Fms-intronic Regulatory Element Research Articles

Therapeutic potential of human microglia transplantation in a chimeric model of CSF1R-related leukoencephalopathy

Microglia replacement strategies are increasingly being considered for the treatment of primary microgliopathies like adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). However, available mouse models fail to recapitulate the diverse neuropathologies and reduced microglia numbers observed in patients. In this study, we generated a xenotolerant mouse model lacking the fms-intronic regulatory element (FIRE) enhancer within Csf1r, which develops nearly all the hallmark pathologies associated with ALSP. Remarkably, transplantation of human induced pluripotent stem cell (iPSC)-derived microglial (iMG) progenitors restores a homeostatic microglial signature and prevents the development of axonal spheroids, white matter abnormalities, reactive astrocytosis, and brain calcifications. Furthermore, transplantation of CRISPR-corrected ALSP-patient-derived iMG reverses pre-existing spheroids, astrogliosis, and calcification pathologies. Together with the accompanying study by Munro and colleagues, our results demonstrate the utility of FIRE mice to model ALSP and provide compelling evidence that iMG transplantation could offer a promising new therapeutic strategy for ALSP and perhaps other microglia-associated neurological disorders.

#2607 Generation of a tissue resident macrophage depleted reporter rat to examine cardiovascular homeostasis and kidney disease

Abstract Cardiovascular disease (CVD) and chronic kidney disease (CKD) are currently the 1st and 10th leading cause of global mortality, respectively. By 2050, CKD is predicted to be the 5th leading cause, with most of those individuals dying of CVD. Recent single cell RNA sequencing data has highlighted the plasticity of macrophage populations in both renal and cardiovascular injury and repair. Many of these subpopulations of macrophages remain uncharacterized. We have generated the Red FIRE rat, which is a cross between the Csf1r-mApple reporter rat and the super-enhancer FIRE knockout rat. The Csf1r-mApple reporter rat introduces a fluorescent signal to cells of the monocyte-macrophage lineage and the KO of FIRE (fms-intronic regulatory element), while the FIRE rat has fms-intronic regulatory element (FIRE), which is the superenhancer in the Csf1r locus knocked out, resulting in depletion of tissue resident macrophages. These red FIRE rats will allow us to visualize all Csf1r expressing cells (macrophages) and in the FIRE rats deplete populations of tissue resident macrophages (TRM) (including those in the kidney and heart). This will enable us to visualize and study TRM populations in homeostasis and injury. We hypothesise that the red FIRE rats will show a more severe phenotype in injury and have altered vascular function (local and systemic). Male and female (8–10-week-old) rats underwent a 6- or 12-week 5/6 subtotal nephrectomy (5/6 STNx) in groups of 4-8. Baseline, midpoint and endpoint urine and serum were collected for analysis, as well as blood pressure measurements and echocardiograms to monitor kidney and heart health/decline. At the end of the experiment, kidneys and the heart were collected for flow cytometry, immunofluorescence, RT-qPCR, and histological staining. The thoracic aorta, mesentery, and common renal arteries were collected for functional analysis using isometric (wire)myography. The knockout of the FIRE super enhancer resulted in depletion of kidney TRMs (Fig. 1) as visualised by immunofluorescent staining of both wild type and macrophage-depleted (red FIRE) rat tissue. Figure 1A shows the composite image and individual stains; DAPI, IBa1 (pan macrophage marker), and Csf1r-mApple (tissue resident macrophages) on a naïve heart. Representative composite images also compare STNx hearts (Fig. 1B) and kidneys (Fig. 1C) in both wild type and macrophage-depleted rats. This allowed for comparison of these rats in both injury and homeostasis. Figure 1: Immunofluorescent staining of the uninjured heart of a FIRE wild type (WT), mApple positive rat (A), the injured kidney (B) and heart (C) of mApple positive, FIRE wild type (Control) or mApple positive, FIRE knock out (FIRE) rats post 12-week 5/6 subtotal nephrectomy (5/6 STNx). DAPI: nuclei, IBa1: all macrophages, csf1r (mApple): tissue resident macrophages. Representative imagining, figure made using FIJI. Our preliminary data suggests previously undescribed roles for tissue-resident macrophages in rats i.e. ion handling, vascular homeostasis and roles in CKD and repair. These results could help reveal new specific druggable targets that could be exploited for treatments which could halt progression of disease and thus reduce end stage renal failure patient numbers, healthcare costs and premature CVD-related deaths.

Resident and recruited macrophages differentially contribute to cardiac healing after myocardial ischemia.

Cardiac macrophages are heterogenous in phenotype and functions, which has been associated with differences in their ontogeny. Despite extensive research, our understanding of the precise role of different subsets of macrophages in ischemia/reperfusion (I/R) injury remains incomplete. We here investigated macrophage lineages and ablated tissue macrophages in homeostasis and after I/R injury in a CSF1R-dependent manner. Genomic deletion of a fms-intronic regulatory element (FIRE) in the Csf1r locus resulted in specific absence of resident homeostatic and antigen-presenting macrophages, without affecting the recruitment of monocyte-derived macrophages to the infarcted heart. Specific absence of homeostatic, monocyte-independent macrophages altered the immune cell crosstalk in response to injury and induced proinflammatory neutrophil polarization, resulting in impaired cardiac remodeling without influencing infarct size. In contrast, continuous CSF1R inhibition led to depletion of both resident and recruited macrophage populations. This augmented adverse remodeling after I/R and led to an increased infarct size and deterioration of cardiac function. In summary, resident macrophages orchestrate inflammatory responses improving cardiac remodeling, while recruited macrophages determine infarct size after I/R injury. These findings attribute distinct beneficial effects to different macrophage populations in the context of myocardial infarction.

Lifelong absence of microglia alters hippocampal glutamatergic networks but not synapse and spine density.

Microglia sculpt developing neural circuits by eliminating excess synapses in a process called synaptic pruning, by removing apoptotic neurons, and by promoting neuronal survival. To elucidate the role of microglia during embryonic and postnatal brain development, we used a mouse model deficient in microglia throughout life by deletion of the fms-intronic regulatory element (FIRE) in the Csf1r locus. Surprisingly, young adult Csf1rΔFIRE/ΔFIRE mice display no changes in excitatory and inhibitory synapse number and spine density of CA1 hippocampal neurons compared with Csf1r+/+ littermates. However, CA1 neurons are less excitable, receive less CA3 excitatory input and show altered synaptic properties, but this does not affect novel object recognition. Cytokine profiling indicates an anti-inflammatory state along with increases in ApoE levels and reactive astrocytes containing synaptic markers in Csf1rΔFIRE/ΔFIRE mice. Notably, these changes in Csf1rΔFIRE/ΔFIRE mice closely resemble the effects of acute microglial depletion in adult mice after normal development. Our findings suggest that microglia are not mandatory for synaptic pruning, and that in their absence pruning can be achieved by other mechanisms.

Putting PLX5622 into perspective: microglia in central nervous system viral infection.

Putting PLX5622 into perspective: microglia in central nervous system viral infection.

CNS macrophages differentially rely on an intronicCsf1renhancer for their development

ABSTRACTThe central nervous system hosts parenchymal macrophages, known as microglia, and non-parenchymal macrophages, collectively termed border-associated macrophages (BAMs). Microglia, but not BAMs, were reported to be absent in mice lacking a conserved Csf1r enhancer: the fms-intronic regulatory element (FIRE). However, it is unknown whether FIRE deficiency also impacts BAM arrival and/or maintenance. Here, we show that macrophages in the ventricular system of the brain, including Kolmer's epiplexus macrophages, are absent in Csf1rΔFIRE/ΔFIRE mice. Stromal choroid plexus BAMs are also considerably reduced. During normal development, we demonstrate that intracerebroventricular macrophages arrive from embryonic day 10.5, and can traverse ventricular walls in embryonic slice cultures. In Csf1rΔFIRE/ΔFIRE embryos, the arrival of both primitive microglia and intracerebroventricular macrophages was eliminated, whereas the arrival of cephalic mesenchyme and stromal choroid plexus BAMs was only partially restricted. Our results provide new insights into the development and regulation of different CNS macrophage populations.

Deletion of a Csf1r enhancer selectively impacts CSF1R expression and development of tissue macrophage populations

The proliferation, differentiation and survival of mononuclear phagocytes depend on signals from the receptor for macrophage colony-stimulating factor, CSF1R. The mammalian Csf1r locus contains a highly conserved super-enhancer, the fms-intronic regulatory element (FIRE). Here we show that genomic deletion of FIRE in mice selectively impacts CSF1R expression and tissue macrophage development in specific tissues. Deletion of FIRE ablates macrophage development from murine embryonic stem cells. Csf1rΔFIRE/ΔFIRE mice lack macrophages in the embryo, brain microglia and resident macrophages in the skin, kidney, heart and peritoneum. The homeostasis of other macrophage populations and monocytes is unaffected, but monocytes and their progenitors in bone marrow lack surface CSF1R. Finally, Csf1rΔFIRE/ΔFIRE mice are healthy and fertile without the growth, neurological or developmental abnormalities reported in Csf1r−/− rodents. Csf1rΔFIRE/ΔFIRE mice thus provide a model to explore the homeostatic, physiological and immunological functions of tissue-specific macrophage populations in adult animals.

The evolution of the macrophage-specific enhancer (Fms intronic regulatory element) within the CSF1R locus of vertebrates

The Csf1r locus encodes the receptor for macrophage colony-stimulating factor, which controls the proliferation, differentiation and survival of macrophages. The 300 bp Fms intronic regulatory element (FIRE), within the second intron of Csf1r, is necessary and sufficient to direct macrophage-specific transcription. We have analysed the conservation and divergence of the FIRE DNA sequence in vertebrates. FIRE is present in the same location in the Csf1r locus in reptile, avian and mammalian genomes. Nearest neighbor analysis based upon this element alone largely recapitulates phylogenies inferred from much larger genomic sequence datasets. One core element, containing binding sites for AP1 family and the macrophage-specific transcription factor, PU.1, is conserved from lizards to humans. Around this element, the FIRE sequence is conserved within clades with the most conserved elements containing motifs for known myeloid-expressed transcription factors. Conversely, there is little alignment between clades outside the AP1/PU.1 element. The analysis favours a hybrid between “enhanceosome” and “smorgasbord” models of enhancer function, in which elements cooperate to bind components of the available transcription factor milieu.

Transcriptional mechanisms that control expression of the macrophage colony-stimulating factor receptor locus.

The proliferation, differentiation, and survival of cells of the macrophage lineage depends upon signals from the macrophage colony-stimulating factor (CSF) receptor (CSF1R). CSF1R is expressed by embryonic macrophages and induced early in adult hematopoiesis, upon commitment of multipotent progenitors to the myeloid lineage. Transcriptional activation of CSF1R requires interaction between members of the E26 transformation-specific family of transcription factors (Ets) (notably PU.1), C/EBP, RUNX, AP-1/ATF, interferon regulatory factor (IRF), STAT, KLF, REL, FUS/TLS (fused in sarcoma/ranslocated in liposarcoma) families, and conserved regulatory elements within the mouse and human CSF1R locus. One element, the Fms-intronic regulatory element (FIRE), within intron 2, is conserved functionally across all the amniotes. Lineage commitment in multipotent progenitors also requires down-regulation of specific transcription factors such as MYB, FLI1, basic leucine zipper transcriptional factor ATF-like (BATF3), GATA-1, and PAX5 that contribute to differentiation of alternative lineages and repress CSF1R transcription. Many of these transcription factors regulate each other, interact at the protein level, and are themselves downstream targets of CSF1R signaling. Control of CSF1R transcription involves feed-forward and feedback signaling in which CSF1R is both a target and a participant; and dysregulation of CSF1R expression and/or function is associated with numerous pathological conditions. In this review, we describe the regulatory network behind CSF1R expression during differentiation and development of cells of the mononuclear phagocyte system.

C/EBPβ Is Required for Survival of Ly6C- Monocytes after Committment to Monocyte Lineage through Upregulation of Csf1r

Monopoiesis is the process in which hematopoietic stem cells (HSCs) continuously give rise to monocytes. Accumulating evidence has identified cellular constituents of monopoiesis. Common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), macrophage-dendritic cell precursors (MDPs) and common monocyte progenitors (cMoPs) are the intermediates during the differentiation of HSCs into mature monocytes. In mice, CD11b+ CD115+ monocytes are further divided into two subsets based on the expression of Ly6C. Classical monocytes express Ly6C on their surface. By contrast, Ly6C− patrolling monocytes have been recently identified, and the molecular mechanisms which regulate the development and homeostasis of Ly6C−monocytes still remain elusive.C/EBPβ is a leucine zipper transcription factor which regulates stress-induced granulopoiesis (Hirai et al. Nat Immunol, 2006, Hayashi et al. Leukemia 2013). We have recently found that peripheral blood (PB) monocytes are significantly reduced in steady-state Cebpb−/− mice (Tamura et al. Biochem Biophys Res Commun, 2015). In addition, last year at this meeting, we have reported that cell death of Ly6C− monocytes was accelerated through reduced expression of Csf1r (encoding a receptor for M-CSF) in Cebpb−/− mice. Here in this study, we determined the precise developmental stage where C/EBPβ is mandatory for survival of Ly6C− monocytes, and investigated the mechanism of Csf1r regulation by C/EBPβ.A recent publication demonstrated that Mx1 is preferentially expressed by monocytes and a Mx1 promoter-mediated conditional system targets monocytes without inoculation of polyI:C (Hashimoto et al. Immunity, 2013), suggesting that Mx1-Cre Cebpbf/f mouse is ideal to evaluate the monocyte-specific requirement for C/EBPβ. We confirmed that upregulation of Cebpb mRNA during monopoiesis was significantly impaired after cMoP stage in Mx1-Cre+Cebpbf/f mice. In order to exclude the possible involvement of Cebpβ deficient microenvironment, bone marrow (BM) cells of Mx1-Cre+Cebpβf/f mice (CD45.2+) were transplanted into lethally irradiated CD45.1+ wild type mice. The frequencies of Ly6C− monocytes in the recipients of Mx1-Cre+Cebpbf/f BM cells were significantly reduced when compared to mice that received Mx1-Cre−Cebpbf/f BM cells (Figure). These results strongly suggest that C/EBPβ is specifically required after commitment to monocytes.In order to investigate the molecular mechanisms involved in the regulation of Csf1r by C/EBPβ, we utilized a combination of a promoter and an enhancer region located in the first intron of Csf1r gene (Fms intronic regulatory element: FIRE) for reporter assay (Pridans et al. Mol Ther Methods Clin Dev, 2014). These regulatory elements contain at least 2 consensus binding sites for C/EBPβ (one in the promoter and the other in the enhancer). C/EBPβ significantly enhanced the reporter activity of the regulatory elements in a dose-dependent manner, and introduction of mutations into either of the consensus binding sites abrogated the reporter activity. Next, we engineered EML cells, a mouse HSC line, to express C/EBPβ-estrogen receptor (ER) fusion protein or ER alone. Nuclear translocation of C/EBPβ-ER in the presence of tamoxifen resulted in significant increase of Csf1r mRNA and protein. Using these cells, we performed chromatin immunoprecipitation PCR. Upon treatment with tamoxifen, significant enrichment of C/EBPβ at the promoter region and the FIRE region was observed. These data indicated that C/EBPβ regulates Csf1r through direct binding to these regulatory elements.Collectively, these results demonstrate that C/EBPβ supports survival of Ly6C− monocytes after commitment to monocyte lineage through direct regulation of Csf1r, which is critical for survival and differentiation of monocytes. [Display omitted] DisclosuresHirai:Kyowa Hakko Kirin: Research Funding; Novartis Pharma: Research Funding. Maekawa:Bristol-Myers K.K.: Research Funding.

A Csf1r-EGFP Transgene Provides a Novel Marker for Monocyte Subsets in Sheep.

Expression of Csf1r in adults is restricted to cells of the macrophage lineage. Transgenic reporters based upon the Csf1r locus require inclusion of the highly conserved Fms-intronic regulatory element for expression. We have created Csf1r-EGFP transgenic sheep via lentiviral transgenesis of a construct containing elements of the mouse Fms-intronic regulatory element and Csf1r promoter. Committed bone marrow macrophage precursors and blood monocytes express EGFP in these animals. Sheep monocytes were divided into three populations, similar to classical, intermediate, and nonclassical monocytes in humans, based upon CD14 and CD16 expression. All expressed EGFP, with increased levels in the nonclassical subset. Because Csf1r expression coincides with the earliest commitment to the macrophage lineage, Csf1r-EGFP bone marrow provides a tool for studying the earliest events in myelopoiesis using the sheep as a model.

Csf1r Is a Downstream Target of C/EBPβ in Ly6C¯ Monocytes

Currently, monocytes are classified into at least two subsets. Classical monocytes, also known as inflammatory monocytes (a Ly6C+ subset in mice and a CD14+ CD16− subset in human), are involved in innate immune responses. On the other hand, patrolling monocytes (a Ly6C− subset in mice and a CD14− CD16+ subset in human) have been recently identified. Ly6C− monocytes are found attached on the luminal side of endothelium and scavenge microparticles. Developmentally, Ly6C+ and Ly6C− monocytes share common monocyte progenitors (cMoPs), or Ly6C− monocytes might be converted from Ly6C+ monocytes. Although involvement of Ly6C− monocytes in various kinds of diseases has been reported, molecular mechanisms which regulate the homeostasis of Ly6C− monocytes are largely unknown.CCAAT/Enhancer Binding Protein β (C/EBPβ) is a leucine zipper type transcription factor. We and others have previously shown that C/EBPβ is required for stress-induced granulopoiesis (Hirai et al. Nat Immunol, 2006, Satake et al. J Immunol, 2012, Hayashi et al. Leukemia 2013). However, its roles in steady state hematopoiesis remain relatively unknown. We have recently found that peripheral blood monocytes are significantly reduced in Cebpb−/− mice (Tamura et al. Biochem Biophys Res Commun, 2015). In addition, last year in this meeting, we have reported that Cebpb mRNA is highly upregulated during differentiation from myeloid progenitors or Ly6C+ monocytes to Ly6C− monocytes, and that Ly6C− monocytes are almost completely absent in Cebpb−/− mice due to enhanced cell death [Abstract #224]. Here, we further investigated the molecular mechanisms underlying C/EBPβ-dependent survival of Ly6C− monocytes.In this study, we focused on the regulation of Csf1r (also known as M-CSF receptor). Csf1r is an essential molecule for the development and survival of monocytes. To determine the developmental stages at which Csf1r plays critical roles, we measured the expressions of Csf1r mRNA in hematopoietic stem/progenitor cells and monocyte subsets obtained from wild-type (WT) mice. Csf1r mRNA was expressed at at low levels in hematopoietic stem/progenitors including macrophage dendritic precursors (MDPs) and cMoPs. Csf1r mRNA started to be upregulated in Ly6C+ monocytes, followed by a drastic increase in Ly6C− monocytes. These expression patterns were quite similar to those of Cebpb, suggesting the close relationship between Csf1r and C/EBPβ. Interestingly, such drastic increase of Csf1r mRNA in Ly6C− monocytes was blunted in Cebpb−/− mice, and protein levels of Csf1r in Cebpb−/− Ly6C− monocytes were significantly lower than those in WT Ly6C− monocytes. In order to evaluate the effect of C/EBPβ overexpression on Csf1r expression, EML cells, a mouse hematopoietic stem cell line, were engineered to express C/EBPβ-estrogen receptor (ER) fusion protein or ER alone. Nuclear translocation of C/EBPβ-ER in the presence of tamoxifen resulted in significantly increased levels of Csf1r mRNA and protein when compared to nuclear translocation of ER alone. Previous reports have demonstrated that a combination of a promoter sequence and an enhancer region located in the first intron of Csf1r gene (Fms intronic regulatory element: FIRE) is enough to recapitulate the endogenous Csf1r expression and that these elements contained consensus binding sites for C/EBP transcription factors. Then, we hypothesized that C/EBPβ binds to these sites, activates transcription of Csf1r gene and promotes survival of Ly6C- monocytes. To evaluate this hypothesis, we utilized an expression vector, in which green fluorescent protein (GFP) is driven by a combination of the Csf1r promoter and FIRE sequences (Csf1r-EGFP-FIRE) (a kind gift from Drs Clare P and David A Hume, University of Edinburgh). When a C/EBPβexpression vector was co-transfected with the vector containing Csf1r-EGFP-FIRE into HEK293 cells, the frequencies of GFP positive cells were significantly higher when compared to a control vector (C/EBPβ vs control; 4.6±0.6 vs 1.6±1.0, p=0.01), suggesting that C/EBPβ regulates Csf1r expression through these elements. We are currently evaluating the significance of C/EBP consensus binding sites in the promoter and the enhancer. ChIP PCR is also in progress to further verify our hypothesis.Collectively, these results suggest that Csf1r is a critical downstream target of C/EBPβ in Ly6C- monocytes. DisclosuresNo relevant conflicts of interest to declare.

Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage.

We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens.

Lentiviral vectors containing mouse Csf1r control elements direct macrophage-restricted expression in multiple species of birds and mammals

The development of macrophages requires signaling through the lineage-restricted receptor Csf1r. Macrophage-restricted expression of transgenic reporters based upon Csf1r requires the highly conserved Fms-intronic regulatory element (FIRE). We have created a lentiviral construct containing mouse FIRE and promoter. The lentivirus is capable of directing macrophage-restricted reporter gene expression in mouse, rat, human, pig, cow, sheep, and even chicken. Rat bone marrow cells transduced with the lentivirus were capable of differentiating into macrophages expressing the reporter gene in vitro. Macrophage-restricted expression may be desirable for immunization or immune response modulation, and for gene therapy for lysosomal storage diseases and some immunodeficiencies. The small size of the Csf1r transcription control elements will allow the insertion of large “cargo” for applications in gene therapy and vaccine delivery.

The function of the conserved regulatory element within the second intron of the mammalian Csf1r locus.

The gene encoding the receptor for macrophage colony-stimulating factor (CSF-1R) is expressed exclusively in cells of the myeloid lineages as well as trophoblasts. A conserved element in the second intron, Fms-Intronic Regulatory Element (FIRE), is essential for macrophage-specific transcription of the gene. However, the molecular details of how FIRE activity is regulated and how it impacts the Csf1r promoter have not been characterised. Here we show that agents that down-modulate Csf1r mRNA transcription regulated promoter activity altered the occupancy of key FIRE cis-acting elements including RUNX1, AP1, and Sp1 binding sites. We demonstrate that FIRE acts as an anti-sense promoter in macrophages and reversal of FIRE orientation within its native context greatly reduced enhancer activity in macrophages. Mutation of transcription initiation sites within FIRE also reduced transcription. These results demonstrate that FIRE is an orientation-specific transcribed enhancer element.

A Two-Step, PU.1-Dependent Mechanism for Developmentally Regulated Chromatin Remodeling and Transcription of the c-fms Gene

Hematopoietic stem cells and multipotent progenitors exhibit low-level transcription and partial chromatin reorganization of myeloid cell-specific genes including the c-fms (csf1R) locus. Expression of the c-fms gene is dependent on the Ets family transcription factor PU.1 and is upregulated during myeloid differentiation, enabling committed macrophage precursors to respond to colony-stimulating factor 1. To analyze molecular mechanisms underlying the transcriptional priming and developmental upregulation of the c-fms gene, we have utilized myeloid progenitors lacking the transcription factor PU.1. PU.1 can bind to sites in both the c-fms promoter and the c-fms intronic regulatory element (FIRE enhancer). Unlike wild-type progenitors, the PU.1(-/-) cells are unable to express c-fms or initiate macrophage differentiation. When PU.1 was reexpressed in mutant progenitors, the chromatin structure of the c-fms promoter was rapidly reorganized. In contrast, assembly of transcription factors at FIRE, acquisition of active histone marks, and high levels of c-fms transcription occurred with significantly slower kinetics. We demonstrate that the reason for this differential activation was that PU.1 was required to promote induction and binding of a secondary transcription factor, Egr-2, which is important for FIRE enhancer activity. These data suggest that the c-fms promoter is maintained in a primed state by PU.1 in progenitor cells and that at FIRE PU.1 functions with another transcription factor to direct full activation of the c-fms locus in differentiated myeloid cells. The two-step mechanism of developmental gene activation that we describe here may be utilized to regulate gene activity in a variety of developmental pathways.

A highly conserved c-fmsgene intronic element controls macrophage-specific and regulated expression

The c-fms gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c-fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSs) were identified within mouse intron 2. Sequences of these DHSs were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fms intronic regulatory element (FIRE), which is even more highly conserved than the c-fms proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Sp1, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high- and low-level c-fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fms gene.