HES6: an emerging player in human hematopoiesis

In this issue of Haematologica, Tamara De Vos et al.¹ describe their study demonstrating that HES6, a member of the Hairy/Enhancer of Split (HES) family,² is required for human hematopoietic precursor cell repopulation and differentiation. Depletion of HES6 in human hematopoietic stem and progenitor cells (HSPC) leads to a remarkable reduction in multilineage differentiation, compromises in vitro hematopoietic progenitor activity and impairs in vivo reconstitution capacity. Transcription factors involved in the Notch signaling pathway play pivotal roles in regulating normal hematopoiesis, and mutations of these factors are commonly associated with hematologic malignancies.^2-5 Notch activation induces expression of downstream target genes, including members of the HES family, which comprises seven members in humans (HES1-7).^2-5 Among these, HES6 has been less explored. The study by De Vos et al. adds important information by unveiling a novel role for HES6 in regulating human HSPC function.

By analyzing published bulk RNA-sequencing and single-cell RNA-sequencing datasets, De Vos et al. found that HES6 is expressed during erythroid/megakaryocyte and plasmacytoid dendritic cell (pDC) development. HES6 is also expressed in multipotent precursors, as well as certain stages of T- and B-cell development following pre-B-cell receptor or pre-T-cell receptor engagement. Knockdown of HES6 in cord blood-derived hematopoietic precursors inhibited the development of T-cell-specified precursors and CD4⁺CD8⁺ double-positive cells, limited the in vitro development of B cells beyond the stage that proliferating large pre-B cells receive pre-B-cell receptor signaling following heavy chain rearrangement, and reduced the frequency and absolute numbers of pDC (CD45RA⁺CD123⁺) in ex vivo differentiation assays, suggesting an important role for HES6 in regulating multilineage differentiation of human HSPC.

A colony-forming unit assay revealed that HES6 knockdown decreased the numbers of myeloid and erythroid colonies. The authors further showed that both the frequencies and absolute cell numbers of megakaryocytes (CD41⁺) and erythroblasts (CD71⁺CD235a^high) differentiated from the HES6 short hairpin (sh)RNA-transduced cells were significantly lower compared to those differentiated from the control cells in liquid culture, confirming that HES6 is essential for human erythrocyte and megakaryocyte development. In keeping with these in vitro findings, HES6 shRNA-transduced HSPC exhibited diminished repopulating potential, leading to an in vivo reduction in the reconstitution of all lineages in the transplanted immunodeficient IL2RG^-/- (NSG) recipient mice compared to reconstitution in the mice transplanted with control HSPC.

By using the sorted cells from HES6 shRNA-transduced human HSPC after 4 days of induction in ex vivo liquid culture, the authors found that HES6 knockdown significantly reduced erythroid-associated genes (TFRC, HBB, EPCAM, E2F4), and affected erythroblast-related gene sets. However, HES6-knockdown erythroid cells failed to downregulate GATA2 and ID2 expression, and had no impact on G ATA1 expression. HES6 knockdown also decreased the expression of genes involved in megakaryocyte development (CD36, DLK1) and proliferation (MKI67, HEMGN). Furthermore, HES6 shRNA-transduced cells demonstrated a remarkable reduction in pDC-associated genes (IRF8, TCF4), B-lineage genes (BLNK, IGLL1, IGHM), as well as hematopoietic precursor cell and pDC genes (TCF3, RUNX2).

Consistent with the reduced cellular output and downregulated genes associated with proliferation, RNA-sequencing illuminated changes in cell cycle-related gene sets in HES6 knockdown cells. Specifically, cell proliferation-related genes, such as TFRC, E2F4 and E2F8, were downregulated upon HES6 knockdown in late erythroblasts. By employing an ethynyl-2´-deoxyuridine incorporation assay, the authors observed a marked decrease in the proportion of cells in the proliferative S phase, which was accompanied by a notable increase in cells in the quiescent G1 phase, in HES6 knockdown cells compared to the control shRNA-transduced cells. These findings imply that HES6 is essential for human HSPC proliferation, potentially through the regulation of genes involved in cell cycle progression.-

Figure 1.Schematic overview of HES6 regulation of hematopoiesis. HES6 modulates the expression of genes involved in lineage differentiation and cell cycle progression, thereby tightly controlling the maintenance and lineage commitment of human hematopoietic stem and progenitor cells. HSPC: hematopoietic stem and progenitor cells; pDC: plasmacytoid dendritic cells.

The study by De Vos et al. highlights the novel role of HES6 in orchestrating human HSPC multilineage development. The transcription factors GATA1 and GATA2 functionally overlap in primitive hematopoietic development.⁶ HES6 has previously been shown to be a pivotal transcriptional cofactor of GATA1. Knockdown of HES6 impaired human erythropoiesis by decreasing the expression of GATA1 as well as GATA1 downstream targets.⁷ It is also known that GATA2 to GATA1 switch is critical for erythroid development.⁸ De Vos et al. offer intriguing new insights into the intricate interplay between HES6 and the G ATA family. Notably, HES6 emerges as a crucial player in erythroid development, functioning independently of GATA2. This implies that HES6 might directly govern the expression of genes vital for HSPC differentiation.

This study also raises an important mechanistic question as to how HES6 regulates HSPC proliferation. The observations that HES6 facilitates HSPC maintenance through mediating G1-to-S transition, suggest that HES6 may be involved in the functional exhaustion of human HSPC. Previous studies have demonstrated a strong association between HES6 and the promyelocytic leukemia nuclear body, highlighting its role in modulating the cell cycle through upregulation of the expression of p21,⁹ a negative regulator of the cell cycle.¹⁰ The current RNA-sequencing and chromatin immunoprecipitation-sequencing data suggest direct regulatory roles for HES6 in positively regulating TFRC and MKI67, two important cell proliferation mediators, in a GATA1-independent manner. It will be important to determine the separated role of HES6 in regulating gene expression uncoupled from GATA transcription factor family members at different stages of HSPC development.

The current study by De Vos et al. provides new insight into the role of HES6 in HSPC maintenance (Figure 1). Their findings add another layer of understanding of the HES family members in regulating hematopoiesis, shedding light on potential lineage- and stage-specific functions of this group of transcription factors in the intricate process of human hemostasis and lineage differentiation.

Footnotes

• Received April 18, 2024
• Accepted April 26, 2024

Correspondence

W. Du duw@upmc.edu

Disclosures

No conflicts of interest to disclose.

Funding

WD is supported by a National Institutes of Health grant R01HL151390 and Children’s Leukemia Research Association (CLRA) research grant.