Altered DNA-binding Specificity Research Articles

IKZF3/Aiolos H195Y mutation identified in a mouse model of B cell leukemia results in altered DNA binding and altered STAT5-dependent gene expression

Precursor B cell acute lymphoblastic leukemia (Pre-B-ALL) arises from developing B cells and frequently involves mutations in genes encoding transcription factors. In this study, we investigated the function of mutations in the transcription factor IKZF3 (Aiolos), R137* and H195Y, discovered in a mouse model of pre-B-ALL. R137* IKZF3 mutation resulted in a truncated protein, while electrophoretic mobility shift assay showed that H195Y IKZF3 mutation resulted in a protein with altered DNA binding. 38B9 pre-B cell lines were generated expressing WT and H195Y IKZF3 proteins. Anti-IKZF3 ChIP-seq showed that H195Y IKZF3 interacted with a larger number of sites that were different than WT IKZF3. Treatment with interleukin-7 induced changes in gene expression in 38B9 cells expressing WT IKZF3, but did not induce any changes in gene expression in cells expressing H195Y IKZF3. Anti-STAT5 ChIP-seq showed that expression of H195Y IKZF3 resulted in redistribution of STAT5 binding sites in the genome. H195Y IKZF3 binding sites overlapped with a subset of STAT5 binding sites, including in the promoter of the Cish gene. These findings suggest that H195Y mutation of IKZF3 results in altered DNA binding specificity and altered binding of STAT5 to target genes.

EKLF/Klf1 Regulates Erythroid Transcription By Its Pioneering Activity and Subsequent Control of RNA Pol II Pause-Release

EKLF/Klf1 is a master transcriptional activator of critical genes that regulate both erythroid fate specification and terminal erythroid maturation. EKLF binds to DNA using three Zn-fingers at its C-terminus while the N-terminus constitutes a transcription activation domain (TAD) that interacts with various transcription co-factors including the protein acetylase CBP. An autosomal semi-dominant mutation at a single residue (E339D) in the mouse EKLF Zn-finger leads to Neonatal anemia (Nan). A mutation at the same residue in human EKLF (E325K) causes Congenital Dyserythropoietic Anemia type IV (CDA IV). Nan/Nan mice show lethality at embryonic day E10-11, in contrast to EKLF-/- homozygotes that survive until E15. Nan/+ heterozygotes survive to adulthood but are severely anemic, unlike EKLF+/- heterozygotes that display no aberrant phenotypes. The Nan-EKLF protein has an altered DNA binding specificity leading to a vastly altered transcriptome by two mechanisms. First, Nan-EKLF binding causes ectopic gene expression that significantly contributes to the severe anemia in Nan/+. Second, a subset of EKLF targets is downregulated in heterozygous Nan/+ mutants despite the presence of one copy of wild type EKLF, exacerbating the anemia. Thus, uncovering the mechanism by which gene expression is altered in Nan/+ may illuminate how EKLF normally activates transcription of its targets in vivo.To this end, we first examined the global occupancy of RNA Pol II phospho-Ser5 (as a paused mark) and phospho-Ser2 (as an elongation mark) in the mouse E13.5 fetal liver as a source of primary definitive erythroid cells. At promoters of ectopically expressed genes, where only Nan-EKLF (but not WT) binding is expected, we predominantly find increased levels of both paused and elongating RNA Pol II suggesting that Nan-EKLF binding activates transcription at ectopic genes by RNA Pol II recruitment and promoter proximal pausing. Further, we find increased levels of H3K27ac and CBP occupancy at these sites indicating that the mechanism of Pol II recruitment relies on CBP-mediated H3K27 acetylation and increased chromatin accessibility. Overall, this suggests robust pioneering activity of Nan-EKLF likely mediated by the interaction of its TAD with the CBP/p300 acetylase complex.At genes downregulated in Nan/+ we find two major patterns of Pol II occupancy. One is the converse of that seen at ectopic genes wherein there is a concomitant decrease in both Pol II p-Ser5 and p-Ser2 levels, along with lower H3K27ac and CBP levels suggesting EKLF gene activation has been lost at these sites in Nan/+. This includes cell cycle EKLF targets such as E2f2 and Rgcc. The second set of genes have comparable levels of p-Ser5 (paused) Pol II in Nan/+ and WT, but lower levels of p-Ser2 (elongating) Pol II in Nan/+. This suggests that although Pol II is being recruited to the TSS and pauses effectively, the pause-release step leading to effective transcription elongation is impaired. This subset includes important EKLF targets such as Bcl11a, Pax7, Xpo7, and several membrane transporters. As expected, CBP and H3K27ac levels are similar in WT and Nan/+ at these sites.To determine the cause of impaired RNA Pol II pause-release we examined the global occupancies of key transcription elongation factors such as P-TEFb and NELF. We find that levels of NELF, a negative elongation factor, remain unchanged in WT and Nan/+. However, levels of the P-TEFb subunit Cdk9, a positive elongation factor that facilitates release of paused RNA Pol II, is significantly lower at the TSS of these genes in Nan/+. This suggests that in Nan/+, possible reduction or loss of EKLF binding at some EKLF target promoters impairs effective recruitment of positive transcription elongation factors, resulting in a failure to efficiently release paused RNA Pol II. This causes downregulation of these EKLF target genes and contributes to the severe anemic phenotypes of the Nan mouse.We conclude that: EKLF exhibits expression control of its target genes at both the transcriptional initiation and elongation steps in vivo; EKLF can act as a pioneer transcription factor and increase chromatin accessibility through H3K27 acetylation by CBP leading to recruitment and pausing of RNA Pol II; and EKLF recruits the positive transcription elongation complex P-TEFb, enabling the controlled release of paused RNA Pol II at transcription start sites of a select group of its targets. DisclosuresNo relevant conflicts of interest to declare.

FOXL2 in adult-type granulosa cell tumour of the ovary: oncogene or tumour suppressor gene?

A recurrent mutation in FOXL2 (c.402C>G; p.C134W) is present in over 95% of adult-type granulosa cell tumours (AGCTs). In contrast, various loss-of-function mutations in FOXL2 lead to the development of blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES). BPES is characterised by an eyelid malformation often accompanied with primary ovarian insufficiency. Two recent studies suggest that FOXL2 C402G is a gain- or change-of-function mutation with altered DNA-binding specificity. Another study proposes that FOXL2 C402G is selectively targeted for degradation, inducing somatic haploinsufficiency, suggesting its role as a tumour suppressor. The latter study relies on data indicative of an FOXL2 allelic imbalance in AGCTs. Here we present RNA-seq data as genetic evidence that no real allelic imbalance is observed at the transcriptomic level in AGCTs. Additionally, there is no loss of protein expression in tumours harbouring the mutated allele. These data and other features of this mutation compared to other oncogenes and tumour suppressor genes argue strongly against FOXL2 being a tumour suppressor in this context. Given the likelihood that FOXL2 C402G is oncogenic, targeting the variant protein or its downstream consequences is the most viable path forward to identifying an effective treatment for this cancer. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Pathognomonic FOXL2 C134W Mutation Alters DNA-Binding Specificity.

The somatic missense point mutation c.402C>G (p.C134W) in the FOXL2 transcription factor is pathognomonic for adult-type granulosa cell tumors (AGCT) and a diagnostic marker for this tumor type. However, the molecular consequences of this mutation and its contribution to the mechanisms of AGCT pathogenesis remain unclear. To explore these mechanisms, we engineered V5-FOXL2WT- and V5-FOXL2C134W-inducible isogenic cell lines and performed chromatin immunoprecipitation sequencing and transcriptome profiling. FOXL2C134W associated with the majority of the FOXL2 wild-type DNA elements as well as a large collection of unique elements genome wide. This model enabled confirmation of altered DNA-binding specificity for FOXL2C134W and identification of unique targets of FOXL2C134W including SLC35F2, whose expression increased sensitivity to YM155. Our results suggest FOXL2C134W drives AGCT by altering the binding affinity of FOXL2-containing complexes to engage an oncogenic transcriptional program. SIGNIFICANCE: A mechanistic understanding of FOXL2C134W-induced regulatory state alterations drives discovery of a rationally designed therapeutic strategy.

Corrupted DNA-binding specificity and ectopic transcription underpin dominant neomorphic mutations in KLF/SP transcription factors

BackgroundMutations in the transcription factor, KLF1, are common within certain populations of the world. Heterozygous missense mutations in KLF1 mostly lead to benign phenotypes, but a heterozygous mutation in a DNA-binding residue (E325K in human) results in severe Congenital Dyserythropoietic Anemia type IV (CDA IV); i.e. an autosomal-dominant disorder characterized by neonatal hemolysis.ResultsTo investigate the biochemical and genetic mechanism of CDA IV, we generated murine erythroid cell lines that harbor tamoxifen-inducible (ER™) versions of wild type and mutant KLF1 on a Klf1−/− genetic background. Nuclear translocation of wild type KLF1 results in terminal erythroid differentiation, whereas mutant KLF1 results in hemolysis without differentiation. The E to K variant binds poorly to the canonical 9 bp recognition motif (NGG-GYG-KGG) genome-wide but binds at high affinity to a corrupted motif (NGG-GRG-KGG). We confirmed altered DNA-binding specificity by quantitative in vitro binding assays of recombinant zinc-finger domains. Our results are consistent with previously reported structural data of KLF-DNA interactions. We employed 4sU-RNA-seq to show that a corrupted transcriptome is a direct consequence of aberrant DNA binding.ConclusionsSince all KLF/SP family proteins bind DNA in an identical fashion, these results are likely to be generally applicable to mutations in all family members. Importantly, they explain how certain mutations in the DNA-binding domain of transcription factors can generate neomorphic functions that result in autosomal dominant disease.

Identification of a transcriptional activation domain in yeast repressor activator protein 1 (Rap1) using an altered DNA-binding specificity variant

Repressor activator protein 1 (Rap1) performs multiple vital cellular functions in the budding yeast Saccharomyces cerevisiae These include regulation of telomere length, transcriptional repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of mRNA-encoding genes, including the highly transcribed ribosomal protein- and glycolytic enzyme-encoding genes. Studies of the contributions of Rap1 to telomere length regulation and transcriptional repression have yielded significant mechanistic insights. However, the mechanism of Rap1 transcriptional activation remains poorly understood because Rap1 is encoded by a single copy essential gene and is involved in many disparate and essential cellular functions, preventing easy interpretation of attempts to directly dissect Rap1 structure-function relationships. Moreover, conflicting reports on the ability of Rap1-heterologous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenge use of this approach to study Rap1. Described here is the development of an altered DNA-binding specificity variant of Rap1 (Rap1AS). We used Rap1AS to map and characterize a 41-amino acid activation domain (AD) within the Rap1 C terminus. We found that this AD is required for transcription of both chimeric reporter genes and authentic chromosomal Rap1 enhancer-containing target genes. Finally, as predicted for a bona fide AD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. In summary, we show here that Rap1 contains an AD required for Rap1-dependent gene transcription. The Rap1AS variant will likely also be useful for studies of the functions of Rap1 in other biological pathways.

Degenerate DNA Binding By Mutant (E339D) KLF1 Dramatically Alters the Erythroid Transcriptome in the Nan Mouse Model

The C2H2 zinc finger transcription factor KLF1 regulates nearly all aspects of erythroid differentiation including iron trafficking, heme synthesis, globin production, membrane skeleton assembly and the cell cycle. KLF1 is primarily, but not exclusively, a transcriptional activator. Previously we showed that the inbred mouse model Nan (neonatal anemia) carries a missense mutation (E339D) in the second zinc finger of KLF1 that causes severe lifelong anemia in heterozygotes and embryonic lethality (E10-11) in homozygotes. We also showed that E339D confers an altered DNA binding specificity such that mutant Nan-KLF1 fails to properly bind numerous known KLF1 target genes. As a result, fetal liver (FL) expression of many genes including globin (Hbb, Hba), dematin (Dmtn/Epb4.9) and the cell cycle regulators E2F2 (E2f2) and p18 (Cdkn2c) are significantly decreased. To examine the Nan global erythroid transcriptome, we performed RNAseq on E14.5 FLs. Fetuses were genotyped for Y chromosome markers and 3 wild-type (WT) and 3 Nan/+ males were analyzed. Using a stringently filtered dataset (CPM > 1 in at least 3 of 6 samples, FDR 0.05, FC ≥ 2), 610 genes were upregulated and 200 downregulated in Nan vs. WT. Notably, only 18% of theupregulated and 52% of the downregulated genes overlapped the 1078 known KLF1 target genes (defined in prior studies as those differentially expressed in Klf1 -/- vs +/+ FL). Moreover, just 3% of the upregulated Nan genes overlapped genes normally activated by KLF1. Thus, significant ectopic gene expression occurs in Nan. Pathway analysis (IPA, PANTHER) identified Toll receptor signaling, acute phase response signaling, and response to interferon as among the top hits in the upregulated gene set in Nan, indicating an overall inflammatory state that would be expected to exacerbate anemia. Indeed, we confirmed significantly increased levels of serum IFNβ and IRF7 in Nan. In the Nan downregulated gene set, cytoskeletal regulation by Rho GTPase and cell cycle regulation were the top hits. Previous studies identified deficiencies of membrane skeleton proteins in Nan. To analyze cell cycle status, we sorted dividing adult spleen and bone marrow (BM) erythroid precursors (proerythroblasts, basophilic and polychromatophilic erythroblasts) using CD44, Ter119 and FSC. Polychromatophilic erythroblasts in Nan BM were arrested in G1 (significantly increased percentage of cells in G0/G1, significantly decreased percentage in S phase; p < 0.05) compared to saline-injected and phenylhydrazine treated WT controls. Surprisingly, however, no other cell cycle defects were seen among other BM compartments and none were seen in the spleen. We next analyzed gene ontology (GO) terms in both the up- and downregulated Nan gene sets (MGI, Mouse Genome Informatics). Ten cell cycle associated genes were downregulated (e.g., Rgcc, Cdc25b, E2f2/4), and 14 were upregulated (e.g., Gadd45b/g, Map3k8, Map3k6, Aim1) indicating a complicated pattern of gene expression differences and physiological outcomes in Nan. Similarly, expression of genes involved in iron regulation were among those most highly affected in Nan and did not predict the observed physiological outcome. The mitochondrial iron transporter SLC25A37 (Mitoferrin) and the transporter SLC25A38, both important in heme synthesis, and the ferrireductase required for transferrin-mediated iron uptake by red cells, STEAP3, are downregulated in Nan. Hamp encoding hepcidin, a negative regulator of the iron importer, ferroportin, is upregulated in Nan >14x. Ferroportin expression is not concomitantly downregulated, however. Despite these changes, no evidence of systemic iron deficiency has been identified in Nan; serum iron levels are normal. Moreover, the zinc protoporphyrin IX to heme ratio is markedly increased in Nan, indicating that a defect in heme biosynthesis up to and including ferrochelatase is unlikely, and uptake of radiolabeled iron by Nan reticulocytes and incorporation into heme are normal. From these studies we conclude: (1) Nan-KLF1 activates and represses a large number of non-KLF1 target genes; (2) Nan-KLF1 inappropriately increases and decreases expression of subsets of normal KLF1 genes resulting in (3) unpredictable physiogical outcomes in Nan. Overall, we conclude that new homeostatic set points arise in Nan as a result of the combined contributions of a large set of differentially expressed genes. DisclosuresNo relevant conflicts of interest to declare.

Is the control of recombination conserved among diverse eukaryotes?

Is the control of recombination conserved among diverse eukaryotes?

Directed Evolution of p53 Variants with Altered DNA-binding Specificities by In Vitro Compartmentalization

The p53 tumour suppressor governs cell fate by differential transactivation of a spectrum of target genes. To further understand how p53 discriminates between target promoters, we have for the first time used in vitro compartmentalization (IVC) to evolve variants with greater affinity for the distal p53 response element in the promoter of the p21 gene involved in cell-cycle arrest, and for the low affinity BS1 response element of the pro-apoptotic PUMA gene. These variants have mutations in the L1 loop of the p53 DNA binding domain and in the N-terminal proline-rich domain. The in vitro binding phenotype of these variants extends to both increased transactivation of promoters containing the response elements in reporter gene studies and increased up-regulation of endogenous p21 as compared to wild-type p53. One variant was co-selected for increased binding to both response elements yet displayed increased apoptotic function. This result supports the notion that prediction of phenotypic outcome based on transcriptional activation of individual genes is confounded by the networked complexity of the p53 response.

Rational design of an estrogen receptor mutant with altered DNA-binding specificity

Although artificial C2-H2 zinc fingers can be designed to recognize specific DNA sequences, it remains unclear to which extent nuclear receptor C4 zinc fingers can be tailored to bind novel DNA elements. Steroid receptors bind as dimers to palindromic response elements differing in the two central base pairs of repeated motifs. Predictions based on one amino acid—one base-pair relationships may not apply to estrogen receptors (ERs), which recognize the two central base pairs of estrogen response elements (EREs) via two charged amino acids, each contacting two bases on opposite DNA strands. Mutagenesis of these residues, E203 and K210 in ERα, indicated that both contribute to ERE binding. Removal of the electric charge and steric constraints associated with K210 was required for full loss of parental DNA-binding specificity and recognition of novel sequences by E203 mutants. Although some of the new binding profiles did not match predictions, the double mutation E203R-K210A generated as predicted a mutant ER that was transcriptionally active on palindromes of PuGCTCA motifs, but not on consensus EREs. This study demonstrates the feasibility of designing C4 zinc finger mutants with novel DNA-binding specificity, but also uncovers limitations of this approach.

Development of Zinc Finger Domains for Recognition of the 5′-CNN-3′ Family DNA Sequences and Their Use in the Construction of Artificial Transcription Factors

Considerable progress has been made in recent years in the design of transcription factors for the directed regulation of endogenous genes. Although many strategies involve selection methods that must be applied for each new target sequence, we have developed an approach based on linkage of predefined zinc finger domains that each recognize a three-base pair DNA sequence to construct artificial transcription factors that bind to a desired sequence. These domains can be assembled to recognize unique 18-base pair DNA sequences with high specificity. Here we report the development and characterization of zinc finger domains that bind to 15 of the 16 5'-CNN-3' subsites. These domains were created through a combination of phage display selection, site-directed mutagenesis, and de novo design. Furthermore, these domains were used to generate a highly specific six-finger protein targeting the ERBB-2 promoter. When fused to regulatory domains, this protein was capable of up- and down-regulating the expression of the endogenous ERBB-2 gene. With the addition of this collection of predefined zinc finger domains, most 5'-CNN-3'-, 5'-GNN-3'-, and 5'-ANN-3'-containing sequences can now be rapidly targeted for directed gene regulation and nuclease cleavage.

Essential structural and functional determinants within the forkhead domain of FOXC1.

The forkhead domain (FHD)-containing developmental transcription factor FOXC1 is mutated in patients presenting with Axenfeld-Rieger malformations. In this paper, we report the introduction of positive, negative or neutral charged amino acids into critical positions within the forkhead domain of FOXC1 in an effort to better understand the essential structural and functional determinants within the FHD. We found that FOXC1 is intolerant of mutations at I87. Additionally, alterations of amino acids within alpha-helix 1 of the FOXC1 FHD affected both nuclear localization and transactivation. Amino acids within alpha-helix 3 were also found to be necessary for transactivation and can have roles in correct localization. Interestingly, changing amino acids within alpha-helix 3, particularly R127, resulted in altered DNA-binding specificity and granted FOXC1 the ability to bind to a novel DNA sequence. Given the limited topological variation of FHDs, due to the high conservation of residues, we anticipate that models of forkhead domain function derived from these data will be relevant to other members of the FOX family of transcription factors.

Altered DNA binding specificity of Arnt by selection of partner bHLH-PAS proteins.

The Ah receptor (AhR) and HLF are transcription factors involved in xenobiotic metabolism and hypoxic response, respectively. AhR and HLF heterodimerize with Arnt as the common partner, and bind to asymmetric E-boxes termed XRE and HRE, respectively. In order to investigate nucleotide preference of the heterodimers, reporter plasmids with oligonucleotides for XREs or HREs with systematic mutations were constructed and their activity was determined. Comparison of the activity revealed that DNA length and nucleotide preference recognized by Arnt subunit in the two heterodimers were largely different between XRE and HRE. We expressed AhR-Arnt and HLF-Arnt in Escherichia coli and used them for DNA binding. The dissociation constant of HLF-Arnt-HRE was 10.4 +/- 1.6 nM. Competition activity of mutated XREs or HREs with wild type was consistent with their transcription activity. Bending of XRE and HRE induced by binding of the relevant heterodimers was observed with stronger bending of XRE than of HRE. By deletional and mutational analyses, an alanine and three arginine (Ala 8, Arg 9, Arg 11 and Arg 12) residues in the basic sequence of HLF were found to be indispensable for the transcriptional activity.

In vivo mutagenesis of the Hoxb8 hexapeptide domain leads to dominant homeotic transformations that mimic the loss-of-function mutations in genes of the Hoxb cluster

Hox proteins are transcription factors that control developmental pathways along the anteroposterior axis of vertebrates. On their own, Hox proteins bind DNA weakly, but they gain specificity and affinity by interaction with members of the PBC subfamily of homeobox proteins. In vitro studies indicate that most of these interactions are mediated by the conserved hexapeptide motif of the Hox proteins. To study the significance of these interactions in vivo, we have generated mice that carry mutations in the Hoxb8 hexapeptide motif. Analysis of skeletal features of these mice reveals the presence of a dominant phenotype consisting of homeotic transformations, similar to those observed in mice with a loss-of-function of Hox genes, such as Hoxa7, Hoxb7, and Hoxb9. Genetic tests demonstrate that the mutations in the Hoxb8 hexapeptide motif are affecting the function of other genes located in the Hoxb cluster. The expression pattern of these genes is not affected; rather it appears that the mutant Hoxb8 protein interferes with the function of other Hox genes by binding to their targets. Our findings suggest that the homeotic transformations result from altered DNA binding specificity of the mutant Hoxb8 protein, implicating the cooperative binding between Hoxb8 hexapeptide motif and cofactors as a critical element in the fine-tuning of Hoxb8 protein target specificity. This is the first time the function of the hexapeptide domain has been evaluated in vivo in mouse development.

In vivo mutagenesis of the Hoxb8 hexapeptide domain leads to dominant homeotic transformations that mimic the loss-of-function mutations in genes of the Hoxb cluster

Hox proteins are transcription factors that control developmental pathways along the anteroposterior axis of vertebrates. On their own, Hox proteins bind DNA weakly, but they gain specificity and affinity by interaction with members of the PBC subfamily of homeobox proteins. In vitro studies indicate that most of these interactions are mediated by the conserved hexapeptide motif of the Hox proteins. To study the significance of these interactions in vivo, we have generated mice that carry mutations in the Hoxb8 hexapeptide motif. Analysis of skeletal features of these mice reveals the presence of a dominant phenotype consisting of homeotic transformations, similar to those observed in mice with a loss-of-function of Hox genes, such as Hoxa7, Hoxb7, and Hoxb9 . Genetic tests demonstrate that the mutations in the Hoxb8 hexapeptide motif are affecting the function of other genes located in the Hoxb cluster. The expression pattern of these genes is not affected; rather it appears that the mutant Hoxb8 protein interferes with the function of other Hox genes by binding to their targets. Our findings suggest that the homeotic transformations result from altered DNA binding specificity of the mutant Hoxb8 protein, implicating the cooperative binding between Hoxb8 hexapeptide motif and cofactors as a critical element in the fine-tuning of Hoxb8 protein target specificity. This is the first time the function of the hexapeptide domain has been evaluated in vivo in mouse development.

Miscoordination of the Iron-Sulfur Clusters of the Anaerobic Transcription Factor, FNR, Allows Simple Repression but Not Activation

The FNR protein of Escherichia coli regulates target genes in response to anaerobiosis. Environmental oxygen is sensed by the acquisition of oxygen-labile [4Fe-4S] clusters that promote dimerization, DNA binding, and productive interactions with RNA polymerase. Three N-terminal cysteine residues (Cys(20), Cys(23), and Cys(29)) and Cys(122) act as ligands for the FNR iron sulfur clusters. An FNR variant, FNR-C20S, that retains only trace activity in vivo can acquire [4Fe-4S] clusters in vitro that enhance site-specific DNA binding. Second site substitutions in activating regions AR1, AR2, and AR3 restore in vivo activity to FNR-C20S, suggesting that the impairment in FNR-C20S activity is due to a failure to communicate with RNA polymerase effectively. Here we show that FNR-C20S can repress a simple FNR-regulated promoter in vivo and that it can form productive heterodimers with an FNR variant with altered DNA binding specificity, FNR-E209V. Transcription studies with FNR-E209V.FNR-C20S heterodimers indicate that the presence of a miscoordinated iron-sulfur cluster (FNR-C20S) in the downstream (but not the upstream) subunit of the FNR dimer impairs activation from a class II promoter and that this impairment can be overcome by amino acid substitutions known to unmask AR2 or improve AR3 in the affected subunit.

Domain III function of Mu transposase analysed by directed placement of subunits within the transpososome.

Assembly of the functional tetrameric form of Mu transposase (MuA protein) at the two att ends of Mu depends on interaction of MuA with multiple att and enhancer sites on supercoiled DNA, and is stimulated by MuB protein. The N-terminal domain I of MuA harbours distinct regions for interaction with the att ends and enhancer; the C-terminal domain III contains separate regions essential for tetramer assembly and interaction with MuB protein (IIIalpha and IIIbeta, respectively). Although the central domain II (the 'DDE' domain) of MuA harbours the known catalytic DDE residues, a 26 amino acid peptide within IIIalpha also has a non-specific DNA binding and nuclease activity which has been implicated in catalysis. One model proposes that active sites for Mu transposition are assembled by sharing structural/catalytic residues between domains II and III present on separate MuA monomers within the MuA tetramer. We have used substrates with altered att sites and mixtures of MuA proteins with either wild-type or altered att DNA binding specificities, to create tetrameric arrangements wherein specific MuA subunits are nonfunctional in II, IIIalpha or IIIbeta domains. From the ability of these oriented tetramers to carry out DNA cleavage and strand transfer we conclude that domain IIIalpha or IIIbeta function is not unique to a specific subunit within the tetramer, indicative of a structural rather than a catalytic function for domain III in Mu transposition.

Functional and Structural Studies of Wild Type SOX9 and Mutations Causing Campomelic Dysplasia

In humans, mutations in SOX9 result in a skeletal malformation syndrome, campomelic dysplasia (CD). The present study investigated two major classes of CD mutations: 1) point mutations in the high mobility group (HMG) domain and 2) truncations and frameshifts that alter the C terminus of the protein. We analyzed the effect of one novel mutation and three other point mutations in the HMG domain of SOX9 on the DNA binding and DNA bending properties of the protein. The F12L mutant HMG domain shows negligible DNA binding, the H65Y mutant shows minimal DNA binding, whereas the A19V mutant shows near wild type DNA binding and bends DNA normally. Interestingly, the P70R mutant has altered DNA binding specificity, but also bends DNA normally. The effects of the point mutations were interpreted using a molecular model of the SOX9 HMG domain. We analyzed the effects upon transcription of mutations resembling the truncation and frameshift mutations in CD patients, and found that progressive deletion of the C terminus causes progressive loss of transactivation. Maximal transactivation by SOX9 requires both the C-terminal domain rich in proline, glutamine, and serine and the adjacent domain composed entirely of proline, glutamine, and alanine. Thus, CD arises by mutations that interfere with DNA binding by SOX9 or truncate the C-terminal transactivation domain and thereby impede the ability of SOX9 to activate target genes during organ development.

Single-chain 434 repressors with altered DNA-binding specificities. Isolation of mutant single-chain repressors by phenotypic screening of combinatorial mutant libraries.

Combinatorial mutant libraries of the single-chain 434 repressor were used to discover novel DNA-binding specificities. Members of the library contain one wild type domain and one mutant domain which are connected by a recombinant peptide linker. The mutant domain contains randomized amino acids in place of the DNA-contacting residues. The single-chain derivatives are expected to recognize artificial operators containing the DNA sequence of ACAA--6 base-pairs--NNNN, where ACAA is bound by the wild-type and NNNN by the mutant domain. An in vivo library screening method was used to isolate mutant DNA-binding domains which recognize the TTAA site of an asymmetric operator. Several mutants showed high affinity binding to the selection target and also strong (up to 80 fold) preference for TTAA over the wild type TTGT sequence. Some of the isolated mutants bound with very high affinities (10-50 pM) to operators containing the TTAC sequence, a close homologue of the TTAA selection target.

Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the beta-globin locus control region in vivo.

The locus control region of the beta-globin cluster contains five DNase I hypersensitive sites (5'HS1-5) required for locus activation. 5'HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5'HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5'HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5'HS3.