ORCID

http://orcid.org/0000-0001-8886-2577

Date of Award

Summer 8-15-2019

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Immunology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Dendritic cells (DCs) comprise an important immune lineage that plays a critical role in initiating and sustaining the proper immune response. They can be divided into two distinct branches, classical/conventional DCs (cDCs) or plasmacytoid DCs (pDCs). cDCs can further be classified as cDC1 or cDC2. Each DC subset exerts unique functions in vivo and are necessary for a complete immune response. The precise transcriptional programs underlying DC specification and commitment remain unclear. cDC1, cDC2, and pDC all arise from the common DC progenitor (CDP) in the bone marrow. How the CDP gives rise to all three DC subsets in an important outstanding question in the field. Several transcription factors have been shown to be important for the development of certain subsets. The transcription factors Irf8, Batf3, Id2, Nfil3, and Bcl6 are required for the cDC1 lineage, while the transcription factors Klf4 and Notch2 are necessary for specific cDC2 subsets. pDCs rely on the transcription factors Tcf4 and Zeb2 for their development. Despite knowing that these factors influence DC development, the interactions between these factors and their timing of action are unclear. Recently, understanding of how the CDP specifies benefited from identifying cDC progenitors (pre-cDCs) that were found to include clonogenic populations separately committed to cDC1 or cDC2 lineages. Two Irf8 enhancers were found to affect cDC1 development in different stages: an E-protein dependent enhancer located 41 kilobases downstream of the transcription start site of IRF8 (+41 kb Irf8 enhancer) is required for the specification of the pre-cDC1, and a BATF-dependent +32 kb Irf8 enhancer required for the maturation to the cDC1. To understand the switch in Irf8 enhancer usage during cDC1 specification, we used single-cell RNA-sequencing of the CDP and identified a cluster of cells that expressed transcription factors that influence cDC1 development, such as Nfil3, Id2, and Zeb2. We then performed genetic epistasis to determine the functional hierarchy of transcription factors involved in cDC1 specification. We organized a transcriptional circuit that explains the switch in Irf8 expression from being Batf3-independent to being Batf3-dependent. The CDP originates in a Zeb2hi and Id2lo state in which Irf8 expression is maintained by the +41 kb Irf8 enhancer. Single-cell RNA-sequencing identified a fraction of the CDP that exclusively possesses cDC1 fate potential. This fraction’s development arises when Nfil3 induces a transition into a Zeb2lo and Id2hi state. A circuit of mutual Zeb2-Id2 repression serves to stabilize states before and after this transition. Id2 expression in the specified pre-cDC1 inhibits E proteins, blocking activity of the +41 kb Irf8 enhancer, and thereby imposing a new requirement for Batf3 for maintaining Irf8 expression via the +32 kb Irf8 enhancer.

Language

English (en)

Chair and Committee

Kenneth M. Murphy Marco Colonna

Committee Members

Takeshi Egawa, Brian T. Edelson, Chyi-Song Hsieh,

Comments

Permanent URL: https://doi.org/10.7936/bjpt-q331

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