Epigenetic Profiling of Single Cells by In Situ Labeling for Studying Hematopoiesis Proper regulation of genome activity and architecture is critical for development, growth, and function of a multicellular organism.1,2 Much of the regulation is believed to involve “epigenetic” modifications on DNA- associated histone proteins,3–5 known as histone marks, which guide the compaction of nucleosomes into higher order chromatin states that in turn regulate the expression of transcription factors that govern cell differentiation and commitment.6,7 Because of limitations in assays used to measure these epigenetic states in single cells, the roles of these histone marks in controlling chromatin structure and gene expression remain poorly understood and vigorously debated. Thus, new methods are required for epigenetic profiling of cells. In Aim 1, I propose to develop a new assay to interrogate the epigenetic state of single mammalian cells with the capability to quantify the abundance of histone marks that inhabit multiple (≤20) distinct genomic loci in situ. In Aim 2, I propose to apply this epigenetic profile assay to study hematopoietic stem cell (HSC) differentiation in order to demonstrate the value of the developed epigenetic profiling method. Hematopoiesis is the formation of blood cells including cells of the immune system which are critical for all animals including humans. In this process, HSCs relinquish pluripotency to commit to unique fates and form blood cells by asynchronously expressing a variety of transcription factors.3,18 Dysregulation at the epigenetic level of hematopoiesis is known to cause blood diseases such as acute myeloid leukemia.19 Because of this, methods capable of detecting the differentiated and epigenetic state of mammalian cells are important for the study of developing HSCs and the progression of leukemic diseases. Using these epigenetic profiles together with transcriptomic (mRNA) profiles that are already established,3 I will determine unique identities for each cell according to their epigenetic marks and differentiated state. I will also identify which epigenetic marks and loci are important for cell fate decision making. The ability to probe epigenetic states by measuring histone marks at many distinct genomic loci (e.g., for transcription factors and their enhancers, etc.) at the single-cell level will be broadly enabling to researchers across a wide range of disciplines. In addition, the method will provide crucial information on the epigenetic modifications present in HSCs. Hypothesis: The epigenetic profile of a cell will reflect its differentiated state and will reveal strong associations between histone marks and expression levels. Aim 1: Develop a multiplexed assay to quantify the levels of five different histone marks present at multiple (≤20) genomic loci. Aim 2: Apply epigenetic profiling assay to the study of hematopoietic stem cell (HSC) differentiation.