Spi-C Has Opposing Effects to PU.1 on Gene Expression in Progenitor B Cells

Schweitzer, Brock L.; Huang, Kuo Chan; Kamath, Meghana B.; Emelyanov, Alexander; Birshtein, Barbara K.; DeKoter, Rodney P.

doi:10.4049/jimmunol.177.4.2195

Cited by 29 publications

(43 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In vitro translations were performed using a coupled transcription/translation kit (Promega). DNA binding reactions and acrylamide gel electrophoresis were performed as previously described (22). Antibody supershift analyses were performed by adding 1 l of anti-PU.1 (Santa Cruz, Santa Cruz CA), anti-GABP␣, or anti-GABP␤1 (gifts from Warren Leonard).…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Interleukin-7 Receptor α Promoter by the Ets Transcription Factors PU.1 and GA-binding Protein in Developing B Cells

DeKoter

Schweitzer

Kamath

et al. 2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Signaling through the IL-7 receptor (IL-7R) is required for development and maintenance of the immune system. The receptor for IL-7 is heterodimeric, consisting of a common ␥ chain (␥c, encoded by Il2rg) and an ␣ subunit (IL-7R␣, encoded by Il7r). The Il7r gene is expressed specifically in the immune system in a developmental stage-specific manner. It is not known how the Il7r gene is transcriptionally regulated during B cell development. The goal of this study is to elucidate the function of the Il7r promoter region in developing B cells. Using a combination of 5 rapid amplification of cDNA ends analysis, transient transfection assays, and DNase I hypersensitivity mapping, we identified the location of the Il7r promoter. Using a combination of electrophoretic mobility shift analysis, chromatin immunoprecipitation experiments, and RNA interference experiments, we found that the Ets transcription factors PU.1 and GA-binding protein (GABP) activate the Il7r promoter by interacting with a highly conserved Ets binding site. In committed B lineage cells, GABP can promote Il7r transcription in the absence of PU.1. However, the results of retroviral gene transfer experiments suggest that PU.1 is uniquely required to initiate transcription of the Il7r locus at the earliest stages of progenitor B cell generation. In summary, these results suggest that Il7r transcription is regulated by both PU.1 and GABP in developing B cells. The cytokine interleukin-7 (IL-7)2 is essential for development and maintenance of the immune system (reviewed in Ref. 1). IL-7 is produced constitutively by stromal cells of the bone marrow, fetal liver, thymus, and by epithelial cells (2). IL-7 promotes both proliferation and differentiation of developing B and T lymphocytes (3, 4). The receptor for IL-7 (IL-7R, encoded by Il7r) is heterodimeric, consisting of an IL-7-specific ␣ chain (IL-7R␣) and a common ␥ chain (␥c, encoded by Il2rg), which is shared with other subunits comprising the receptors for IL-2, IL-4, IL-9, IL-15, and IL-21. The Il2rg gene is expressed by all hematopoietic cells (5). Transcription of the Il7r gene is initiated in common lymphoid progenitor cells (6). IL-7R␣ is expressed in developing B cells but is down-regulated upon B cell maturation (7,8). In developing T cells, IL-7R␣ is initially down-regulated at the CD4 and CD8 double negative 3 stage of thymic development (9). However, Il7r transcription is re-activated after the CD4 and CD8 double-positive stage and is expressed on peripheral CD4 or CD8 single-positive T cells (7). Therefore, the Il7r gene is expressed in a cell type and developmental stage-specific manner (7, 10). Targeted null mutation of either the Il7r (11, 12) or the Il2rg gene (13, 14) results in a profound block to early B and T cell development in mice. In human patients, mutations in the IL7RA gene cause a type of severe combined immunodeficiency in which the major deficiencies are in T cell development, whereas B and NK cells are relatively normal in number (15). Therefore, the IL-7R␣ is critically ...

show abstract

Section: Methodsmentioning

confidence: 99%

Regulation of the Interleukin-7 Receptor α Promoter by the Ets Transcription Factors PU.1 and GA-binding Protein in Developing B Cells

DeKoter

Schweitzer

Kamath

et al. 2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…An 1817 bp FcγRIIb promoter fragment was PCR amplified from C57Bl/6 genomic DNA and cloned into the pGL3-basic luciferase reporter vector (Promega, Madison, WI) as previously described [15]. A single nucleotide in the PU.1 binding site was mutated using the Stratagene QuickChange XL kit (Stratagene), using the oligonucleotides 5′-GACCGTTTCTTTTCACGTCCCCATTTGGACTTCA-3′ and 5′-TGAAGTCCAAATGGGGACGTGAAAAGAAACGGTC-3′ (PU.1 binding site italicized, mutated nucleotide underlined).…”

Section: Transfection and Reporter Assaysmentioning

confidence: 99%

“…Day 14.5 Sfpi1 +/+ , Sfpi1 −/− , Sfpi1 +/BN , or Sfpi1 BN/BN FLPs were generated by timed matings of Sfpi1 +/BN or Sfpi1 +/− mice as previously described [17]. For rescue experiments, Sfpi1 −/− FLPs were infected by spin-infection and analyzed by flow cytometry after two days of culture, as described [15]. Colony forming assays were performed as previously described [16] using recombinant murine GM-CSF (1 ng/ml), G-CSF (10 ng/ml), M-CSF (10 ng/ml) or IL-3 (5 ng/ml) + IL-6 (10 ng/ml) + SCF (100 ng/ml) (Peprotech).…”

Section: Cell Culturementioning

confidence: 99%

“…Primers used were as follows: wild-type forward (5′-ACCTGGAGCTCAGCTGGATGTTA-3′), BN forward (5′-CGTATCGATTGTACACTTAAGGGCCG-3′), common reverse (5′ GGGCGACGGGTTAATGCTAT-3′). HA-PU.1ΔN31 retrovirus was constructed by insertion of ΔN31 transcript into the Not I and Apa I sites to replace the Spi-C sequence of MIG-Spi-C vector [15]. ΔN31 transcripts were amplified from pBluescript plasmid containing PU.1 cDNA lacking the N-terminal 31 amino acids and cloned as above.…”

Section: Construction Of Retroviral Vectorsmentioning

confidence: 99%

See 1 more Smart Citation

Reduction in PU.1 activity results in a block to B-cell development, abnormal myeloid proliferation, and neonatal lethality

Houston

Kamath

Schweitzer

et al. 2007

Experimental Hematology

Self Cite

View full text Add to dashboard Cite

Objective-It has been demonstrated that high concentration of the transcription factor PU.1 (encoded by Sfpi1) promotes macrophage development, whereas low concentration induces B cell development in vitro. This has led to the hypothesis that lower levels of PU.1 activity are required for B cell than for macrophage development in vivo. We utilized an allele of Sfpi1 (termed BN) with a mutation in the first coding exon which resulted in a reduction of PU.1 expression in order to test this hypothesis. Methods-Using gene targeting in ES cells, two ATG-start site codons of PU.1 were mutated, resulting in reduced PU.1 expression originating from a third start codon. Mice were assayed for phenotypic abnormalities using fluorescence activated cell sorting, microscopy, and colony forming ability. In addition, isolated cells were tested for their differentiation potential in vitro and in vivo.Results-Lymphoid and myeloid cells derived from cultured Sfpi1 BN/BN fetal liver cells had reduced levels of PU.1 expression and activity. B cell development was intrinsically blocked in cells isolated from Sfpi1 BN/BN mice. In addition, myeloid development was impaired in Sfpi1 BN/BN fetal liver. However, neonatal Sfpi1 BN/BN mice had a dramatic expansion and infiltration of immature myeloid cells.Conclusion-Contrary to our original hypothesis, high levels of PU.1 activity are required to induce both myeloid and B cell development. In addition, neonatal mice homozygous for the hypomorphic allele acquire a myeloproliferative disorder and die within 1 month of age.

show abstract

“…Spic is a member of the ets gene family and its gene product has been described as having opposing effects to PU.1 during B-cell maturation. 60,61 Spic has also been implicated, with STAT-6, in regulating germline IgE transcription. 62 Mef2c (myocyte enhancer factor 2C) has previously been defined as a transcription factor found in B cells following inflammatory stimuli.…”

Section: Discussionmentioning

confidence: 99%

Defining a transcriptional fingerprint of murine splenic B-cell development

et al. 2008

View full text Add to dashboard Cite

B-cell development occurs in a stepwise fashion that can be followed by the expression of B cell-specific surface markers. In this study, we wished to identify proteins that could contribute to the changes in expression of such markers. By using RNA from freshly isolated B220 þ cells, we hoped to reduce the effect of artifacts that occur during the isolation and amplification steps necessary to use flow cytometry analysis-sorted subsets in microarray experiments. Analyses comparing expression patterns from B220 þ 2-week bone marrow (pro-B, pre-B, immature B cells), 2-week spleen (predominantly transitional cells) and 8-week spleen (mainly mature B cells) yielded hundreds of genes. We also examined the B cell-activating factor (BAFF)-dependent effects on immature splenic B cells by comparing expression patterns in the spleen between 2-week A/J vs 2-week A/WySnJ mice, which lack functional BAFF receptor signaling. Genes that showed the expression differences between spleen and bone marrow samples were then analyzed through quantitative PCR on B-cell subsets isolated using two different sorting protocols. A comparison of the results from our study with the results from other analyses showed not only some overlap of preferentially expressed genes but also an expansion of other genes potentially involved in B-cell development.

show abstract

Spi-C Has Opposing Effects to PU.1 on Gene Expression in Progenitor B Cells

Cited by 29 publications

References 64 publications

Regulation of the Interleukin-7 Receptor α Promoter by the Ets Transcription Factors PU.1 and GA-binding Protein in Developing B Cells

Regulation of the Interleukin-7 Receptor α Promoter by the Ets Transcription Factors PU.1 and GA-binding Protein in Developing B Cells

Reduction in PU.1 activity results in a block to B-cell development, abnormal myeloid proliferation, and neonatal lethality

Defining a transcriptional fingerprint of murine splenic B-cell development

Contact Info

Product

Resources

About