GA-binding Protein-dependent Transcription Initiator Elements

CK2␣ is one of two isoforms of protein kinase CK2, a highly conserved, ubiquitous, and vital phosphotransferase whose expression is kept at constant cellular levels and whose dysregulated expression has been linked to malignant diseases. The upstream sequence of the gene coding for human CK2␣ (CSNK1A1, chromosomal location 20p13) has been examined for promoter location and transcription factor interactions using reporter gene assays (luciferase; HeLa cells), site-directed mutagenesis, electrophoretic mobility shift assays, super-shifts, UV cross-linking, Western blotting, and DNA affinity chromatography. Highest promoter activity has been found in a region comprising positions ؊9 to 46. Factors Sp1, Ets-1, and NF-B have been identified as interaction partners and, by mutation of individual sites and simultaneous mutations of two or more sites, shown to cross-talk to each other. At least two of the factors (Sp1; NF-B) were susceptible to phosphorylation by CK2 holoenzyme, a tetramer composed of two CK2␣ and two regulatory CK2␤ proteins, but not by individual CK2␣. Because the phosphorylation decreases promoter binding and repeated immunoprecipitation reveals presence of "free" CK2␤ in cell extracts, it is tempting to speculate that the gene product CK2␣ might readily form CK2 holoenzyme and feed back onto gene transcription. The data represent the first promoter control analysis of a mammalian CK2␣ gene and provide a hypothesis of how the constant expression level of CK2␣ may be achieved. Protein kinase CK21 (also named casein kinase II) is a pleiotropic, ubiquitous, and conserved Ser/Thr kinase that is essential for viability of eukaryotes. CK2 occurs in two highly related isoforms, CK2␣ and CK2␣Ј. Both of these occur as tetrameric holoenzymes complexed stoichiometrically to regulatory CK2␤ proteins. This tetrameric structure is also highly conserved and required for appropriate control of substrate specificity. Although a considerable number of substrates has been documented, comprising proteins involved in processes such as transcription, replication, translation, and signaling, the exact physiological role of CK2 remains poorly understood. However, CK2 has been linked to proliferation, transformation, and cell cycle regulation (reviewed in Refs.

Section: Discussionmentioning

confidence: 99%

Transcription Factors Ets1, NF-κB, and Sp1 Are Major Determinants of the Promoter Activity of the Human Protein Kinase CK2α Gene

Krehan¹,

Ansuini²,

Böcher³

et al. 2000

“…Further mutations that destroy SPy's ability to interact with TC1, TC2, and TC3 in a single-stranded manner could also prove to be most informative. The association of transcription sites with the Pu:Py tracts (35) suggests they may be acting as initiator (Inr) elements, possibly similar to the atypical Inrs detailed in the majority of ribosomal protein (36) as well as other promoters (37). These Inrs are characterized as a 12-20-bp Pu:Py tract often buried within a GC-rich sequence.…”

Section: Spy and Sp1 Act Together In The Transactivation Of The C-srcmentioning

confidence: 99%

Transcription of the Human c-Src Promoter Is Dependent on Sp1, a Novel Pyrimidine Binding Factor SPy, and Can Be Inhibited by Triplex-forming Oligonucleotides

Ritchie¹,

Boyd²,

Wong³

et al. 2000

The tyrosine kinase pp60 c-src has been implicated in the regulation of numerous normal physiological processes as well the development of several human cancers. However, the mechanisms regulating its expression have not been addressed. In the present study, we report the presence of two Sp1/Sp3 binding sites and three polypurine:polypyrimidine (Pu:Py) tracts in the c-Src promoter that are essential for controlling expression. We demonstrate that Sp1, but not Sp3, is capable of activating the c-Src promoter and that Sp3 is also capable of inhibiting Sp1-mediated transactivation. The presence of multiple Pu:Py tracts conferred S1 sensitivity on plasmids in vitro, suggesting they are capable of adopting non B-DNA conformations. These tracts specifically bind a nuclear factor we named SPy (Src pyrimidine binding factor), which demonstrates both novel double-and single-stranded binding specificities. Mutations eliminating SPy binding compromised Src transcriptional activity, especially in concert with additional mutations affecting Sp1 binding, suggesting the two factors may cooperate in regulating c-Src expression. Finally, we demonstrate that triplex-forming oligonucleotides designed to target both Sp1 and SPy binding sites can down-regulate c-Src expression in vitro, suggesting a potential therapeutic approach to controlling c-Src expression in diseases where aberrant expression or activity has been documented.

“…1C) has the initiator consensus sequence TCATTC (31). This raises the possibility that function of the Il7r promoter utilizes interactions between the Ets binding site and initiator sequences, a combination that has been previously described (32,33). To test if initiator sequences are utilized in the Il7r promoter, we used site-directed mutagenesis to change the DNA sequence at Ϫ120 (see Fig.…”

Section: An Intact Ets Site Is Essential For Activity Of the Il7r Promentioning

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

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 ...