Transcription Factor Repertoire of Homeostatic Eosinophilopoiesis

Abstract

The production of mature eosinophils is a tightly orchestrated process with the aim to sustain normal eosinophil levels in tissues while also maintaining low numbers of these complex and sensitive cells in the blood. To identify regulators of homeostatic eosinophilopoiesis in mice, we took a global approach to identify genome-wide transcriptome and epigenome changes that occur during homeostasis at critical developmental stages, including eosinophil-lineage commitment and lineage maturation. Our analyses revealed a markedly greater number of transcriptome alterations associated with eosinophil maturation (1199 genes) than with eosinophil-lineage commitment (490 genes), highlighting the greater transcriptional investment necessary for differentiation. Eosinophil progenitors (EoPs) were noted to express high levels of granule proteins and contain granules with an ultrastructure distinct from that of mature resting eosinophils. Our analyses also delineated a 976-gene eosinophil-lineage transcriptome that included a repertoire of 56 transcription factors, many of which have never previously been associated with eosinophils. EoPs and eosinophils, but not granulocyte-monocyte progenitors (GMPs) or neutrophils, expressed Helios and Aiolos, members of the Ikaros family of transcription factors, which regulate gene expression via modulation of chromatin structure and DNA accessibility. Epigenetic studies revealed a distinct distribution of active chromatin marks between genes induced with lineage commitment and genes induced with cell maturation during eosinophil development. In addition, Aiolos and Helios binding sites were significantly enriched in genes expressed by EoPs and eosinophils with active chromatin, highlighting a potential novel role for Helios and Aiolos in regulating gene expression during eosinophil development.

IL-33 Precedes IL-5 in Regulating Eosinophil Commitment and Is Required for Eosinophil Homeostasis

“…While the difference in granularity we see by cytology is consistent with the differences in side scatter we observe in our flow cytometry analysis, it contrasts to the previously described eosinophil precursors, which have been reported to possess granule proteins (29). Moreover, IL-5Rα can also be expressed on neutrophils under certain conditions (30).…”

“…Interestingly, they expressed mRNA for eosinophil granule proteins and eosinophil-associated transcription factors, but lacked eosin-positive granules. Because of this and the clearly defined nuclear morphology of an eosinophil, we postulate that these cells represent an alternative precursor state than those defined from long-term IL-5 culture approaches (29). Initially, when these cultures were switched into IL-5, many of these expanded cells (>50%) were dying by apoptosis at day 7, even when a 10-fold higher concentration of IL-5 was used (data not shown).…”

The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan

“…As such, we investigated whether lncRNAs control the function or lifespan of short-lived myeloid cells in response to extracellular cues. We first analyzed multiple RNA-seq datasets for lncRNAs preferentially expressed by mature short-lived myeloid cells 5,6 . We identified an uncharacterized lncRNA ( Gm14005 ) that we termed Morrbid (MyelOid Rna Regulator of Bim-Induced Death).…”

Chromatin Preparation from Murine Eosinophils for Genome-Wide Analyses

“…We performed chromatin immunoprecipitation coupled with massively parallel sequencing (ChlP-seq) to identify genetic regulatory elements that are active during homeostatic eosinophil production in the bone marrow [10]. Our studies provided the first demonstration of stage-specific epigenomic signatures in the eosinophil lineage [10].…”

“…We performed chromatin immunoprecipitation coupled with massively parallel sequencing (ChlP-seq) to identify genetic regulatory elements that are active during homeostatic eosinophil production in the bone marrow [10]. Our studies provided the first demonstration of stage-specific epigenomic signatures in the eosinophil lineage [10]. Previously, molecular assays involving chromatin required large numbers of purified cells (often as many as 10–50 × 106), which made studies focused on transcriptional regulation in rare cell populations, like eosinophils, very challenging and expensive to perform.…”