Integration of Cut&Run, atac-seq and RNA-seq analyses defines the impact of germline ETV6 mutations on chromatin binding and gene expression in megakaryocytes Free

Germline mutations in ETV6 cause thrombocytopenia and predisposition to malignancies. ETV6 (ETS Variant 6) is a transcriptional repressor essential for embryonic development and bone marrow hematopoiesis. Several variants have been identified in ETV6 and while mutations in the ETS DNA-binding domain of ETV6 impair direct DNA binding, the P214L mutation in the central domain leads to aberrant cellular localization of ETV6, decreasing transcriptional repression, and impairing megakaryocyte (MK) maturation. Despite its critical role in hematopoiesis, very few direct transcriptional targets of ETV6 have been identified in MK development. We sought to define ETV6's genome-wide binding sites and the impact of ETV6 P214L variant in differentiated MK.

Using CRISPR/Cas9, we generated ETV6 knockout (KO) and P214L knock-in variants in human CD34⁺ hematopoietic stem cells, derived from cord blood, and then differentiated into megakaryocytes over 14 days. We performed Cleavage Under Target and Release Under Nuclease (CUT&RUN) to map ETV6 binding, assay for transposase-accessible chromatin sequencing (ATAC-seq) to assess chromatin accessibility, and bulk RNA-seq to profile transcriptional changes.

ATAC-seq profiling demonstrated that the overall chromatin accessibility remained unchanged between controls and ETV6 KO cells, as well as between silent control and P214L mutants, indicating that ETV6 loss or mutation affects transcriptional regulation without altering global chromatin accessibility.

CUT&RUN analysis revealed ETV6 binding activity in both wild-type and silent control (CRISPR/Cas9) megakaryocytes. In wild-type cells, we detected 1,757 peaks in replicate 1 and 3,570 peaks in replicate 2, with 974 target genes shared between both replicates. Similarly, silent control megakaryocytes identified 2,853 and 3,104 peaks in replicates 1 and 2, respectively, with 1,592 shared target genes. 722 genes were commonly bound by ETV6 in both WT and silent control megakaryocytes. ETV6 binding was drastically reduced in ETV6 KO cells (181 peaks in Rep 1 and 67 peaks in Rep 2) and in P214L mutants (326 peaks in Rep1 and 266 peaks in Rep 2). Over 60% of ETV6 peaks localized to promoters in controls, whereas ETV6 peaks in KO and mutants were largely in non-promoter regions. Motif analysis (MEME suite) of ETV6 binding sites revealed that ETS motifs were most significantly enriched in WT (E-value= 2.0e-424) and silent control sample (E-value= 4.2e-180), with ETV6 as the predominant ETS transcription factor. This ETS motif enrichment was lost in both ETV6 KO and P214L mutant cells. We also performed immunofluorescence and cell spreading assays in ETV6 knockout and wild-type megakaryocytes to assess their ability to form proplatelets. Both experiments showed that loss of ETV6 disrupts cytoskeletal organization in ETV6 KO megakaryocytes, preventing proper proplatelet formation indicating a critical role for ETV6 in late-stage megakaryocyte maturation.

RNA-seq analysis revealed that among the common 722 target genes, transcripts for 44 of the genes were altered (41 upregulated and 3 downregulated) in the ETV6 KO; and 21 were altered in the P214L variant (18 upregulated and 3 down-regulated) with 18 of these up-regulated gene in P214L variant common among ETV6 KO as well. Pathway analysis identified significant enrichment of the estrogen signaling pathway (p = 0.0008), interleukin-2 signaling pathway (p = 0.0003), and MAP kinase pathway (p = 0.000008). A subset of genes, including ESR2, SHC1, SYK, and GNAI2, were commonly represented across these pathways, suggesting that disruption of ETV6 function may converge on these signaling networks to impair megakaryocyte development. We compared our data with previously published datasets on ETV6 mutations and, while we observed several overlapping genes, we also identified novel targets in our dataset including ESR2. When we analyzed its expression, ESR2 levels were ~7-fold higher in ETV6 KO megakaryocytes compared to wild type and ~5-fold higher in the P214L variant compared to the silent control. Prior studies have shown that estrogen supports megakaryocyte growth and maturation by activating GATA1 through ERβ, which in turn triggers STAT1 signaling. Our findings suggest that altered ESR2 expression may influence signaling pathway, offering new insights into how estrogen signaling might impact platelet production and how its disruption could impair megakaryocyte development.