Transcription factor complex formation and chromatin fine structure alterations at the murine c-fms (CSF-1 receptor) locus during maturation of myeloid precursor cells

Abstract

Expression of the gene for the macrophage colony stimulating factor receptor (CSF-1R), c-fms, has been viewed as a hallmark of the commitment of multipotent precursor cells to macrophages. Lineage-restricted expression of the gene is controlled by conserved elements in the proximal promoter and within the first intron. To investigate the developmental regulation of c-fms at the level of chromatin structure, we developed an in vitro system to examine the maturation of multipotent myeloid precursor cells into mature macrophages. The dynamics of chromatin fine structure alterations and transcription factor occupancy at the c-fms promoter and intronic enhancer was examined by in vivo DMS and UV-footprinting. We show that the c-fms gene is already transcribed at low levels in early myeloid precursors on which no CSF-1R surface expression can be detected. At this stage of myelopoiesis, the formation of transcription factor complexes on the promoter was complete. By contrast, occupancy of the enhancer was acutely regulated during macrophage differentiation. Our data show that cell-intrinsic differentiation decisions at the c-fms locus precede the appearance of c-fms on the cell surface. They also suggest that complex lineage-specific enhancers such as the c-fms intronic enhancer regulate local chromatin structure through the coordinated assembly and disassembly of distinct transcription factor complexes.

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

“…1A). Based on in vivo footprinting experiments we have previously reported the presence of two PU.1 sites in FIRE (31). We have revisited this issue using EMSA and found that the downstream site is bound by another factor forming a complex that is not competed by a PU.1 consensus sequence (data not shown).…”

“…were described previously (31,32,34). PCR products were labeled by primer extension using ␥-32 P-labeled nested primers and were analyzed on 6% denaturing polyacrylamide gels.…”

“…In contrast, cell surface expression of CSF-1 receptor protein and high levels of mRNA are readily detected only in committed macrophage precursors (31) and their progeny. An initial mechanistic explanation of why this was the case was provided by in vivo footprinting studies demonstrating that the increase in c-fms mRNA expression during macrophage differentiation correlates with a dynamic assembly and disassembly of transcription factor complexes on the FIRE enhancer (31). However, the molecular details of this dynamic behavior are unknown because the identities of the specific factors and cofactors recruited were not determined in the previous study.…”

“…We previously showed by in vivo footprinting that the c-fms locus is already partly occupied by transcription factors in HSCs and becomes fully occupied in committed myeloid progenitor cells (34). In contrast, cell surface expression of CSF-1 receptor protein and high levels of mRNA are readily detected only in committed macrophage precursors (31) and their progeny. An initial mechanistic explanation of why this was the case was provided by in vivo footprinting studies demonstrating that the increase in c-fms mRNA expression during macrophage differentiation correlates with a dynamic assembly and disassembly of transcription factor complexes on the FIRE enhancer (31).…”

E2a/Pbx1 oncogene inhibits terminal differentiation but not myeloid potential of pro-T cells

“…Mice with targeted disruption of the c-fms gene show increased splenic erythoid burst-forming units (BFU-E) and highproliferative potential colony-forming cells, which suggested that M-CSF-R might play a role in primitive hematopoietic cells (Dai et al, 2002). Indeed, low levels of c-fms transcripts have been detected in pluripotent hematopoietic cells, but cell surface expression of the receptor is detectable only later in the monocytic lineage (Tagoh et al, 2002;Follows et al, 2005). M-CSF-R transcript expression has been also detected within the thymus in the earliest sub-population that exhibits both T-lymphoid and macrophage, but no B-lymphoid potentials (Balciunaite et al, 2005), and it has been proposed that a critical step in lymphoid commitment is downregulation of cytokine receptors that drive myeloid differentiation, including the M-CSF-R (Kondo et al, 2000;King et al, 2002).…”