Blood stem cell PU.1 upregulation is a consequence of differentiation without fast autoregulation

Abstract

Transcription factors (TFs) regulate cell fates, and their expression must be tightly regulated. Autoregulation is assumed to regulate many TFs’ own expression to control cell fates. Here, we manipulate and quantify the (auto)regulation of PU.1, a TF controlling hematopoietic stem and progenitor cells (HSPCs), and correlate it to their future fates. We generate transgenic mice allowing both inducible activation of PU.1 and noninvasive quantification of endogenous PU.1 protein expression. The quantified HSPC PU.1 dynamics show that PU.1 up-regulation occurs as a consequence of hematopoietic differentiation independently of direct fast autoregulation. In contrast, inflammatory signaling induces fast PU.1 up-regulation, which does not require PU.1 expression or its binding to its own autoregulatory enhancer. However, the increased PU.1 levels induced by inflammatory signaling cannot be sustained via autoregulation after removal of the signaling stimulus. We conclude that PU.1 overexpression induces HSC differentiation before PU.1 up-regulation, only later generating cell types with intrinsically higher PU.1.

PU.1 Expression Defines Distinct Functional Activities in the Phenotypic HSC Compartment of a Murine Inflammatory Stress Model

“…To test our hypothesis, we treated PU.1-EYFP reporter mice with IL-1β or PBS (hereafter referred to as -IL-1β) daily for 20 days to simulate a chronic inflammatory state. The PU.1-EYFP reporter mouse line harbors EYFP fused to the C-terminus of PU.1 and is knocked into the endogenous PU.1 locus, allowing for prospective isolation of cells based on PU.1 protein levels [ 3 , 7 , 8 , 9 , 15 , 17 ]. Following IL-1β treatment, we verified increased numbers of neutrophils and platelets in the peripheral blood by CBC, and as we previously reported, we noted increased overall numbers of phenotypic HSC-enriched SLAM cells in the BM ( Figure S1A,B ) [ 6 , 7 ].…”

“…First, increased PU.1 expression following inflammatory stimulation is dynamic. We and others have shown that elevated PU.1 expression is reversible and is not sustained in HSC following the removal of IL-1β or other cytokines [ 3 , 9 ]. Second, the ‘myeloid bias’ phenotype in the setting of transplantation appears closely linked to high levels of HSC activity, and the original characterizations of ‘myeloid-biased’ HSC interpret the elevated myeloid lineage output to be at least in part related to the engraftment of highly potent primitive HSC [ 24 , 25 ].…”

“…Previously, we and others have shown that IL-1β activates expression of the transcription factor PU.1, both at the RNA and protein levels, via direct IL-1R signaling in HSC [ 3 , 5 , 6 , 7 , 8 , 9 ]. PU.1/Spi1 is an ETS-family transcription factor that serves as a ‘master regulator’ of blood lineage output [ 10 ].…”

Open-source personal pipetting robots with live-cell incubation and microscopy compatibility

“…Immunostaining of VENUS fluorescent protein in myeloid progenitors was performed according to protocols as described 31 , 34 , 39 , 55 . Briefly, different subsets of myeloid progenitors based on VENUS expression were sorted by FACS (BD ARIA III), directly seeded on (10 µg/mL) anti CD43-biotin antibody-coated plastic-bottom 96-well plates (Greiner Bio-one), stored for 30 min at 4 °C and fixed with 4% paraformaldehyde (Sigma Aldrich) for 10 min at RT.…”

Patterning and growth are coordinated early in the cell cycle

“…Quantitative time-lapse imaging of transcription factor dynamics has provided key insights into gene regulatory network function in single cell organisms and mammalian cell lines [33–36]. For example, through observation of PU.1 and GATA1 dynamics in mouse cell lines, Hoppe et al [37] challenged an existing model that early myeloid lineage choice is determined by random fluctuations of these mutually antagonistic transcription factors.…”