Chromatin Modifier Hmga1 Maintains Hematopoietic Stem Cell Integrity in Stress Conditions

Hematopoietic stem cells (HSCs) respond to various stresses, such as inflammation, and expand hematopoietic stem and progenitor cells (HSPCs) to produce mature blood cells; however, the mechanisms by which HSCs maintain hematopoiesis in differential responses to homeostatic and stress conditions have yet to be elucidated. High-mobility group AT-hook 1 (Hmga1), a chromatin modifier, opens and closes the chromatin and modulates the transcription. Hmga1 is highly expressed in somatic stem cells including HSC. Hmga1 is shown to promote the development of myeloid malignancies via driving the proliferation (Li, et al. Blood 2022); however, it is unclear how Hmga1 regulates normal HSC and the hematopoiesis in homeostatic and stress conditions.

In this study, we assessed the function of Hmga1 in HSCs by generating a new Hmga1/Hmga1a knock-out (KO) mouse in responses to bone marrow transplantation and treatment of 5-FU. In the homeostatic condition, primary Hmga1a KO mice did not show changes in blood cell counts in the peripheral blood (PB) and HSC counts in the bone marrow (BM), compared to wild-type (WT) mice, in one year observation period. A previous report demonstrated that Hmga1 KO mice developed diabetes (Foti, et al. Nature Med 2005); however, our Hmga1 KO male and female mice both did not increase level of blood glucose in the PB plasma, compared to WT mice. In order to understand the function of Hmga1 in hematopoiesis in stress conditions, we performed a competitive serial transplantation by using Hmga1 KO and WT BM cells. We found that WT cells maintained the repopulation capacity at the secondary transplantation, while Hmga1 KO cells markedly decreased chimerism in both myeloid and lymphoid cells in the PB and those in HSPCs in the BM. While WT HSCs mostly maintained the quiescent status, Hmga1 KO HSCs drove the cell cycle, showing 50% cells at G1, S, and G2/M stages. In addition, Hmga1 KO mice delayed recovery of blood cell counts in the PB and HSCs after injection of 5-FU and showed a lower survival rate than WT mice. Therefore, the Hmga1 gene was critical for the maintenance of adult HSC and regeneration of blood cells in stress conditions. By performing RNA-sequencing of HSCs and granulocyte/monocyte progenitor (GMP) cells 4-5 months post the transplantation, we found that Hmga1 KO HSC showed positive enrichments in cell cycle regulator genes and negative enrichments in stem cell signature and polycomb-repressive complex 2 (PRC2)-target genes, compared to WT HSC. By using our dataset of the transcriptome of Hmga2 KO HSC (Kubota, et al. EMBO J 2024), Hmga1 KO HSCs showed changes in the transcription of genes different from those in Hmga2 KO HSCs, relative to WT HSCs, suggesting that Hmga1 and Hmga2 bound to distinct target genes in HSC. Because Hmga1 can directly bind to DNA via its AT-hook domains, we performed Hmga1-ChIP-sequencing in c-Kit+ WT BM cells and found that Hmga1 was mostly bound to intergenic and intron regions. Among 11787 Hmga1-binding peaks in WT cells, DNA motif enrichment analysis revealed GC-rich binding sequences of CTCF and CTCFL, chromatin structure regulators, and AT-rich sequences, which might be bound by homeobox genes. By using annotated genes proximally bound by Hmga1, we found that Hmga1 KO HSCs formed a cluster of genes, of which expression was significantly repressed, compared to WT HSCs. Gene ontology analysis revealed that genes in the cluster repressed in Hmga1 KO HSCs were involved in stem cell genes, such as Hlf, Nr4a2, and Ndn, and canonical PRC2-target genes. In contrast, Hmga1 KO GMP cells showed similar expression levels of Hmga1-binding genes to those in WT GMP cells. Therefore, Hmga1 appeared to directly activate the transcription of genes to maintain the integrity of HSC in response to stresses. Since Hmga1 has been shown to modulate chromatin structures, we performed ATAC-sequencing analyses in HSCs and found that Hmga1 KO HSCs showed 2952 open chromatins and 56986 closed chromatins, compared to WT HSCs. DNA motif enrichment analysis revealed enrichments of binding sequences of CTCF/CTCFL in closed chromatin regions, suggesting that the Hmga1 gene was required to open chromatins including binding sites of CTCF/CTCFL in HSC.

In conclusion, we demonstrated that Hmga1 was required to maintain the integrity of HSC in stress conditions. Hmga1 ensured chromatin accessibility including the CTCF-binding sites and activated the transcription of stem cell genes.