FKLF-2: a novel Krüppel-like transcriptional factor that activates globin and other erythroid lineage genes

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3578-3584 ◽  
Author(s):  
Haruhiko Asano ◽  
Xi Susan Li ◽  
George Stamatoyannopoulos
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Zinc Finger Protein ◽  
Globin Gene ◽  
Northern Blot Hybridization ◽  
Gene Promoters ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Wide Range ◽  
Carboxyl Terminal

Abstract FKLF-2, a novel Krüppel-type zinc finger protein, was cloned from murine yolk sac. The deduced polypeptide sequence of 289 amino acids has 3 contiguous zinc fingers at the near carboxyl-terminal end, an amino-terminal domain characterized by its high content of alanine and proline residues and a carboxyl-terminal domain rich in serine residues. By Northern blot hybridization, the human homologue of FKLF-2 is expressed in the bone marrow and striated muscles and not in 12 other human tissues analyzed. FKLF-2 is constitutively expressed in established cell lines with an erythroid phenotype, but it is inconsistently expressed in cell lines with myeloid or lymphoid phenotypes. The expression of FKLF-2 messenger RNA (mRNA) is up-regulated after induction of mouse erythroleukemia cells. In luciferase assays, FKLF-2 activates predominantly the γ, and to a lesser degree, the ɛ and β globin gene promoters. The activation of γ gene promoter does not depend on the presence of an HS2 enhancer. FKLF-2 activates the γ promoter predominantly by interacting with the γ CACCC box, and to a lesser degree through interaction with the TATA box or its surrounding DNA sequences. FKLF-2 also activated all the other erythroid specific promoters we tested (GATA-1, glycophorin B, ferrochelatase, porphobilinogen deaminase, and 5-aminolevulinate synthase). These results suggest that in addition to globin, FKLF-2 may be involved in activation of transcription of a wide range of genes in the cells of the erythroid lineage.

FKLF-2: a novel Krüppel-like transcriptional factor that activates globin and other erythroid lineage genes

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3578-3584 ◽  
Author(s):  
Haruhiko Asano ◽  
Xi Susan Li ◽  
George Stamatoyannopoulos
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Zinc Finger Protein ◽  
Globin Gene ◽  
Northern Blot Hybridization ◽  
Gene Promoters ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Wide Range ◽  
Carboxyl Terminal

FKLF-2, a novel Krüppel-type zinc finger protein, was cloned from murine yolk sac. The deduced polypeptide sequence of 289 amino acids has 3 contiguous zinc fingers at the near carboxyl-terminal end, an amino-terminal domain characterized by its high content of alanine and proline residues and a carboxyl-terminal domain rich in serine residues. By Northern blot hybridization, the human homologue of FKLF-2 is expressed in the bone marrow and striated muscles and not in 12 other human tissues analyzed. FKLF-2 is constitutively expressed in established cell lines with an erythroid phenotype, but it is inconsistently expressed in cell lines with myeloid or lymphoid phenotypes. The expression of FKLF-2 messenger RNA (mRNA) is up-regulated after induction of mouse erythroleukemia cells. In luciferase assays, FKLF-2 activates predominantly the γ, and to a lesser degree, the ɛ and β globin gene promoters. The activation of γ gene promoter does not depend on the presence of an HS2 enhancer. FKLF-2 activates the γ promoter predominantly by interacting with the γ CACCC box, and to a lesser degree through interaction with the TATA box or its surrounding DNA sequences. FKLF-2 also activated all the other erythroid specific promoters we tested (GATA-1, glycophorin B, ferrochelatase, porphobilinogen deaminase, and 5-aminolevulinate synthase). These results suggest that in addition to globin, FKLF-2 may be involved in activation of transcription of a wide range of genes in the cells of the erythroid lineage.

scholarly journals The major low-molecular-weight heat shock protein in chloroplasts shows antigenic conservation among diverse higher plant species.

1989 ◽  
Vol 9 (2) ◽  
pp. 461-468 ◽  
Author(s):  
E Vierling ◽  
L M Harris ◽  
Q Chen
Keyword(s):  
Molecular Weight ◽  
Heat Shock ◽  
Plant Species ◽  
Fusion Proteins ◽  
Higher Plants ◽  
Low Molecular Weight ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Wide Range ◽  
Carboxyl Terminal

Several plant species are known to synthesize low-molecular-weight nucleus-encoded heat shock proteins (HSPs) which localize to chloroplasts. DNA sequence analysis of chloroplast HSP cDNAs from pea (Pisum sativum) and soybean (Glycine max) has shown that the carboxyl-terminal halves of these proteins are homologous to low-molecular-weight HSPs from a wide range of eucaryotes (E. Vierling, R. T. Nagao, A. E. DeRocher, and L. M. Harris, EMBO J. 7:575-581, 1988). We used a pea cDNA to construct fusion proteins containing either the carboxyl-terminal heat shock domain or the amino-terminal domain of the chloroplast HSP. The fusion proteins were overexpressed in Escherichia coli and used to produce choloroplast HSP-specific polyclonal antibodies. The carboxyl-terminal antibodies recognized chloroplast HSP precursor proteins from pea and from three divergent plant species, Arabidopsis thaliana, petunia (Petunia hybrida), and maize (Zea mays). The amino-terminal antibodies recognized effectively only the pea precursor. When intact plants of each species were subjected to a heat stress regime mimicking field growth conditions, significant levels of the mature forms of the chloroplast HSPs accumulated in pea, A. thaliana, and maize. The levels of accumulated HSPs remained unchanged for 12 h following the stress treatment. We conclude that the synthesis of chloroplast-localized HSPs is an important component of the stree response in all higher plants and that chloroplast HSPs from dicotyledonous and monocotyledonous plants have a conserved carboxyl-terminal domain.

scholarly journals FKLF, a Novel Krüppel-Like Factor That Activates Human Embryonic and Fetal β-Like Globin Genes

1999 ◽  
Vol 19 (5) ◽  
pp. 3571-3579 ◽  
Author(s):  
Haruhiko Asano ◽  
Xi Susan Li ◽  
George Stamatoyannopoulos
Keyword(s):  
Zinc Finger Protein ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Globin Gene ◽  
Transcriptional Activator ◽  
Gene Promoters ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Rt Pcr ◽  
Amino Terminal

ABSTRACT A cDNA encoding a novel Krüppel-type zinc finger protein, FKLF, was cloned from fetal globin-expressing human fetal erythroid cells. The deduced polypeptide sequence composed of 512 amino acids revealed that, like Sp1 and EKLF, FKLF has three contiguous zinc fingers at the near carboxyl-terminal end. A long amino-terminal domain is characterized by the presence of two acidic and two proline-rich regions. Reverse transcription (RT)-PCR assays using various cell lines demonstrated that the FKLF mRNA is expressed predominantly in erythroid cells. FKLF message is detectable by RT-PCR in fetal liver but not in adult bone marrow cells. As predicted from its structural features, FKLF is a transcriptional activator. In luciferase assays FKLF activated the γ- and ɛ-globin gene promoters, and, to a much lower degree, the β-globin promoter. Studies of HS2-γ gene reporter constructs carrying CACCC box deletions revealed that the CACCC box sequence of the γ gene promoter mediates the activation of the γ gene by FKLF. Other erythroid promoters (GATA-1, glycophorin B, ferrochelatase, porphobilinogen deaminase, and 5-aminolevulinate synthase) containing CACCC elements or GC-rich potential Sp1-binding sites were activated minimally, if at all, by FKLF, indicating that FKLF is not a general activator of genes carrying the CACCC motifs. Transfection of K562 cells with FKLF cDNA enhanced the expression of the endogenous ɛ- and γ-globin genes, suggesting an in vivo role of FKLF in fetal and embryonic globin gene expression. Our results indicate that the protein potentially encoded by the FKLF cDNA acts as a transcriptional activator of embryonic and fetal β-like globin genes.

The major low-molecular-weight heat shock protein in chloroplasts shows antigenic conservation among diverse higher plant species

1989 ◽  
Vol 9 (2) ◽  
pp. 461-468
Author(s):  
E Vierling ◽  
L M Harris ◽  
Q Chen
Keyword(s):  
Molecular Weight ◽  
Heat Shock ◽  
Plant Species ◽  
Fusion Proteins ◽  
Higher Plants ◽  
Low Molecular Weight ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Wide Range ◽  
Carboxyl Terminal

Several plant species are known to synthesize low-molecular-weight nucleus-encoded heat shock proteins (HSPs) which localize to chloroplasts. DNA sequence analysis of chloroplast HSP cDNAs from pea (Pisum sativum) and soybean (Glycine max) has shown that the carboxyl-terminal halves of these proteins are homologous to low-molecular-weight HSPs from a wide range of eucaryotes (E. Vierling, R. T. Nagao, A. E. DeRocher, and L. M. Harris, EMBO J. 7:575-581, 1988). We used a pea cDNA to construct fusion proteins containing either the carboxyl-terminal heat shock domain or the amino-terminal domain of the chloroplast HSP. The fusion proteins were overexpressed in Escherichia coli and used to produce choloroplast HSP-specific polyclonal antibodies. The carboxyl-terminal antibodies recognized chloroplast HSP precursor proteins from pea and from three divergent plant species, Arabidopsis thaliana, petunia (Petunia hybrida), and maize (Zea mays). The amino-terminal antibodies recognized effectively only the pea precursor. When intact plants of each species were subjected to a heat stress regime mimicking field growth conditions, significant levels of the mature forms of the chloroplast HSPs accumulated in pea, A. thaliana, and maize. The levels of accumulated HSPs remained unchanged for 12 h following the stress treatment. We conclude that the synthesis of chloroplast-localized HSPs is an important component of the stree response in all higher plants and that chloroplast HSPs from dicotyledonous and monocotyledonous plants have a conserved carboxyl-terminal domain.

The coordinate replication of the human beta-globin gene domain reflects its transcriptional activity and nuclease hypersensitivity

1988 ◽  
Vol 8 (11) ◽  
pp. 4958-4965
Author(s):  
V Dhar ◽  
D Mager ◽  
A Iqbal ◽  
C L Schildkraut
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Globin Gene ◽  
K562 Cells ◽  
Globin Genes ◽  
Beta Globin ◽  
Beta Globin Gene ◽  
Hypersensitive Sites ◽  
Flanking Sequences ◽  
Globin Gene Domain

The temporal order of replication of DNA sequences in the chromosomal domain containing the human beta-globin gene cluster and its flanking sequences (140 kilobases) was measured and compared in two different human cell lines. In human erythroleukemia (K562) cells, in which embryonic and fetal globin genes are transcribed, all of the sequences we examined from the beta-globin domain replicated early during S phase, while in HeLa cells, in which globin genes are transcriptionally silent, these sequences replicated late during S. Potential sites of initiation of DNA replication within this domain were identified. The beta-globin gene domain was also found to differ with respect to the nuclease sensitivity of the chromatin in these two cell lines. In K562 cells, hypersensitive sites for endogenous nucleases and DNase I were present in the chromatin near the earliest-replicating segments in the beta-globin domain.

Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes

1983 ◽  
Vol 3 (4) ◽  
pp. 596-604
Author(s):  
C A Whitlock ◽  
S F Ziegler ◽  
O N Witte
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Lymphoid Cells ◽  
Growth Potential ◽  
Malignant Growth ◽  
Wide Range ◽  
Feeder Layers ◽  
Growth Phenotype ◽  
Abelson Virus

Some molecular changes which correlate with the tumorigenic progression of neoplastic cells can best be studied with in vitro cell lines that represent each stage in the progression. Lymphoid cells infected by Abelson murine leukemia virus exhibit a wide range of growth potential in vitro and in vivo. Uncloned populations that are poorly oncogenic early after infection become progressively more oncogenic with successive passages of the cells in culture. In such mass cultures, it is difficult to evaluate whether a rare subpopulation of highly oncogenic cells becomes dominant in the culture or whether the individual cells progress in oncogenic phenotype. To examine this latter possibility, Abelson virus-infected lymphoid cells were cloned by limiting-dilution culture 10 days postinfection. We isolated two clones that grew poorly in agar, required feeder layers of adherent bone marrow cells for growth in liquid culture, and were extremely slow to form tumors in syngeneic animals. Both clones, after passage in the presence of adherent feeder layers for 3 months, grew well in liquid and agar-containing cultures in the absence of feeder layers and formed tumors in animals at a rapid rate. The progression of these clonal cell lines to a more malignant growth phenotype occurred in the absence of detectable changes in the concentration, half-life, phosphorylation, in vitro kinase activity, or cell localization of the Abelson virus-encoded transforming protein. No change in the concentration or arrangement of integrated Abelson viral DNA sequences was detected in either clone. Thus, perhaps changes in the expression of cellular genes would appear to alter the growth properties of lymphoid cells after their initial transformation by Abelson virus. Such cellular changes could complement the activity of the Abelson virus transforming protein in producing the fully malignant growth phenotype.

scholarly journals Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane.

1995 ◽  
Vol 130 (1) ◽  
pp. 15-27 ◽  
Author(s):  
B Soullam ◽  
H J Worman
Keyword(s):  
Membrane Protein ◽  
Nuclear Membrane ◽  
Structural Features ◽  
Inner Nuclear Membrane ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Integral Protein ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Amino Terminal Domain

We have examined transfected cells by immunofluorescence microscopy to determine the signals and structural features required for the targeting of integral membrane proteins to the inner nuclear membrane. Lamin B receptor (LBR) is a resident protein of the nuclear envelope inner membrane that has a nucleoplasmic, amino-terminal domain and a carboxyl-terminal domain with eight putative transmembrane segments. The amino-terminal domain of LBR can target both a cytosolic protein to the nucleus and a type II integral protein to the inner nuclear membrane. Neither a nuclear localization signal (NLS) of a soluble protein, nor full-length histone H1, can target an integral protein to the inner nuclear membrane although they can target cytosolic proteins to the nucleus. The addition of an NLS to a protein normally located in the inner nuclear membrane, however, does not inhibit its targeting. When the amino-terminal domain of LBR is increased in size from approximately 22.5 to approximately 70 kD, the chimeric protein cannot reach the inner nuclear membrane. The carboxyl-terminal domain of LBR, separated from the amino-terminal domain, also concentrates in the inner nuclear membrane, demonstrating two nonoverlapping targeting signals in this protein. Signals and structural features required for the inner nuclear membrane targeting of proteins are distinct from those involved in targeting soluble polypeptides to the nucleoplasm. The structure of the nucleocytoplasmic domain of an inner nuclear membrane protein also influences targeting, possibly because of size constraints dictated by the lateral channels of the nuclear pore complexes.

scholarly journals Ty3 Capsid Mutations Reveal Early and Late Functions of the Amino-Terminal Domain

2007 ◽  
Vol 81 (13) ◽  
pp. 6957-6972 ◽  
Author(s):  
Liza S. Z. Larsen ◽  
Min Zhang ◽  
Nadejda Beliakova-Bethell ◽  
Virginia Bilanchone ◽  
Anne Lamsa ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Helical Structure ◽  
Spherical Particles ◽  
P Bodies ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Fluorescence And Electron Microscopy ◽  
Carboxyl Terminal ◽  
Rna Protection ◽  
Amino Terminal Domain

ABSTRACT The Ty3 retrotransposon assembles into 50-nm virus-like particles that occur in large intracellular clusters in the case of wild-type (wt) Ty3. Within these particles, maturation of the Gag3 and Gag3-Pol3 polyproteins by Ty3 protease produces the structural proteins capsid (CA), spacer, and nucleocapsid. Secondary and tertiary structure predictions showed that, like retroviral CA, Ty3 CA contains a large amount of helical structure arranged in amino-terminal and carboxyl-terminal bundles. Twenty-six mutants in which alanines were substituted for native residues were used to study CA subdomain functions. Transposition was measured, and particle morphogenesis and localization were characterized by analysis of protein processing, cDNA production, genomic RNA protection, and sedimentation and by fluorescence and electron microscopy. These measures defined five groups of mutants. Proteins from each group could be sedimented in a large complex. Mutations in the amino-terminal domain reduced the formation of fluorescent Ty3 protein foci. In at least one major homology region mutant, Ty3 protein concentrated in foci but no wt clusters of particles were observed. One mutation in the carboxyl-terminal domain shifted assembly from spherical particles to long filaments. Two mutants formed foci separate from P bodies, the proposed sites of assembly, and formed defective particles. P-body association was therefore found to be not necessary for assembly but correlated with the production of functional particles. One mutation in the amino terminus blocked transposition after cDNA synthesis. Our data suggest that Ty3 proteins are concentrated first, assembly associated with P bodies occurs, and particle morphogenesis concludes with a post-reverse transcription, CA-dependent step. Particle formation was generally resistant to localized substitutions, possibly indicating that multiple domains are involved.

Sign in / Sign up

Export Citation Format

Share Document