Abstract
The transcription factor PU.1 is predominantly expressed in dendritic cells (DCs) and is essential for DC differentiation. Although there are several reports that PU.1 positively regulates the expression of DC-specific genes, whether PU.1 also has a suppressive effect on DCs is largely unknown. Here we demonstrate that PU.1 suppresses the expression of Th2 cytokines including IL-13 and IL-5 in bone marrow-derived DCs (BMDCs), through repression of the expression of GATA3, which is a master regulator of Th2 differentiations. When PU.1 siRNA was introduced into BMDCs, LPS-induced expression of IL-13 and IL-5 was increased along with upregulation of the constitutive expression of GATA2 and GATA3. The additional introduction of GATA3 siRNA but not of GATA2 siRNA abrogated PU.1 siRNA-mediated upregulation of IL-13 and IL-5. A chromatin immunoprecipitation assay showed that PU.1 bound to Gata3 proximal promoter region, which is more dominant than the distal promoter in driving GATA3 transcription in DCs. The degree of histone acetylation at the Gata3 promoter was decreased in PU.1 siRNA-introduced DCs, suggesting the involvement of PU.1 in chromatin modification of the Gata3 promoter. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, increased the degree of histone H3 acetylation at the Gata3 promoter and induced the subsequent expression of GATA3. Experiments using HDAC inhibitors and siRNAs showed that HDAC3 suppressed GATA3 expression. The recruitment of HDAC3 to the Gata3 promoter was decreased by PU.1 knockdown. LPS-induced IL-13 expression was dramatically reduced in BMDCs generated from mice lacking the conserved GATA3 response element, termed CGRE, which is an essential site for the binding of GATA3 on the Il-13 promoter. The degree of H3K4me3 at CGRE was significantly increased in PU.1 siRNA-transfected stimulated DCs. Our results indicate that PU.1 plays pivotal roles in DC development and function, serving not only as a transcriptional activator but also as a repressor.
Highlights
PU.1 is a hematopoietic lineage-specific transcription factor that belongs to the Ets family
Western blotting of whole cell lysates of these PU.1 siRNA-transfected cells using specific antibodies indicated that the protein level of GATA3 was apparently increased by PU.1 knockdown compared to control siRNA-transfected cells, whereas the amount of GATA1 or GATA2 protein was not affected by PU.1 knockdown (Fig 2B). These results suggest that PU.1 represses the expression of GATA3 but not of GATA1 and GATA2 by bone marrow-derived dendritic cell (DC) (BMDCs)
The GATAfamily consists of 6 molecules, of which GATA1, GATA2, and GATA3 are expressed in a hematopoietic cell-specific manner: GATA1 and GATA2 are essential for the erythroid/megakaryocyte development and are involved in the development of granulocytes; GATA3 is a master regulator of Th2 cells
Summary
PU. is a hematopoietic lineage-specific transcription factor that belongs to the Ets family. PU. knockout mice lack mature cells of the monocyte, neutrophil, and B and T lineages [1,2,3,4], demonstrating the requirement for this factor in myeloid and lymphoid development. It has been proposed that graded levels of PU. expression in hematopoietic progenitors are determinative of their lineage commitment, as high PU. level directs macrophage differentiation, a low level is sufficient for fetal B cell development [5, 6], and an intermediate level of PU. is required for granulocyte differentiation [7]. PU. regulates gene expression by binding to canonical ETS motifs through its interaction with other transcription factors, including interferon regulatory factor 4 (IRF4), IRF8, C/EBPα and β, and c-Jun [11]. PU. down-regulates its target genes through epigenetic modifications including histone deacetylation and DNA methylation, by interacting with HDAC1 or Dnmt3a/b respectively in murine erythroleukemia cells [14,15,16]
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