Pursuing the Spatial Organization of Lineage-Determining Genes of Hematopoiesis

Abstract

Hematopoiesis is the continuous, highly regulated process, during which all mature blood cell types are generated from a small pool of hematopoietic stem cells (HSCs). The commitment of HSCs and multipotent progenitors to specific lineages, namely, myeloid, erythroid, and lymphoid, is governed by a network of lineage-determining transcription factors, including PU.1 (encoded by Spi1), GATA-1 (encoded by Gata1), and TCF1 (encoded by Tcf7). The activities of these transcription factors and their roles in hematopoietic cell differentiation are closely related to the epigenetic states of the chromatin at their gene loci. While sequencing methods such as ChIP-seq and CUT&RUN have established the genome-wide distribution of histone modifications in blood cells, these approaches are inherently limited by their inability to resolve cell-to-cell variation or to provide spatial information at the single-cell level. This thesis applies a recently developed method, Single Cell Evaluation of Post-TRanslational Epigenetic Encoding (SCEPTRE), to murine hematopoietic cells. SCEPTRE integrates Expansion Microscopy (ExM), DNA fluorescence in situ hybridization (FISH), and immunofluorescence to simultaneously visualize histone modifications and specific gene loci in intact single cells. The first goal of this thesis was to validate ExM and DNA FISH as robust and reproducible methods for studying murine bone marrow derived macrophages (BMDMs), as SCEPTRE had not previously been applied to this primary cell type. The second goal was to begin the characterization of the spatial distribution of histone modifications and chromatin structures relative to the three lineage-defining gene loci: Spi1, Gata1, and Tcf7.