Characterization of a Colony Stimulating Factor Receptor (CSF1R) Upstream Regulatory Element in Haematopoiesis

Colony stimulating factor 1 receptor (CSF1R) signalling regulates the development and differentiation of myelomonocytic cells. In the embryo, Csf1r mRNA is expressed by placental trophoblasts, but otherwise expression is restricted to myeloid lineages. During myeloid differentiation, CSF1R is first expressed in multipotent progenitors (MPP), particularly MPP3. CSF1R expression provides a marker for cells of the mononuclear phagocyte system. We have identified multiple regulatory elements within the mouse Csf1r locus that contribute to lineage-specific expression. We previously demonstrated that the deletion of a conserved intronic enhancer, the fms-intronic regulatory element, led to selective loss of tissue-resident macrophages (TRM), in a transgenic mouse model. Here we focus on a second 150 bp conserved element lying immediately 5’ of the first coding exon which we have called CSF1R Upstream Element A (CUREA). Previous studies using multicopy transgenic reporters indicated that CUREA was required for expression in TRM.

To investigate the function of CUREA in myelopoiesis, we generated CUREA^-/- mice using ribonucleoprotein complex (CRISPR-Cas9 and synthetic SgRNAs) injected into zygotes from the Csf1r-T2A-FusionRed reporter. Homozygous mutant mice were healthy, fertile, and did not exhibit any of the deleterious phenotypes found in Csf1r knockout mice. CUREA^-/- mice showed an accumulation of long-term haematopoietic stem cells (HSCs) (13.3% vs. 10.4% in controls, p=0.0046) and short-term HSCs (15.5% vs. 18.2% in controls, p=0.013), associated with a concomitant decrease in the MPP3 population (57.6% vs. 62.9% in controls, p=0.0026). qPCR analysis showed decreased Csf1r transcript levels in bone marrow Lin-SCA+C-KIT+ and MPP3 cells in CUREA mutants (Δ52.3% vs. control, p=0.0058 and Δ23.3% vs. control, p=0.011, respectively) and splenocytes (Δ14.7% vs. control, p<0.0001) from mutant mice. Despite the reduction in mRNA levels, no significant changes in CSF1R expression between CUREA^+/+ and CUREA^-/- were detected in HSPCs or MNCs by flow cytometry. Accordingly, TRM density in the brain, lung, kidney, heart, spleen, liver, and fat did not differ between CUREA^+/+ and CUREA^-/- mice. In vitro, responses to CSF1, assessed by proliferation, differentiation, and CSF1R protein expression, were not altered in bone marrow-derived macrophages. We observed no differences in the accumulation and mobilization of monocytes following in vivo injection of CSF1 in the bone marrow, blood, and spleen of CUREA^+/+ and CUREA^-/- mice. To evaluate the impact of CUREA deletion on stress haematopoiesis, we administered cyclophosphamide to mice, but there was no significant change in monocyte mobilization following chemotherapy administration in the mutant mice. In overview, the CUREA element contributes to the early induction of Csf1r mRNA during myeloid differentiation but is redundant for sustained expression in macrophages.

In line with the previously reported role of this element, in vitro osteoclast differentiation from bone marrow precursors was delayed in CUREA^-/- mice, associated with decreased Csf1r transcript levels after 7 days of culture (Δ17.6% vs. control, p=0.0254). 5’ RACE confirmed the disappearance of the osteoclast-specific CSF1R transcription start site (TSS) in CUREA^-/- mice. To assess if the delayed osteoclast growth impacts bone remodelling in vivo, we performed micro-CT scans on the femurs of 12-week-old mice. We observed changes in trabecular bone complexity, highlighted by increased trabecular bone and thickness, associated with reduced space within trabecular bone in CUREA^-/- mice.

Collectively, our data shows that CUREA enhances myeloid differentiation of HSCs. CUREA deletion leads to accumulation of long term-HSCs and short term-HSCs and is associated with a decrease in MPP3. 5’ RACE demonstrated that CUREA functions as an enhancer but not as a promoter in HSCs and myelomonocytic cells. In contrast, CUREA is required to generate additional TSS in osteoclasts and promote osteoclast differentiation in vitro and function in vivo. This work provides new insight into the understanding of the function of CUREA and more generally, provides evidence that HSPCs' stemness and differentiation can be modulated by targeting selected regulatory elements.