The Ewing Sarcoma Protein (EWS) Binds Directly to the Proximal Elements of the Macrophage-Specific Promoter of the CSF-1 Receptor (csf1r) Gene

Abstract

Many macrophage-specific promoters lack classical transcriptional start site elements such as TATA boxes and Sp1 sites. One example is the CSF-1 receptor (CSF-1R, CD115, c-fms), which is used as a model of the transcriptional regulation of macrophage genes. To understand the molecular basis of start site recognition in this gene, we identified cellular proteins binding specifically to the transcriptional start site (TSS) region. The mouse and human csf1r TSS were identified using cap analysis gene expression (CAGE) data. Conserved elements flanking the TSS cluster were analyzed using EMSAs to identify discrete DNA-binding factors in primary bone marrow macrophages as candidate transcriptional regulators. Two complexes were identified that bind in a highly sequence-specific manner to the mouse and human TSS proximal region and also to high-affinity sites recognized by myeloid zinc finger protein 1 (Mzf1). The murine proteins were purified by DNA affinity isolation from the RAW264.7 macrophage cell line and identified by mass spectrometry as EWS and FUS/TLS, closely related DNA and RNA-binding proteins. Chromatin immunoprecipitation experiments in bone marrow macrophages confirmed that EWS, but not FUS/TLS, was present in vivo on the CSF-1R proximal promoter in unstimulated primary macrophages. Transfection assays suggest that EWS does not act as a conventional transcriptional activator or repressor. We hypothesize that EWS contributes to start site recognition in TATA-less mammalian promoters.

Regulated Expression of PTPRJ/CD148 and an Antisense Long Noncoding RNA in Macrophages by Proinflammatory Stimuli

“…The presence of a conserved MZF1 binding site in the Ptprj promoter is consistent with its expression in myeloid cells. The RNA binding zinc finger proteins EWS and FUS/TLS bind to the consensus binding sites for MZF1, implicating their significance in the assembly of the basal transcriptional complex [74]. Taken together, the bioinformatic analyses of the Ptprj putative promoter revealed that the Ptprj gene is highly regulated in macrophages and the Ptprj promoter resembles a macrophage-specific promoter.…”

FUsed in Sarcoma Is a Novel Regulator of Manganese Superoxide Dismutase Gene Transcription

“…It has been demonstrated that ACTGGCTC (reverse sequence) in the form of double stranded DNA in the macrophage specific cerebrospinal fluid receptor-1 gene can bind with FUS protein (25). Blast homology search analysis identified that the GGCTC sequence was present in MnSOD promoter at about 2.8-kb upstream from the start site.…”

Maintaining Cell Identity through Global Control of Genomic Organization

“…Moreover, at the gene that encodes defensin-1, a PU.1 site adjacent to a TTTAAA element (which may represent a weak TATA box) is required to recruit the transcriptional machinery; however, if the TTTAAA element is replaced by a canonical TATA box, the PU.1 site is no longer required for gene activation, suggesting that PU.1 is essential to recruit and position the transcriptional machinery in the absence of a strong core promoter (Yaneva et al, 2006). More in general, the promoters of many macrophage-specific genes do not contain canonical core promoter elements (like a TATA box or the Inr) (Hume et al, 2008); conversely, they contain clusters of PU.1 sites, the last one of such sites being often located just upstream of the mapped TSS (Tenen et al, 1997). Overall, these data suggest that in the absence of strong core promoter elements, PU.1 may directly control the site of transcriptional initiation.…”