HLTF Regulates Erythroid Differentiation through Transcription Factors and Chromatin Landscape Modulation

Erythroid development is a critical process in which multipotent hematopoietic stem cells differentiate into mature red blood cells. This process is tightly regulated by transcription factors (TF), with GATA1 playing a central role as a master TF. While previous studies have demonstrated that GATA1 expression changes dynamically during erythroid development, the precise mechanisms regulating GATA1 in erythroid cells remain incompletely understood. To identify regulators of GATA1 in erythroid differentiation, we conducted DNA pull-down experiments coupled with mass spectrometry at different time points (Day 7, Day 9, and Day 11). Our findings revealed the presence of several previously reported TFs, including GATA1, STAT1, BRG1, and HEMGN. Notably, comparative analysis of the binding profiles across time points revealed that HLTF exhibited high binding affinity to the GATA1 promoter, with a gradually increasing trend over time. We further conducted a comprehensive analysis of HLTF expression in the bone marrow microenvironment. Integrated the bulk RNA-seq, scRNA-seq and ATAC-seq data analyses revealed HLTF was predominantly expressed in erythroblasts. These findings suggest a potentially crucial significant role for HLTF in regulating GATA1 expression and erythroid differentiation.

Subsequently, we employed ChIP-qPCR experiments confirmed HLTF binding to the GATA1 promoter. Dual luciferase reporter and DNA pull-down assays further demonstrated HLTF's specific binding to the wild-type GATA1 promoter, but not to a mutant version. CRISPR-Cas9-mediated HLTF knockout (KO-HLTF) resulted in significant reductions in GATA1 mRNA and protein levels, accompanied by inhibited cell growth, blocked erythroid differentiation, and increased apoptosis in CD34+ cells. Importantly, GATA1 overexpression in HLTF knockout cells rescued the impaired erythroid differentiation.

To further explore the regulatory mechanism of HLTF, we performed RNA-seq analysis. We identified 952 significantly dysregulated genes, with downregulation of erythroid-related genes such as GATA1, TFRC, EPB41, and SLC2A1. Enrichment analysis confirmed downregulation of the erythroid differentiation pathway. Given these transcriptomic changes, we investigated potential alterations in global chromatin accessibility using ATAC-seq. Results showed that HLTF knockdown greatly reduced chromatin accessibility, particularly in promoter regions of target genes like GATA1 and SLC2A1. Intriguingly, footprinting analysis revealed that HLTF loss led to diminished GATA1 binding to its target genes.

Next, we performed CUT&Tag to further characterize the direct targets of HLTF. This revealed significant HLTF binding to gene promoter regions, with direct binding to targets such as GATA1 and SLC2A1. Motif analysis showed significant enrichment of GATA1 and KLF1 motifs in HLTF-bound regions. Integrating these findings with footprinting results, we hypothesized that HLTF may form a transcriptional complex with GATA1. Indeed, CUT&Tag data indicated extensive co-binding of HLTF and GATA1 on known target genes. To corroborate this hypothesis, we performed immunofluorescence experiments, which demonstrated nuclear co-localization of HLTF and GATA1. Furthermore, their physical interaction was confirmed through immunoprecipitation assays.

In conclusion, our findings reveal a novel HLTF-mediated erythropoiesis mechanism, which may open a new window for the treatment of erythroid-related diseases.