Subtraction Cloning and Initial Characterization of Novel Epo-Immediate Response Genes

Gregory, Richard C.; Lord, K. A.; Panek, Leigh B.; Gaines, Peter; Dillon, Susan B.; Wojchowski, Don M.

doi:10.1006/cyto.2000.0686

Cited by 26 publications

(27 citation statements)

References 53 publications

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“…To identify potential EKLF target genes, subtractive hybridization was performed with total RNA isolated from day 13.5 fetal livers of wild-type (WT) and EKLF-deficient mice (78). This method has successfully been applied to a large number of applications, including gene expression in cancer, development, and hematopoiesis (28,30,62). In the subtraction utilizing wild-type RNA as the tester population and EKLF-deficient fetal liver RNA as the driver, ϳ175 differentially expressed clones were identified and subjected to sequence analysis.…”

Section: Resultsmentioning

confidence: 99%

Alterations in Expression and Chromatin Configuration of the Alpha Hemoglobin-Stabilizing Protein Gene in Erythroid Krüppel-Like Factor-Deficient Mice

Pilon

Nilson

Zhou

et al. 2006

Molecular and Cellular Biology

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Erythroid Krüppel-like factor (EKLF) is an erythroid zinc finger protein identified by its interaction with a CACCC sequence in the ␤-globin promoter, where it establishes local chromatin structure permitting ␤-globin gene transcription. We sought to identify other EKLF target genes and determine the chromatin status of these genes in the presence and absence of EKLF. We identified alpha hemoglobin-stabilizing protein (AHSP) by subtractive hybridization and demonstrated a 95 to 99.9% reduction in AHSP mRNA and the absence of AHSP in EKLF-deficient cells. Chromatin at the AHSP promoter from EKLF-deficient cells lacked a DNase I hypersensitive site and exhibited histone hypoacetylation across the locus compared to hyperacetylation of wild-type chromatin. Wild-type chromatin demonstrated a peak of EKLF binding over a promoter region CACCC box that differs from the EKLF consensus by a nucleotide. In mobility shift assays, the AHSP promoter CACCC site bound EKLF in a manner comparable to the ␤-globin promoter CACCC site, indicating a broader recognition sequence for the EKLF consensus binding site. The AHSP promoter was transactivated by EKLF in K562 cells, which lack EKLF. These results support the hypothesis that EKLF acts as a transcription factor and a chromatin modulator for the AHSP and ␤-globin genes and indicate that EKLF may play similar roles for other erythroid genes.

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Section: Resultsmentioning

confidence: 99%

Alterations in Expression and Chromatin Configuration of the Alpha Hemoglobin-Stabilizing Protein Gene in Erythroid Krüppel-Like Factor-Deficient Mice

Pilon

Nilson

Zhou

et al. 2006

Molecular and Cellular Biology

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“…To the best of our knowledge, Rcd-1 is the first arm repeat protein exhibiting this property, adding to the protein family's characterized potential to interact with other proteins or small ligands (Huber et al 1997;Conti et al 1998;Conti and Kuriyan 2000;Daniels and Weis 2002;Ha et al 2004). Rcd-1's nucleic acid binding suggests an additional mechanistic feature by which it could exert its role in cellular differentiation (Okazaki et al 1998;Gregory et al 2000;Chen et al 2001;Friedman 2002;Hiroi et al 2002). However, understanding the process of cellular differentiation lies not in any single protein but, rather, in the way larger protein complexes and signaling pathways orchestrate the changes in transcription and translation, thereby bringing about changes to the cell as a whole; whether Rcd-1's nucleotide interactions occur in vivo, regulated by other proteins, represents an attractive hypothesis but remains to be investigated further.…”

Section: Discussionmentioning

confidence: 99%

“…Northern blots using human Rcd-1 as a probe showed significant expression in human testes, ovaries, and thymus (Okazaki et al 1998). mrcd1 was also isolated as an erythropoietin-responsive gene potentially involved in hematopoetic cell development (Gregory et al 2000). A recent finding reports that mRcd-1 interacts with the c-Myb protein (Haas et al 2004), which, in turn, is required for the generation of monocytes from a myeloid progenitor (for review, see Friedman 2002).…”

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confidence: 99%

Atomic model of human Rcd‐1 reveals an armadillo‐like‐repeat protein with in vitro nucleic acid binding properties

2007

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Rcd-1, a protein highly conserved across eukaryotes, was initially identified as a factor essential for nitrogen starvation-invoked differentiation in fission yeast, and its Saccharomyces cerevisiae homolog, CAF40, has been identified as part of the CCR4-NOT transcription complex, where it interacts with the NOT1 protein. Mammalian homologs are involved in various cellular differentiation processes including retinoic acid-induced differentiation and hematopoetic cell development. Here, we present the 2.2 Å X-ray structure of the highly conserved region of human Rcd-1 and investigate possible functional abilities of this and the full-length protein. The monomer is made up of six armadillo repeats forming a solventaccessible, positively-charged cleft 21-22 Å wide that, in contrast to other armadillo proteins, stays fully exposed in the dimer. Prompted by this finding, we established that Rcd-1 can bind to single-and double-stranded oligonucleotides in vitro with the affinity of G/C/T ) A. Mutation of an arginine residue within the cleft strongly reduced or abolished oligonucleotide binding. Rcd-1's ability to bind to nucleic acids, in addition to the previously reported protein-protein interaction with NOT1, suggests a new feature in Rcd-1's role in regulation of overall cellular differentiation processes.Keywords: Rcd-1; transcription; X-ray crystallography; structure Supplemental material: see www.proteinscience.org Cellular differentiation is an essential characteristic of cells required for the formation of multicellular organisms. However, many unicellular organisms, such as yeast, also differentiate in order to survive in hostile environments. Recent studies suggest that the mechanisms and factors involved in differentiation are not very cell type specific, let alone organism specific (Yamamoto et al. 1999;Hiroi et al. 2002).Schizosaccharomyces pombe, a fission yeast, resembles higher eukaryotes in several ways including genetic structure, cell cycle control, and various signaling pathways. It also undergoes cellular differentiation during sexual development involving conjugation, meiosis, and sporulation. Sexual differentiation is controlled primarily through two signals: nutrient starvation and the presence of mating pheromone. Transcriptional factor Ste11 is required and activates several genes involved in conjugation and meiosis (Kelly et al. 1988;Watanabe et al. 1988;Hughes et al. 1990;Sugimoto et al. 1991;Willer et al. 1995). Included are the mei2, rep1, and ste genes (including ste11), and other mating-type genes (Kelly et al. 1988;Watanabe et al. 1988;Hughes et al. 1990; Reprint requests to: Emil F. Pai, Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada; e-mail: pai@hera.med.utoronto.ca; fax: (416) 581-7545.Abbreviations: Rcd-1, required for cell differentiation. Article published online ahead of print. Article and publication date are at

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“…To test whether this bias in signaling perhaps reflected an unusual property of FDCER cells, Epo and SCF activation of ERKs also was assayed in primary erythroid cells expanded from marrow, and in splenocytes from mice treated with thiamphenicol. For each preparation, using transgenic mice expressing an EGF receptor-Epo receptor chimera from a GATA-1 gene-derived expression vector, we have shown previously that erythroid progenitor cells comprise Ն50% of expanded populations (13,39). As shown in Fig.…”

Section: Fig 1 Epo Receptor Forms Er-hy343 and Er-hy343f Expressionmentioning

confidence: 99%

Integrative Signaling by Minimal Erythropoietin Receptor Forms and c-Kit

Pircher¹,

Geiger²,

Zhang

et al. 2001

Journal of Biological Chemistry

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Erythroid homeostasis depends critically upon erythropoietin (Epo) and stem cell factor cosignaling in late progenitor cells. Epo bioresponses are relayed efficiently by minimal receptor forms that retain a single Tyr-343 site for STAT5 binding, while forms that lack all cytoplasmic Tyr(P) sites activate JAK2 and the transcription of c-Myc plus presumed additional target genes. In FDCER cell lines, which express endogenous c-Kit, the signaling capacities of such minimal Epo receptor forms (ER-HY343 and ER-HY343F) have been dissected to reveal: 1) that Epo-dependent mitogenesis, survival, and bcl-x gene expression via ER-HY343 depend upon the intactness of the Tyr-343 STAT5 binding site; 2) that ER-HY343-dependent bcl-x L gene transcription is enhanced markedly via c-Kit; 3) that socs-3, plfap, dpp-1, and cacy-bp gene transcription is induced via ER-HY343, whereas dpp-1 and cacy-bp gene expression is also supported by ER-HY343F; 4) that ectopically expressed SOCS-3 suppresses proliferative signaling by not only ER-HY343 but also c-Kit; and 5) that in FDCER and primary erythroid cells, c-Kit appears to provide the primary route to MAPK activation. Thus, integration circuits exist in only select downstream pathways within Epo and stem call factor receptor signaling. Epo,1 the prime hormonal regulator of red cell development, initiates its effects by binding to receptor dimers on the surface of erythroid burst-and colony-forming units and activating the tethered Janus family kinase (JAK) 2 (1, 2). JAK2 then mediates the phosphorylation of eight cytoplasmic tyrosine sites within the Epo receptor, and via these sites, a complex set of Src homology 2 domain-encoding effectors (and associated cofactors) are engaged. These include STAT 5A and B; Grb2/ mSOS/Raf/Ras; phosphatidylinositol 3-kinase, phospholipase-␥1; and SHIP; Lyn, Syk, and Tec; SHPTP-1 and 2; the nucleotide exchange factors Vav and C3G (via Cbl); Cis and SOCS-3; and the adaptors Shc, Gab1, Gab2, CrkL, APS, and IRS-2 (reviewed by Wojchowski et al. (Ref. 3)). Although many of these are proto-oncogenic growth regulators, others are negative effectors whose action in terminating Epo-stimulated events is likewise crucial to regulated erythropoiesis. These include Cis, which appears to compete with STAT5 for binding at Tyr-343 (4, 5); SHPTP-1, which acts to dephosphorylate JAK2 (6), and the suppressor of cytokine signaling, SOCS-3, which also binds and inhibits JAK2 (7). In SOCS-3 Ϫ/Ϫ mice, in fact, a fatal erythrocytosis is precipitated (8).Despite the complexity of this signaling network, studies of tyrosine-mutated and -truncated Epo receptors in cell lines (9, 10), murine fetal liver (11, 12), and adult murine marrow and spleen (13) 2)), definitive erythropoiesis fails and lethal embryonic anemias are engendered. In at least certain systems, however, Epo receptor forms that lack all cytoplasmic Tyr(P) sites (yet activate JAK2) also have been reported to retain significant bioactivity (4), and among STAT5 a Ϫ/Ϫ and b Ϫ/Ϫ mice those which survive embryonic stre...

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Subtraction Cloning and Initial Characterization of Novel Epo-Immediate Response Genes

Cited by 26 publications

References 53 publications

Alterations in Expression and Chromatin Configuration of the Alpha Hemoglobin-Stabilizing Protein Gene in Erythroid Krüppel-Like Factor-Deficient Mice

Alterations in Expression and Chromatin Configuration of the Alpha Hemoglobin-Stabilizing Protein Gene in Erythroid Krüppel-Like Factor-Deficient Mice

Atomic model of human Rcd‐1 reveals an armadillo‐like‐repeat protein with in vitro nucleic acid binding properties

Integrative Signaling by Minimal Erythropoietin Receptor Forms and c-Kit

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