Functional domains of the TGF-β-inducible transcription factor Tieg3 and detection of two putative nuclear localization signals within the zinc finger DNA-binding domain

2007 ◽  
Vol 101 (3) ◽  
pp. 712-722 ◽  
Author(s):  
Björn Spittau ◽  
Ziyuan Wang ◽  
Dagmara Boinska ◽  
Kerstin Krieglstein
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Zinc Finger ◽  
Nuclear Localization ◽  
Binding Domain ◽  
Functional Domains ◽  
Dna Binding Domain ◽  
Nuclear Localization Signals

scholarly journals Regions outside the DNA-binding domain are critical for proper in vivo specificity of an archetypal zinc finger transcription factor

2013 ◽  
Vol 42 (1) ◽  
pp. 276-289 ◽  
Author(s):  
J. Burdach ◽  
A. P. W. Funnell ◽  
K. S. Mak ◽  
C. M. Artuz ◽  
B. Wienert ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Zinc Finger ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Zinc Finger Transcription Factor

New Insights into the Mechanism of Dominant Anemia Caused By Zinc Finger Mutations in KLF1

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 740-740
Author(s):  
Andrew C Perkins ◽  
Kevin R Gillinder ◽  
Graham Magor ◽  
Mathieu Lajoie ◽  
Timothy L Bailey ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Zinc Finger ◽  
Fetal Liver ◽  
Binding Domain ◽  
Erythroid Cell ◽  
Dna Binding Domain ◽  
Rna Seq ◽  
Wild Type ◽  
Cell Biol

Abstract Krûppel-like factor-1 (KLF1) is an essential erythroid-specific transcription factor [1, 2]. A number of studies have shown up to ~700 genes are poorly expressed when KLF1 is absent [3-6]. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice, and partly responsible for congenital dyserythropoietic anemia type IV (CDA-IV) observed in humans with dominant mutations in the DNA-binding domain of KLF1 [7]. Recently an ENU-generated mouse model of neonatal anemia, ‘nan’, was also reported to harbour a mutation in the second zinc-finger of KLF1 [8]. Remarkably, the ‘nan’ mutation (E339D) resides at exactly the same amino acid which results in human CDA IV (= E325 in humans). Unlike loss of function point mutations in KLF1, this mutation leads to a more severe phenotype than the KLF1 null allele, suggesting it is an unusual dominant mutation [9]. To investigate how this mutation might cause disease, we introduced tamoxifen-inducible versions of KLF1 and KLF1nan into an erythroid cell line derived from Klf1-/- fetal liver cells [10]. We performed ChIP-seq to determine differences in genome occupancy in vivo, and identified novel sites occupied by EKLF-E339D but not by wild type KLF1. Using de novo motif discovery [11], we find KLF1nan binds a slightly degenerate CACC box element (CCMNGCCC) in comparison with wild type KLF1 (CCMCRCCC). This specificity is novel with respect to any known TFs, so we think it represents a sequence specificity not normally encoded in mammals. Ectopic binding to non-erythroid gene promoters is accompanied by aberrant gene expression as determined by 4sU labelling and deep sequencing of tamoxifen-induced primary nuclear RNAs. We find a 4-fold greater number of genes induced by KLF1-nan compared with wild type KLF1 which is consistent with degenerate genome occupancy. We compared the KLF1-nan dependent genes with RNA-seq performed in primary fetal liver for KLF1+/nan versus KLF1+/- mice. We confirmed aberrant binding using EMSA and surface plasmon resonance (SPR) using recombinant GST-Klf1 zinc finger domains expressed in E.coli. The degenerate motif is consistent with structural models of how the second zinc finger of KLF1 specifically interacts with its binding site [12, 13]. We are undertaking structural studies to confirm this modelling. Together RNA-seq, ChIP-seq and SPR studies have provided a novel explanation for how mutations in KLF1 result in dominant anemia in mice and man. To our knowledge this mechanism, whereby a transcription factor DNA-binding domain mutation leads to promiscuous binding, activation of an aberrant transcriptional program and subsequent derailing of co-ordinated differentiation, is novel. References: 1.Perkins, A.C., A.H. Sharpe, and S.H. Orkin. Nature, 1995. 375(6529): p. 318-22. 2.Nuez, B., et al., Nature, 1995. 375(6529): p. 316-8. 3.Pilon, A.M., et al., Mol Cell Biol, 2006. 26(11): p. 4368-77. 4.Drissen, R., et al., Mol Cell Biol, 2005. 25(12): p. 5205-14. 5.Hodge, D., et al., Blood, 2006. 107(8): p. 3359-70. 6.Tallack, M.R., et al., Genome Res, 2012. 22(12):2385-98 7.Arnaud, L., et al., Am J Hum Genet. 87(5): p. 721-7. 8.Siatecka, M., et al., Proc Natl Acad Sci U S A. 2010. 107(34):15151-6 9.Heruth, D.P., et al., Genomics, 2010. 96(5): p. 303-7. 10.Coghill, E., et al., Blood, 2001. 97(6): p. 1861-1868. 11.Bailey, T.L., et al., Nucleic Acids Res, 2009. 37(Web Server issue): p. W202-8. 12.Schuetz, A., et al., Cell Mol Life Sci, 2011. 68(18): p. 3121-31. 13.Oka, S., et al., Biochemistry, 2004. 43(51): p. 16027-35. Disclosures No relevant conflicts of interest to declare.

scholarly journals The recessive phenotype displayed by a dominant negative microphthalmia-associated transcription factor mutant is a result of impaired nucleation potential.

1996 ◽  
Vol 16 (3) ◽  
pp. 1203-1211 ◽  
Author(s):  
K Takebayashi ◽  
K Chida ◽  
I Tsukamoto ◽  
E Morii ◽  
H Munakata ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
T Antigen ◽  
Binding Domain ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Negative Effect

In the DNA binding domain of microphthalmia-associated transcription factor (MITF), four mutations are reported: mi, Mi wh, mi ew, and mi or. MITFs encoded by the mi, Mi wh, mi ew, and Mi or mutant alleles (mi-MITF, Mi wh-MITF, Mi ew-MITF, and Mi or-MITF, respectively) interfered with the DNA binding of wild-type MITF, TFE3, and another basic helix-loop-helix leucine zipper protein in vitro. Polyclonal antibody against MITF was produced and used for investigating the subcellular localization of mutant MITFs. Immunocytochemistry and immunoblotting revealed that more than 99% of wild-type MITF and Mi wh-MITF located in nuclei of transfected NIH 3T3 and 293T cells. In contrast, mi-MITF predominantly located in the cytoplasm of cells transfected with the corresponding plasmid. When the immunoglobulin G (IgG)-conjugated peptides representing a part of the DNA binding domain containing mi and Mi wh mutations were microinjected into the cytoplasm of NRK49F cells, wild-type peptide and Mi wh-type peptide-IgG conjugate localized in nuclei but mi-type peptide-IgG conjugate was detectable only in the cytoplasm. It was also demonstrated that the nuclear translocation potential of Mi or-MITF was normal but that Mi ew-MITF was impaired as well as mi-MITF. In cotransfection assay, a strong dominant negative effect of Mi wh-MITF against wild-type MITF-dependent transactivation system on tyrosinase promoter was observed, but mi-MITF had a small effect. However, by the conjugation of simian virus 40 large-T-antigen-derived nuclear localization signal to mi-MITF, the dominant negative effect was enhanced. Furthermore, we demonstrated that the interaction between wild-type MITF and mi-MITF occurred in the cytoplasm and that mi-MITF had an inhibitory effect on nuclear localization potential of wild-type MITF.

scholarly journals Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

2012 ◽  
Vol 11 (12) ◽  
pp. 1441-1450 ◽  
Author(s):  
Hong-Ming Hsu ◽  
Yu Lee ◽  
Dharmu Indra ◽  
Shu-Yi Wei ◽  
Hsing-Wei Liu ◽  
...  
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Trichomonas Vaginalis ◽  
Structural Integrity ◽  
Nuclear Translocation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Nuclear Localization Signals ◽  
Localization Signal

ABSTRACTInTrichomonas vaginalis, a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNA-binding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals.

scholarly journals Metal-Responsive Transcription Factor 1 (MTF-1) Activity Is Regulated by a Nonconventional Nuclear Localization Signal and a Metal-Responsive Transactivation Domain

2009 ◽  
Vol 29 (23) ◽  
pp. 6283-6293 ◽  
Author(s):  
Uschi Lindert ◽  
Mirjam Cramer ◽  
Michael Meuli ◽  
Oleg Georgiev ◽  
Walter Schaffner
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Zinc Fingers ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Localization Signal ◽  
Transcription Factor 1

ABSTRACT Metal-responsive transcription factor 1 (MTF-1) mediates both basal and heavy metal-induced transcription of metallothionein genes and also regulates other genes involved in the cell stress response and in metal homeostasis. In resting cells, MTF-1 localizes to both the cytoplasm and the nucleus but quantitatively accumulates in the nucleus upon metal load and under other stress conditions. Here we show that within the DNA-binding domain, a region spanning zinc fingers 1 to 3 (amino acids [aa] 137 to 228 in human MTF-1) harbors a nonconventional nuclear localization signal. This protein segment confers constitutive nuclear localization to a cytoplasmic marker protein. The deletion of the three zinc fingers impairs nuclear localization. The export of MTF-1 to the cytoplasm is controlled by a classical nuclear export signal (NES) embedded in the acidic activation domain. We show that this activation domain confers metal inducibility in distinct cell types when fused to a heterologous DNA-binding domain. Furthermore, the cause of a previously described stronger inducibility of human versus mouse MTF-1 could be narrowed down to a 3-aa difference in the NES; “humanizing” mouse MTF-1 at these three positions enhanced its metal inducibility to the level of human MTF-1.

NMR Structure of Transcription Factor Sp1 DNA Binding Domain†,‡

Biochemistry ◽  
2004 ◽  
Vol 43 (51) ◽  
pp. 16027-16035 ◽  
Author(s):  
Shinichiro Oka ◽  
Yasuhisa Shiraishi ◽  
Takuya Yoshida ◽  
Tadayasu Ohkubo ◽  
Yukio Sugiura ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Domain ◽  
Nmr Structure ◽  
Dna Binding Domain ◽  
Transcription Factor Sp1

scholarly journals Solution Structure of the DNA-Binding Domain of the Tomato Heat-Stress Transcription Factor HSF24

1996 ◽  
Vol 236 (3) ◽  
pp. 911-921 ◽  
Author(s):  
Jurgen Schultheiss ◽  
Olaf Kunert ◽  
Uwe Gase ◽  
Klaus-Dieter Scharf ◽  
Lutz Nover ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Heat Stress ◽  
Dna Binding ◽  
Solution Structure ◽  
Binding Domain ◽  
Dna Binding Domain

scholarly journals The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

1990 ◽  
Vol 10 (10) ◽  
pp. 5128-5137 ◽  
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Binding Specificity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Transcription Activator ◽  
Activator Proteins ◽  
Dna Binding Specificity

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

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