Amino-terminal Zinc Finger Research Articles

Absence of the Rpb9 subunit of RNA polymerase II reduces the chronological life span in fission yeast.

Fission yeast RNA polymerase II consists of 12 subunits, Rpb1-Rpb12. Among these subunits, Rpb9 is the only subunit whose absence does not cause lethality under optimum growth conditions in fission yeast. However, an rpb9 null fission yeast mutant exhibits a slow-growth phenotype under optimum growth conditions and a defect in survival under environmental and genotoxic stress conditions. To further gain an understanding of its physiological roles, in the present study we have elucidated the role of the Rpb9 subunit in chronological aging using fission yeast as the model organism. Our results provide evidence that the absence of Rpb9 reduces the chronological life span in fission yeast. Our data further shows that lack of Rpb9 in fission yeast causes oxidative stress sensitivity and accumulation of reactive oxygen species during the stationary phase. Our domain mapping experiments have demonstrated that the Rpb9 region encompassing its amino-terminal zinc finger domain and the central linker region is important for the role of Rpb9 in chronological aging. Finally, we also show that expression of the budding yeast or human Rpb9 ortholog can functionally complement the reduced chronological life span phenotype of the fission yeast rpb9 deletion mutant. Taken together, our study has identified a new role of the Rpb9 subunit in chronological aging.

Deletion of the non-essential Rpb9 subunit of RNA polymerase II results in pleiotropic phenotypes in Schizosaccharomyces pombe

Schizosaccharomyces pombe RNA polymerase II comprises twelve different subunits. Its Rpb9 subunit comprises 113 amino acids, and is the only non-essential subunit of S. pombe RNA polymerase II. However, its functions have not been studied in S. pombe. The results presented in this study demonstrate that Rpb9 is involved in regulating growth under optimum and certain stress conditions in S. pombe. To further address the role (s) of various domains of this subunit in regulating these phenotypes, deletion mutant analysis was done. We observed that the region spanning 1–74 amino acids, encompassing the amino-terminal zinc finger domain and the linker region of Rpb9 was able to rescue the phenotypes associated with rpb9+deletion. We also demonstrate that the functions of this subunit are only partially conserved among yeast and humans. Our computational biology approaches provide a structural basis for the differential role of various Rpb9 domains in S. pombe. Furthermore, using these tools we show that there has been a co-evolution of the interaction residues between the Rpb9 subunit and the two largest subunits of RNA polymerase II, allowing for a more stringent organism-specific packing. Taken together, our results have provided functional and structural insights into the Rpb9 subunit of S. pombe.

GATA1 mutations in red cell disorders.

GATA1 is an essential regulator of erythroid cell gene expression and maturation. In its absence, erythroid progenitors are arrested in differentiation and undergo apoptosis. Much has been learned about GATA1 function through animal models, which include genetic knockouts as well as ones with decreased levels of expression. However, even greater insights have come from the finding that a number of rare red cell disorders, including Diamond-Blackfan anemia, are associated with GATA1 mutations. These mutations affect the amino-terminal zinc finger (N-ZF) and the amino-terminus of the protein, and in both cases can alter the DNA-binding activity, which is primarily conferred by the third functional domain, the carboxyl-terminal zinc finger (C-ZF). Here we discuss the role of GATA1 in erythropoiesis with an emphasis on the mutations found in human patients with red cell disorders.

Human leukemia mutations corrupt but do not abrogate GATA-2 function

By inducing the generation and function of hematopoietic stem and progenitor cells, the master regulator of hematopoiesis GATA-2 controls the production of all blood cell types. Heterozygous GATA2 mutations cause immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. GATA2 disease mutations commonly disrupt amino acid residues that mediate DNA binding or cis-elements within a vital GATA2 intronic enhancer, suggesting a haploinsufficiency mechanism of pathogenesis. Mutations also occur in GATA2 coding regions distinct from the DNA-binding carboxyl-terminal zinc finger (C-finger), including the amino-terminal zinc finger (N-finger), and N-finger function is not established. Whether distinct mutations differentially impact GATA-2 mechanisms is unknown. Here, we demonstrate that N-finger mutations decreased GATA-2 chromatin occupancy and attenuated target gene regulation. We developed a genetic complementation assay to quantify GATA-2 function in myeloid progenitor cells from Gata2 -77 enhancer-mutant mice. GATA-2 complementation increased erythroid and myeloid differentiation. While GATA-2 disease mutants were not competent to induce erythroid differentiation of Lin-Kit+ myeloid progenitors, unexpectedly, they promoted myeloid differentiation and proliferation. As the myelopoiesis-promoting activity of GATA-2 mutants exceeded that of GATA-2, GATA2 disease mutations are not strictly inhibitory. Thus, we propose that the haploinsufficiency paradigm does not fully explain GATA-2-linked pathogenesis, and an amalgamation of qualitative and quantitative defects instigated by GATA2 mutations underlies the complex phenotypes of GATA-2-dependent pathologies.

GATA1 Binding Kinetics on Conformation-Specific Binding Sites Elicit Differential Transcriptional Regulation.

GATA1 organizes erythroid and megakaryocytic differentiation by orchestrating the expression of multiple genes that show diversified expression profiles. Here, we demonstrate that GATA1 monovalently binds to a single GATA motif (Single-GATA) while a monomeric GATA1 and a homodimeric GATA1 bivalently bind to two GATA motifs in palindromic (Pal-GATA) and direct-repeat (Tandem-GATA) arrangements, respectively, and form higher stoichiometric complexes on respective elements. The amino-terminal zinc (N) finger of GATA1 critically contributes to high occupancy of GATA1 on Pal-GATA. GATA1 lacking the N finger-DNA association fails to trigger a rate of target gene expression comparable to that seen with the wild-type GATA1, especially when expressed at low level. This study revealed that Pal-GATA and Tandem-GATA generate transcriptional responses from GATA1 target genes distinct from the response of Single-GATA. Our results support the notion that the distinct alignments in binding motifs are part of a critical regulatory strategy that diversifies and modulates transcriptional regulation by GATA1.

Dissecting the Molecular Pathways That Underlie Disease-Causing GATA1 Mutations

AbstractAbstract 3439Missense mutations in the gene encoding hematopoietic transcription factor GATA1 cause congenital anemias and/or thrombocytopenias. All seven reported mutations give rise to amino acid substitutions within the amino-terminal zinc finger (NF), but produce a range of phenotypes. The clinical severity depends on the site and type of substitution, and different substitutions of the same residue can produce disparate phenotypes. We combined structural, biochemical, in vivo conditional rescue approaches, and genomic analyses to systematically characterize all known GATA1 mutations with the goal of determining how they alter GATA1 function to result in disease.Introducing mutant forms of GATA1 into GATA1-null erythroid or bipotential erythromegakaryocytic cell lines essentially recapitulated patient phenotypes. The V205M, G208S, G208R, and D218Y mutations severely impaired both erythroid and megakaryocyte maturation, while the R216Q, R216W, and D218G mutations had only a mild effect on the maturation of these lineages. Global differentiation defects were reproduced at the level of individual GATA1 target genes. Moreover, the former mutants greatly impaired both the transcriptional activation and repression functions of GATA1, while the latter moderately impaired gene activation but had no effect on repression.It had been suggested previously that GATA1 mutations could be categorized into two classes, those that impair binding of the NF to the essential GATA1 cofactor FOG1 (V205M, G208S, G208R) and those that diminish binding of the NF to DNA (R216Q and R216W). The impact of the final two mutations (D218G and D218Y) remained uncertain, as this residue is not part of any known interaction face.Our work led to the following novel conclusions: 1.Binding studies using isothermal titration calorimetry (ITC) and chromatin immunoprecipitation (ChIP) produced concurrent results showing that the V205M, G208S, G208R, and D218Y mutations diminish the GATA1-FOG1 interaction in vitro and FOG1 recruitment to GATA1 target genes in vivo. Interestingly, in contrast to D218Y, D218G did not affect FOG1 binding in vitro or in vivo. Furthermore, G208S had a less pronounced impact on FOG1 binding than the other three mutations, thus correlating the severity of the clinical presentation with the degree of FOG1 disruption. This confirms and extends previous work linking impaired FOG1 binding to the disease phenotypes associated with this class of mutations.2.ITC showed that R216Q and R216W disrupt DNA binding in vitro, consistent with previous in vitro studies. However, remarkably, ChIP assays revealed that neither mutation impaired in vivo GATA1 target site occupancy at any examined simple or palindromic GATA elements, suggesting that failure to bind DNA does not account for the associated clinical phenotypes.3.Notably, the R216Q and D218G mutations selectively diminished recruitment of Tal1/SCL without affecting the interaction with FOG1 or DNA. This implicates for the first time the Tal1/SCL complex in the pathogenesis of disorders caused by GATA1 mutations. Since the Tal1/SCL complex functions mostly during GATA1 gene activation, this also explains the observation that these GATA1 mutants largely retain their ability to repress transcription. Moreover, changes in the gene expression profiles of R216Q and D218G expressing cells are highly correlated with each other but clearly distinct from the gene expression changes associated with different substitutions at the same residues (R216W or D218Y), revealing a specific subset of genes that are most sensitive to disruption of the GATA1-Tal1/SCL interaction.4.An unexpected finding from our studies is that different substitutions of the same residue can disrupt binding to distinct cofactors (e.g. D218G impairs Tal1/SCL binding while D218Y impairs FOG1 binding), thus accounting for variable disease presentation.In concert, our work on GATA1 mutations in their native environment reveals critical new insights not obtainable from in vitro studies. This highlights the usefulness of gene complementation studies in the relevant lineages for the dissection of transcription pathways to better understand and ultimately diagnose and treat hematologic disease. Disclosures:No relevant conflicts of interest to declare.

The 3'-5' Exonuclease, TREX1, Interacts with Poly(ADP-ribose) Polymerase-1 (PARP1) in Response to DNA Damage

AbstractAbstract 1046 Background:Recognition of nucleic acids plays a key role in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). The major 3’-5’ DNA exonuclease, TREX1, degrades single- and double-stranded DNA to minimize potential immune activation by persistent self or retroelement DNA. Mutations in the human TREX1gene have been linked to four diseases: SLE; Aicardi-Goutières syndrome (AGS); retinal vasculopathy and cerebral leukodystrophy (RVCL); and familial chilblain lupus (CBL). SLE, AGS and CBL have overlapping features characterized by expression of interferon-alpha and autoantibody production, but are distinct clinical conditions. RVCL is a non-inflammatory endotheliopathy. Methods:As part of our efforts to determine the molecular mechanisms underlying TREX1 involvement in the pathogenesis of these diseases, we identified a novel TREX1-binding partner protein by Mass Spectrometry and Co-Immunoprecipitation (Co-IP) assays. We examined the interaction domains and the effects of mutations in TREX1 associated with these diseases. Results:We found that TREX1 interacted with poly(ADP-ribose) polymerase-1 (PARP1), a nuclear DNA repair enzyme involved in the DNA damage response. The interaction domain of PARP1 was mapped to the amino-terminal zinc finger domains. Subcellular localization analysis showed that TREX1 normally localized to the cytoplasm and cytomembranes, but translocated to the nucleus (with some bound to chromatin) in response to sublethal DNA damage. Co-IP assays revealed that the interaction was increased following DNA damage in concert with the post-translational PARlation of PARP1. Functional studies showed that TREX1 induced the PARlation of PARP1 in a TREX1 dose-dependent manner. Furthermore, siRNA knockdown of TREX1 promoted PARP1 cleavage and loss of PARP1 enzymatic activity, resulting in the induction of apoptosis. We further analyzed the effects of TREX1-PARP1 interaction in relation to the pathogenesis of AGS or RVCL. Co-IP assays revealed that the RVCL-related TREX1-mutant protein, V235fs, bound to PARP1 as well as wild-type TREX1, whereas the AGS-related R114H mutant protein did not bind PARP1. Conclusion:These findings demonstrate that TREX1 contributes to the regulation of PARP1 in the DNA damage response and that mutation-induced alterations in the function of TREX1 may be a factor in the development or progression of these autoimmune diseases by affecting PARP1 activity. Disclosures:No relevant conflicts of interest to declare.

Linking Transcription Factor Pathways to Disease-Causing GATA1 Mutations

Abstract 2371Missense mutations in the gene encoding hematopoietic transcription factor GATA1 cause congenital anemias and/or thrombocytopenias. Seven such mutations are reported. All of these give rise to amino acid substitutions within the amino terminal zinc finger (NF) of GATA1, producing a range of clinical phenotypes. Thus, V205M, G208R, and D218Y cause severe anemia and thrombocytopenia; G208S, R216Q, and D218G cause thrombocytopenia with minimal anemia; R216W gives rise to thrombocytopenia and congenital erythropoietic porphyria. One of these mutations, R216Q, occurs at the DNA binding interface and alters the ability of GATA1 to recognize a subset of cis motifs in vitro. Other mutations, including V205M, G208S, D218G, and D218Y, occur outside the DNA binding domain of the NF and inhibit interactions with the GATA1 cofactor FOG1 as determined by in vitro binding assays. However, these two mechanisms do not easily explain the broad spectrum of phenotypes associated with the mutations. For example, how do two substitutions of the same residue bring about disparate phenotypes?We examined the effects of each mutation on erythroid maturation, lineage-specific gene expression, in vivo target gene occupancy, and cofactor recruitment by introducing altered forms of GATA1 into murine GATA1-null proerythroblasts. The V205M, G208R, and D218Y mutations severely impaired erythroid maturation, recapitulating patient phenotypes. The G208S mutation also severely impaired erythroid maturation, causing a more pronounced defect than that expected from the clinical presentation. In contrast, R216Q and D218G produced mild effects in erythroid cells consistent with patient phenotypes. The porphyria-associated mutation R216W also produced relatively subtle effects in erythroid cells. We note that among the mutants, failure to activate gene expression strongly correlated with failure to repress gene expression. ChIP assays revealed that the V205M, G208R, and D218Y mutations impaired GATA1 target site occupancy. This indicates that despite normal DNA binding in vitro, the association with cofactor complexes is required for stable binding to chromatinized target sites in vivo. In contrast, the G208S mutant exhibited relatively normal chromatin occupancy, but reduced recruitment of FOG1 and SCL/Tal1 to GATA1-bound sites at erythroid genes. D218G also perturbed cofactor recruitment without greatly affecting GATA1 binding to its target genes. Notably, this mutation diminished SCL/Tal1 recruitment without significantly altering FOG1 occupancy. This implicates the SCL/Tal1 transcription complex in the pathogenesis of disorders caused by certain GATA1 mutations. Moreover, by uncoupling GATA1 chromatin occupancy and cofactor recruitment, G208S and D218G offer potentially useful tools for unraveling site-specific mechanisms of GATA1-regulated gene expression. Finally, both the R216Q and R216W mutants displayed relatively normal GATA1 chromatin occupancy and FOG1 and SCL/Tal1 recruitment at most sites. R216W presents as porphyria, and selective defects in the regulation of heme biosynthetic genes have yet be uncovered. Given that R216Q presents as thrombocytopenia, defects caused by this mutation may be revealed only in the context of megakaryocytes. Studies using similar rescue assays of a GATA1-null megakaryocyte-erythroid progenitor line are underway and will be discussed.In concert, our results reveal that in vivo analysis of GATA1 in its native environment provides mechanistic insights not obtainable from in vitro studies. Moreover, they demonstrate the usefulness of gene complementation assays for the dissection of transcription pathways surrounding normal and altered GATA1 to improve our understanding of disease. Disclosures:No relevant conflicts of interest to declare.

A Novel GATA1 Mutation (Ter414Arg) in a Family with the Rare X-Linked Blood Group Lu(a-b-) Phenotype.

Abstract 1979Poster Board I-1001The X-borne transcription factor GATA-1 is essential for erythroid and megakaryocyte development. In 1986, Norman et al. (Vox Sang 51:49) described a large family in which the rare Lu(a-b-) blood group phenotype is inherited as the result of an X-borne gene. Serologic and flow cytometric analyses confirmed the suppression of Lutheran blood group antigens on the red blood cells from the original male propositus. Analysis of DNA from the propositus revealed a mutation (1240T>C) in the termination codon of GATA1 converting TGA to a codon for arginine (CGA). The mutation predicts a translated GATA-1 protein containing an additional 41 amino acids at the carboxy terminus. DNA from an unaffected sister of the propositus had a wild-type GATA1 sequence. The GATA1 mutation was not present in 78 random blood donors as determined by restriction fragment length polymorphism (RFLP) analysis using BspHI. The propositus, who is now 64, has an Hb of 122 g/l, a low platelet count (86 × 109/l) with occasional macrothrombocytes (diameter 4-5 um) and a history of bruising easily. Several mutations in the amino terminal zinc finger domain of GATA-1 have been linked with thrombocytopenia and thalassemia or dsyerythropoietic anemia and the presence of macrothrombocytes. Our data provide evidence that mutations outside the zinc finger domain can affect GATA-1 functions in erythroid and megakaryocyte differentiation. In this case, the predicted extended carboxy terminus may interfere with GATA-1 interactions involving other DNA-binding proteins. Disclosures:No relevant conflicts of interest to declare.

GATA-factor dependence of the multitype zinc-finger protein FOG-1 for its essential role in megakaryopoiesis.

The function of GATA transcription factors in diverse developmental contexts depends in part on physical interaction with cofactors of the Friend of GATA (FOG) family. However, previous studies indicate that FOG-1 may play a GATA-1-independent role in early megakaryopoiesis, suggesting that FOG proteins might act in a GATA factor-independent manner. Here, we have generated mouse knock-in (KI) mutants harboring a critical valine-to-glycine substitution in the amino-terminal zinc fingers of GATA-1 and GATA-2 to ablate FOG interaction. In contrast to male GATA-1(KI) (GATA-1 is located on the X-chromosome) or GATA-2(KI/KI) mice, compound GATA-1(KI) GATA-2(KI/KI) mutant mice display complete megakaryopoietic failure, a phenocopy of FOG-1(-/-) mice. We conclude that FOG-1 requires an interaction with either GATA-1 or -2 as part of its essential role in early megakaryopoiesis. On the basis of these and previous reports, we infer that GATA factor dependence is a critical aspect of FOG protein function.

Platelet characteristics in patients with X-linked macrothrombocytopenia because of a novel GATA1mutation

AbstractA new mutation is described in the X-linked gene GATA1, resulting in macrothrombocytopenia and mild dyserythropoietic features but no marked anemia in a 4-generation family. The molecular basis for the observed phenotype is a substitution of glycine for aspartate in the strictly conserved codon 218 (D218G) of the amino-terminal zinc finger loop of the transcription factor GATA1. Zinc finger interaction studies demonstrated that this mutation results in a weak loss of affinity of GATA1 for its essential cofactor FOG1, whereas direct D218G-GATA1 binding to DNA was normal. The phenotypic effects of this mutation in the patients' platelets have been studied. Semiquantitative RNA analysis, normalized for β-actin messenger RNA, showed extremely low transcription of the GATA1 target genes GPIbβ and GPIXbut also a significantly lower expression of the nondirectly GATA1-regulated Gsα gene, suggestive of incomplete megakaryocyte maturation. In contrast, GPIIIa expression was close to normal in agreement with its early appearance during megakaryocyte differentiation. Flow cytometric analysis of patient platelets confirmed the existence of a platelet population with abnormal size distribution and reduced GPIb complex levels but with normal GPIIIa expression. It also showed the presence of very immature platelets lacking almost all membrane glycoproteins studied (GPIbα, GPIbβ, GPIIIa, GPIX, and GPV). Patients' platelets showed weak ristocetin-induced agglutination, compatible with the disturbed GPIb complex. Accordingly, electron microscopy of the patients' platelets revealed giant platelets with cytoplasmic clusters consisting of smooth endoplasmic reticulum and abnormal membrane complexes. In conclusion,GATA1 mutations can lead to isolated X-linked macrothrombocytopenia without anemia.

NMR and molecular dynamics studies of the hydration of a zinc finger-DNA complex

The hydration of a high-affinity protein-DNA complex involving the three amino terminal zinc finger domains of transcription factor IIIA (TFIIIA) and a 15-base-pair DNA duplex was investigated by NMR spectroscopy and molecular dynamics (MD) simulations. Intermolecular nuclear Overhauser effects (NOEs) between protein and water provided an experimental basis for identifying potential sites of hydration. These initial assignments were evaluated with the aid of two, 2 ns MD simulations of the protein-DNA complex conducted with the explicit inclusion of water solvent. The two independent simulations produced similar trends in terms of water residence times around the solute, and these results were used to separate protein-water NOEs from alternate exchange-relayed cross peaks. Furthermore, only six of the 170 protons which failed to show intermolecular NOEs to solvent showed nearby long-resident water molecules in the MD simulations, illustrating an impressive level of agreement between theory and experiment. Analyses of the MD trajectories also allowed an examination of the role of water in recognition and binding affinity of the zinc fingers with DNA. The interface is well hydrated, characterized by direct contacts between the protein and DNA, as well as mediating water bridges. Approximately 18 water-mediated hydrogen bonds between the protein and DNA were observed on average. Roughly half of these were water molecules with long residence times that are most likely to be important for binding, since they involve residues which have been shown through biochemical studies to be crucial for protein-DNA binding. This level of atomic detail could not otherwise be established through the existing NMR and crystal structures of the TFIIIA-DNA complex.

Inactivation of HIV-1 Nucleocapsid Protein P7 by Pyridinioalkanoyl Thioesters: CHARACTERIZATION OF REACTION PRODUCTS AND PROPOSED MECHANISM OF ACTION

The synthesis and antiviral properties of pyridinioalkanoyl thioester (PATE) compounds that target nucleocapsid p7 protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) have been described previously (Turpin, J. A., Song, Y., Inman, J. K., Huang, M., Wallqvist, A., Maynard, A., Covell, D. G., Rice, W. G., and Appella, E. (1999) J. Med. Chem. 42, 67-86). In the present study, fluorescence and electrospray ionization-mass spectrometry were employed to determine the mechanism of modification of NCp7 by two lead compounds, N-[2-(5-pyridiniovaleroylthio)benzoyl]sulfacetamide bromide and N-[2-(5-pyridiniovaleroylthio)benzoyl]-4-(4-nitrophenylsulfonyl )anili ne bromide (compounds 45 and 47, respectively). Although both compounds exhibit antiviral activity in cell-based assays, we failed to detect appreciable ejection of zinc from NCp7 under conditions in which previously described NCp7-active disulfides readily eject zinc. However, upon "activation" by Ag(+), compound 45 reacted with NCp7 resulting in the zinc ejection from both zinc fingers. The reaction followed a two-step mechanism in which zinc was ejected from the carboxyl-terminal zinc finger faster than from the amino-terminal zinc finger. Both compounds covalently modified the protein with pyridinioalkanoyl groups. Compound 45 modified cysteines 36 and 49 of the carboxyl-terminal zinc finger. The results obtained herein demonstrate that PATE compounds can be constructed that selectively target only one of the two zinc fingers of NCp7, thus providing an impetus to pursue development of highly selective zinc finger inhibitors.

ClpE, a novel type of HSP100 ATPase, is part of the CtsR heat shock regulon of Bacillus subtilis.

Clp ATPases, which include the ubiquitous HSP100 family, are classified according to their structural features and sequence similarities. During the course of the Bacillus subtilis genome sequencing project, we identified a gene encoding a new member of the HSP100 family. We designated this protein ClpE, as it is the prototype of a novel subfamily among the Clp ATPases, and have identified homologues in several bacteria, including Listeria monocytogenes, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, Lactobacillus sakei and Clostridium acetobutylicum. A unique feature of these Hsp100-type Clp ATPases is their amino-terminal zinc finger motif. Unlike the other class III genes of B. subtilis (clpC and clpP ), clpE does not appear to be required for stress tolerance. Transcriptional analysis revealed two sigmaA-type promoters, expression from which was shown to be inducible by heat shock and puromycin treatment. Investigation of the regulatory mechanism controlling clpE expression indicates that this gene is controlled by CtsR and is thus a member of the class III heat shock genes of B. subtilis. CtsR negatively regulates clpE expression by binding to the promoter region, in which five CtsR binding sites were identified through DNase I footprinting and sequence analysis.

Fzf1p of Saccharomyces cerevisiae is a positive regulator of SSU1 transcription and its first zinc finger region is required for DNA binding

The FZF1 gene of Saccharomyces cerevisiae encodes a five-zinc-finger transcription factor involved in sulphite tolerance. Previous work based on multicopy suppression analysis placed FZF1 upstream of SSU1, which encodes a plasma membrane protein and putative transporter also implicated in sulphite detoxification. Consistent with this analysis, Fzf1p was found to be a positive regulator of SSU1 transcription. The SSU1 promoter region involved in activation by FZF1 was defined, and the Fzf1 protein was shown to bind to it directly in vitro. Deletion of a single, amino-terminal zinc finger of Fzf1p resulted in loss of DNA binding, while the fourth and fifth zinc fingers were found to be dispensable. Copyright © 1999 John Wiley & Sons, Ltd.

Fzf1p of Saccharomyces cerevisiae is a positive regulator of SSU1 transcription and its first zinc finger region is required for DNA binding.

The FZF1 gene of Saccharomyces cerevisiae encodes a five-zinc-finger transcription factor involved in sulphite tolerance. Previous work based on multicopy suppression analysis placed FZF1 upstream of SSU1, which encodes a plasma membrane protein and putative transporter also implicated in sulphite detoxification. Consistent with this analysis, Fzf1p was found to be a positive regulator of SSU1 transcription. The SSU1 promoter region involved in activation by FZF1 was defined, and the FZF1 protein was shown to bind to it directly in vitro. Deletion of a single, amino-terminal zinc finger of Fzf1p resulted in loss of DNA binding, while the fourth and fifth zinc fingers were found to be dispensable.

Differential transcriptional activation by the N-terminal region of BRCA1 splice variants BRCA1a and BRCA1b.

The breast and ovarian cancer susceptibility gene BRCA1, is a nuclear phosphoprotein which functions as a tumor suppressor in human breast cancer cells. BRCA1 protein contains an amino-terminal zinc finger motif and a carboxy-terminal acidic region. Recently, the carboxy-terminal region of BRCA1 and the amino-terminal region of BRCA2 proteins were shown to function as transactivation domains when fused to GAL4 DNA binding domain. We have recently isolated and characterized two new naturally occurring variants of BRCA1 (BRCA1a/p110 and BRCA1b/p100) which are phosphoproteins containing phosphotyrosine that associate with E2F transcriptional factors, cyclins and cyclin dependent kinases indicating a role for BRCA1 proteins in cell-cycle regulation. Here we show for the first time that the amino-terminal region of BRCA1a (BNT) but not BRCA1b can also function as a transcriptional activator when fused to GAL4 DNA binding domain. Thus, BRCA1/1a proteins contain two autonomous transcriptional activation domains, one at the amino-terminal region (BNT) and the other at the carboxy-terminal region (BCT). BRCA1b retains only the BCT domain since it has lost part of the potential BNT domain as a result of alternative splicing. Our results also suggest the presence of an inhibitory domain at the carboxy terminal region of BRCA1 and BRCA1a proteins (BID). Thus, BRCA1b protein may function as a dominant negative variant that could regulate the transcriptional activity of BRCA1/BRCA1a proteins and hence may serve as a marker for identifying individuals with greater potential for developing breast cancer. It may be possible that loss of transcriptional activation or protein-protein interactions in patients with mutations in the amino terminal zinc finger domain could deprive the cell of an important mechanism for regulating cell proliferation leading to the development of breast cancer.

Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA.

We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone 1HN, 15N, 13C alpha and 13C beta and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.

Transcriptional activity of pannier is regulated negatively by heterodimerization of the GATA DNA-binding domain with a cofactor encoded by the u-shaped gene of Drosophila.

The genes pannier (pnr) and u-shaped (ush) are required for the regulation of achaete-scute during establishment of the bristle pattern in Drosophila. pnr encodes a protein belonging to the GATA family of transcription factors, whereas ush encodes a novel zinc finger protein. Genetic interactions between dominant pnr mutants bearing lesions situated in the amino-terminal zinc finger of the GATA domain and ush mutants have been described. We show here that both wild-type Pannier and the dominant mutant form activate transcription from the heterologous alpha globin promoter when transfected into chicken embryonic fibroblasts. Furthermore, Pnr and Ush are found to heterodimerize through the amino-terminal zinc finger of Pnr and when associated with Ush, the transcriptional activity of Pnr is lost. In contrast, the mutant pnr protein with lesions in this finger associates only poorly with Ush and activates transcription even when cotransfected with Ush. These interactions have been investigated in vivo by overexpression of the mutant and wild-type proteins. The results suggest an antagonistic effect of Ush on Pnr function and reveal a new mode of regulation of GATA factors during development.

Solution structure of the first three zinc fingers of TFIIIA bound to the cognate DNA sequence: determinants of affinity and sequence specificity

The high resolution solution structure of a protein containing the three amino-terminal zinc fingers of Xenopus laevis transcription factor IIIA (TFIIIA) bound to its cognate DNA duplex was determined by nuclear magnetic resonance spectroscopy. The protein, which is designated zf1-3, binds with all three fingers in the DNA major groove, with a number of amino acids making base-specific contacts. The DNA structure is close to B-form. Although the mode of interaction of zf1-3 with DNA is similar to that of zif268 and other structurally characterized zinc finger complexes, the TFIIIA complex exhibits several novel features. Each zinc finger contacts four to five base-pairs and the repertoire of known base contact residues is extended to include a tryptophan at position +2 of the helix (finger 1) and arginine at position +10 (finger 3). Sequence-specific base contacts are made over virtually the entire length of the finger 3 helix. Lysine and histidine side-chains involved in base recognition are dynamically disordered in the solution structure; in the case of lysine, in particular, this could significantly decrease the entropic cost of DNA binding. The TGEKP(N) linker sequences, which are highly flexible in the unbound protein, adopt ordered conformations on DNA binding. The linkers appear to play an active structural role in stabilization of the protein-DNA complex. Substantial protein-protein contact surfaces are formed between adjacent fingers. As a consequence of these protein-protein interactions, the orientation of finger 1 in the major groove differs from that of the other fingers. Contributions to high affinity binding by zf1-3 come from both direct protein-DNA contacts and from indirect protein-protein interactions associated with structural organization of the linkers and formation of well-packed interfaces between adjacent zinc fingers in the DNA complex. The structures provide a molecular level explanation for the large body of footprinting and mutagenesis data available for the TFIIIA-DNA complex.