A lncRNA identifies an IRF8 enhancer element in negative feedback control of dendritic cell differentiation

Xu, Huaming; Zenke, Martin (Thesis advisor); Zimmer-Bensch, Geraldine Marion (Thesis advisor)

Aachen : RWTH Aachen University (2023)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2023

Abstract

Hematopoietic stem cells (HSC) give rise to all blood cells, including antigen-presenting dendritic cells (DC). DC are highly specialized immune cells with a pivotal function in immunity and tolerance that link innate and adaptive immune responses. DC develop from HSC via successive steps of lineage commitment and differentiation. More specifically, HSC develop into multipotent progenitors (MPP) that are committed to DC restricted common DC progenitors (CDP) in the bone marrow (BM), and differentiate into classic DC type 1 and type 2 (cDC1 and cDC2, respectively) and plasmacytoid DC (pDC). Interferon regulatory factor 8 (IRF8) is a hematopoietic transcription factor (TF) positioned at the center of the regulatory gene network and is subject of complex epigenetic regulation in DC differentiation. IRF8 expression starts at the CDP stage and is high in pDC and cDC1. However, how IRF8 expression is regulated and eventually limited, and the epigenetic mechanisms involved is largely unknown. In the present study, we used an integrated approach with ATAC-seq, ChIP-seq, chromatin conformation capture (3C), and RNA-seq to track gene expression and the epigenetic dynamics of IRF8 during DC differentiation. The IRF8 promoter was found to interact with its flanking enhancers already at the CDP stage, and then more frequent interactions with its upstream and downstream sequences were found in pDC and cDC1, respectively. Interestingly, we identified a novel long non-coding RNA (lncRNA) transcribed from the +32 kb enhancer downstream of IRF8 transcription start site (TSS) and expressed specifically in pDC, referred to as lncIRF8. The +32 kb enhancer locus also shows differential epigenetic signatures in pDC versus cDC1, indicating a potential role of lncIRF8 for DC subsets specification. The limited lifespan of BM cells in vitro makes genome editing or generation of stable transgenic cells difficult. To this end, we described conditionally immortalized HoxB8 multipotent hematopoietic progenitors (HoxB8 MPP) cultured with a four-cytokine cocktail of stem cell factor (SCF), Flt3 ligand (Flt3L), insulin-like growth factor-1 (IGF-1), and IL-6/soluble IL-6 receptor fusion protein (hyper-IL-6), which upon differentiation faithfully recapitulate DC development and allow genome editing by CRISPR/Cas9. We found that the lncIRF8 promoter but not lncIRF8 itself is crucial for pDC and cDC1 development, as demonstrated by lncIRF8-promoter knockout (KO) by CRISPR/Cas9, lncIRF8 overexpression and rescue experiments. Thus, lncIRF8 acts as an indicator of IRF8 +32 kb enhancer activity. CRISPR activation and CRISPR interference of lncIRF8 and IRF8 promoters by dCas9-VP64 and dCas9-KARB, respectively, revealed that IRF8 expression is activated by the +32 kb enhancer and limited through the same enhancer element by a negative feedback loop during DC differentiation. CRISPR activation of lncIRF8 promoter dramatically promotes cDC1 development, while CRISPR interference of lncIRF8 and IRF8 promoters compromises both pDC and cDC1 development. Taken together, we proposed a model where IRF8 expression during DC development is first activated by its flanking enhancers, including the +32 kb enhancer, and then second limited by feedback inhibition through the lncIRF8 promoter element in the +32 kb enhancer. Our work reveals a previously unrecognized negative feedback loop of IRF8 that orchestrates its own expression and thus controls DC differentiation.

Institutions

• Department of Biology [160000]
• Neuroepigenetics Teaching and Research Unit [164620]
• [531040-2]