type controls along the granulocytic lineage 6 months post induction of recombination. Network with a reduced FDR threshold (<0.025) shown to reduce complexity for the purposes of visualization. D. Differential network activation in granulocyte progenitor population 6 months post induction of recombination. E. Feature plots of Myc expression in Asxl1^WT and Asxl1^Δ/Δ mice. Note the increased density and intensity of blue MYC expressing cells in Asxl1^Δ/Δ mice. F. Myc expression along granulocyte trajectory. G. Myc expression in terminal granulocyte progenitors (Pseudotime 15–20).

Figure 3.. ASXL1 Deletion Does Not Substantially Impact the Landscape of Covalent Histone Modifications in Developing Myeloid Cells.

A. Bone marrow from Asxl1^WT and Asxl1^Δ/Δ mice was harvested 6 months after induction of recombination via poly I:C administration, maturing granulocyte lineage cells isolated by FACS, and subjected to CUT&Tag for H3K27me3. Spearman Correlation of Genome-wide H3K27me3 signal in indicated bone marrow sub-populations. B. Representative tracks of H3K27me3 signal in indicated bone marrow sub-populations. C. Mean normalized read density at H3K27me3 peaks in indicated bone marrow sub populations. D. RPKM values for reads in peaks for indicated histone marks in CD133 purified granulocytic progenitors. E. Mean normalized read density for indicated histone marks at Myc target genes as measured by CUT&Tag in CD133-granulocytic progenitors from CausalPath analysis in Figure 2.

Figure 4.. ASXL1 Deletion Leads to an Increase in RNAPII Pause-Release.

A. Spearman correlation of histone mark or RNAPII CUT&Tag in CD133 positive granulocyte progenitors from Asxl1^WT mice (n=2–4/group) and published ASXL1 ChIP-seq. B. Schematic depicting the calculation of RNAPII pause-index. C. Cumulative distribution of RNAPII pause-indices at genes bound by Myc and RNAPII in Asxl1^WT and Asxl1^Δ/Δ mice (n=4/group). D. Cumulative distribution of RNAPII pause-indices at genes bound by RNAPII and lacking a Myc peak in Asxl1^WT and Asxl1^Δ/Δ mice (n=4/group). Median pause index difference between Asxl1^WT and Asxl1^Δ/Δ conditions. Statistical significance between RNAPII pause-index distributions in Asxl1^WT and Asxl1^Δ/Δ conditions evaluated using a KS-test. E. Mean signal at all genes bound by RNAPII. Representative RNAPII tracks from genes with differential RNAPII pausing in from Asxl1^WT and Asxl1^Δ/Δ mice.