Normal Myeloid Development Requires Both the Glutamine-Rich Transactivation Domain and the PEST Region of Transcription Factor PU.1 but Not the Potent Acidic Transactivation Domain

Abstract

؊/؊ ES cell rescue system to genetically test which previously identified PU.1 functional domains are necessary for the development of mature macrophages. PU.1 functional domains include multiple N-terminal acidic and glutamine-rich transactivation domains, a PEST domain, several serine phosphorylation sites, and a C-terminal Ets DNA binding domain, all delineated and characterized by using standard biochemical and transactivational assays. By using the production of mature macrophages as a functional readout in our assay system, we have established that the glutamine-rich transactivation domain, a portion of the PEST domain, and the DNA binding domain are required for myelopoiesis. Deletion of three acidic domains, which exhibit potent transactivation potential in vitro, had no effect on the ability of PU.1 to promote macrophage development. Furthermore, mutagenesis of four independent sites of serine phosphorylation also had no effect on myelopoiesis. Collectively, our results indicate that PU.1 interacts with important regulatory proteins during macrophage development via the glutamine-rich and PEST domains. The PU

In vivo complex formation of PU.1 with HDAC1 associated with PU.1-mediated transcriptional repression

“…We ®rst analysed the e ects of the deletion mutants D1 ± 70, D74 ± 122 and D1 ± 100, on c-myc promoter activity. The N-terminal aa 1 ± 70 of PU.1 have been shown to encode the N-terminal TBP/RB binding domain (Hagemeier et al, 1993), and 1 ± 122 the transactivation and CBP binding domain (Fisher et al, 1998;Yamamoto et al, 1999). All these deletion mutants reduced c-myc promoter activity like the wild type PU.1 (Figure 5b).…”

Transcriptional regulation of granulocyte and monocyte development

“…The DNA-binding domain of PU.1 and its N-terminal, glutamine-rich trans-activating domain were both required to rescue myelopoiesis in PU.1 (7/7) ES cells, whereas its acidic trans-activating domain was dispensable (Fisher et al, 1998).…”

IFN-Stimulated Gene 15 Is Synergistically Activated Through Interactions Between the Myelocyte/Lymphocyte-Specific Transcription Factors, PU.1, IFN Regulatory Factor-8/IFN Consensus Sequence Binding Protein, and IFN Regulatory Factor-4: Characterization of a New Subtype of IFN-Stimulated Response Element

“…The plasmids pCB6PU.1⌬PEST and pCB6PU.1S148A were obtained from Dr. M. Atchison (University of Pennsylvania, Philadelphia, PA) (22). Site-directed mutagenesis of tyrosine residues Y23 and Y48 within the DNA binding domain (DBD) of IRF-8\ICSBP (ICSBPY23F and ICSBPY48F) to phenylalanine were generated using the plasmid pTarget-ICSBP (7) and the GeneEditor kit (Promega, Madison, WI) with the primers Y23F (5Ј-GACAGTAGCATGTTTCCAGGCCTGATTTGGG-3Ј) and Y48F (5Ј-GGAAACACGCCGGCAAGCAAGATTTTAAT-3Ј).…”