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Abstract

Most mature hematopoietic cell types must be continuously replenished by hematopoietic stem and progenitor cells (HSPCs) to compensate for cell loss. In adult mice and humans, rare and largely quiescent hematopoietic stem cells (HSCs) in the bone marrow infrequently divide or differentiate into rapidly proliferating multipotent progenitors (MPPs). These MPPs then differentiate into progenitors with more restricted hematopoietic fates. In this thesis, I present analyses of single-cell transcriptome and chromatin accessibility data, which suggest that cellcycle progression influences lineage onset in adult murine hematopoietic progenitors in vivo.

By analyzing single-cell RNA sequencing data, I characterized transcriptome profiles of those hematopoietic progenitor cells that appear to transition from the multipotent state to a lineage-restricted state. To this end, I identified transcriptome profiles of progenitors that were classified as MPPs based on fluorescence-activated cell sorting (FACS), but already exhibited weak transcription of genes that are specifically upregulated in more differentiated, lineagerestricted progenitors. Specifically, I identified three MPP-subsets that are myeloid-biased, erythroid/megakaryocyte-biased, or lymphoid-biased. I found that myeloid-biased MPPs—in contrast to unbiased and other lineage-biased MPP subsets—are mostly in the S or G2/M phase of the cell-cycle. The robustness of this association between myeloid bias and proliferation was further supported by the analysis of single-cell RNA data from mice in which USP22, a regulator of emergency myelopoiesis, was depleted in hematopoietic cells. Loss of USP22 caused an increased fraction of myeloid-biased cells within MPPs and simultaneously increased proliferation and mitochondrial respiration in all MPP-subsets.

To further examine the association of progenitor proliferation and differentiation, I analyzed joint single-cell RNA and chromatin accessibility data. While transcriptome profiles allowed me to assign cells to cell-cycle phases, I analyzed chromatin accessibility to detect early lineage-specification events. By comparing chromatin accessibility between progenitors of different cell-cycle phases, I found that, during S phase, hematopoietic progenitors open genomic regions associated with myeloid differentiation. This opening occurs prior to the transcription of myeloid genes. Experimental acceleration of progenitor proliferation and myeloid differentiation, by induction of polymicrobial sepsis, enhanced the opening of myeloid regulatory regions in transcriptionally uncommitted progenitors during S phase. In both steadystate and perturbed hematopoiesis, S phase-coupled opening of myeloid regulatory regions primarily occurred in progenitors with reduced accessibility at stemness-associated regions. Together, these findings led me to conclude that progression through S phase induces or facilitates myeloid differentiation of progenitors – especially if proliferation is accelerated or if stem cell programs are no longer sustained.