DNA Binding Domain Amino Acids Research Articles

Posttranscriptional regulation of the T-box gene midline via the 3'UTR in Drosophila is complex and cell- and tissue-dependent.

The T-box (Tbx) proteins have a 180-230 amino acid DNA-binding domain, first reported in the Brachyury (T) protein. They are highly conserved among metazoans. They regulate a multitude of cellular functions in development and disease. Here, we report posttranscriptional and translational regulation of midline (mid), a Tbx member in Drosophila. We found that the 3'UTR of mid has mRNA degradation elements and AT-rich sequences. In Schneider S2 cells, mid-mRNA could be detected only when the transgene was without the 3'UTR. Similarly, the 3'UTR linked to the Renilla luciferase reporter significantly reduced the activity of the Luciferase, whereas deleting only the degradation elements from the 3'UTR resulted in reduced activity, but not as much. Overexpression of mid in MP2, an embryonic neuroblast, showed no significant difference in the levels of mid-mRNA between the 2 transgenes, with and without the 3'UTR, indicating the absence of posttranscriptional regulation of mid in MP2. Moreover, while elevated mid-RNA was detected in MP2 in nearly all hemisegments, only a fifth of those hemisegments had elevated levels of the protein. Overexpression of the 2 transgenes resulted in MP2-lineage defects at about the same frequency. These results indicate a translational/posttranslational regulation of mid in MP2. The regulation of ectopically expressed mid in the wing imaginal disc was complex. In the wing disc, where mid is not expressed, the ectopic expression of the transgene lacking the 3'UTR had a higher level of mid-RNA and the protein had a stronger phenotypic effect. These results indicate that the 3'UTR can subject mid-mRNA to degradation in a cell- and tissue-specific manner. We further report a balancer-mediated transgenerational modifier effect on the expression and gain of function effects of the 2 transgenes.

40 years of the homeobox: either it is wrong or it is quite interesting.

Many genes that regulate development share a 180 bp DNA sequence, called the homeobox, encoding a 60 amino acid DNA-binding domain ( McGinnis et al., 1984c; Scott and Weiner, 1984). Because the homeobox is long enough to hybridize to related, but different, genes, it has been a powerful tool for discovering developmental regulators. This year is the 40th anniversary of the first homeobox report. Here, I describe work carried out at Indiana University that led to the discovery of the homeobox. The accompanying Perspective from McGinnis and Levine describes the independent discovery made at the Biozentrum in Basel ( McGinnis and Levine, 2024). At the time, the competition was lively but, as we all met each other - and realized that no one cares more about your work than competitors - we fortunately became friends and have enjoyed many years of following and respecting each other's work.

Follow the Mutations: Toward Class-Specific, Small-Molecule Reactivation of p53.

The mutational landscape of p53 in cancer is unusual among tumor suppressors because most of the alterations are of the missense type and localize to a single domain: the ~220 amino acid DNA-binding domain. Nearly all of these mutations produce the common effect of reducing p53’s ability to interact with DNA and activate transcription. Despite this seemingly simple phenotype, no mutant p53-targeted drugs are available to treat cancer patients. One of the main reasons for this is that the mutations exert their effects via multiple mechanisms—loss of DNA contacts, reduction in zinc-binding affinity, and lowering of thermodynamic stability—each of which involves a distinct type of physical impairment. This review discusses how this knowledge is informing current efforts to develop small molecules that repair these defects and restore function to mutant p53. Categorizing the spectrum of p53 mutations into discrete classes based on their inactivation mechanisms is the initial step toward personalized cancer therapy based on p53 allele status.

1HN, 13C, and 15N backbone resonance assignments of the human DNA ligase 3 DNA-binding domain (residues 257-477).

In mammalian cells, the process of DNA ligation is necessary during DNA replication to create an intact "lagging" strand from a series of smaller Okazaki fragments and to repair DNA strand breaks that arise either due to the direct action of a DNA damaging agent or as a consequence of DNA damage excision during DNA repair. In humans, there are three genes that encode for members of the DNA ligase family (LIG1, LIG3 and LIG4) (Ellenberger and Tomkinson in Ann Rev Biochem 77:313-338. 2008). Although these genes code for polypeptides with overlapping functions in the nucleus, the only mitochondrial DNA ligase (DNA ligase IIIα), which is essential for mitochondrial genome maintenance, is encoded by the LIG3 gene (Lakshmipathy and Campbell in Mol Cell Biol 19:3869-3876, 1999; Zong et al. in Mol Cell 61:667-676, 2016) Because mitochondria play a central and multifunctional role in malignant tumor progression, there is emerging interest in targeting key mitochondrial proteins. Notably, there is evidence in pre-clinical models that inhibitors of DNA ligase IIIα, which is frequently up-regulated in cancer, preferentially target cancer cells via their effect on mitochondria (Zong et al. 2016). Since NMR spectroscopy provides unique capabilities for identifying small molecules that bind specifically to DNA ligase IIIα versus the other DNA ligases), the backbone 1HN, 13C, and 15N NMR resonance assignments were completed for a 222 amino acid DNA-binding domain of human DNA ligase III. These NMR assignments represent a vital first step towards developing DNA ligase III-selective inhibitors.

Distinctive structural basis for DNA recognition by the fission yeast Zn2Cys6 transcription factor Pho7 and its role in phosphate homeostasis

Pho7, a member of the Zn2Cys6 family of fungal transcription factors, is the key transcriptional activator underlying fission yeast phosphate homeostasis, a physiological response to phosphate starvation in which the pho1, pho84 and tgp1 genes are upregulated. Here, we delineated a minimized 61-amino-acid Pho7 DNA-binding domain (DBD) and determined the 1.7 Å crystal structure of the DBD at its target site in the tgp1 promoter. Two distinctive features of the Pho7 DBD are: it binds DNA as a monomer, unlike most other fungal zinc-cluster factors that bind as homodimers; and it makes extensive interactions with its asymmetric target sequence over a 14-bp footprint that entails hydrogen bonding to 13 individual bases within, and remote from, the CGG triplet typically recognized by other Zn2Cys6 DBDs. Base pair substitutions at Pho7 sites in the tgp1 and pho1 promoters highlight the importance of the 5′-CGG triplet for Pho7 binding in vitro and Pho7-dependent gene expression in vivo. We identify several DBD amino acids at which alanine substitution effaced or attenuated the pho1 phosphate starvation response and concordantly reduced Pho7 binding to a pho1 promoter site.

A genome-wide analysis of MADS-box genes in peach [Prunus persica (L.) Batsch

BackgroundMADS-box genes encode a family of eukaryotic transcription factors distinguished by the presence of a highly-conserved ~58 amino acid DNA-binding and dimerization domain (the MADS-box). The central role played by MADS-box genes in peach endodormancy regulation led us to examine this large gene family in more detail. We identified the locations and sequences of 79 MADS-box genes in peach, separated them into established subfamilies, and broadly surveyed their tissue-specific and dormancy-induced expression patterns using next-generation sequencing. We then focused on the dormancy-related SVP/AGL24 and FLC subfamilies, comparing their numbers and phylogenetic relationships with those of other sequenced woody perennial genomes.ResultsWe identified 79 MADS-box genes distributed across all eight peach chromosomes and frequently located in clusters of two or more genes. They encode proteins with a mean length of 248 ± 72 amino acids and include representatives from most of the thirteen Type II (MIKC) subfamilies, as well as members of the Type I Mα, Mβ, and Mγ subfamilies. Most Type I genes were present in species-specific monophyletic lineages, and their expression in the peach sporophyte was low or absent. Most Type II genes had Arabidopsis orthologs and were expressed at much higher levels throughout vegetative and fruit tissues. During short-day-induced growth cessation, seven Type II genes from the SVP/AGL24, AGL17, and SEP subfamilies showed significant changes in expression. Phylogenetic analyses indicated that multiple, independent expansions have taken place within the SVP/AGL24 and FLC lineages in woody perennial species.ConclusionsMost Type I genes appear to have arisen through tandem duplications after the divergence of the Arabidopsis and peach lineages, whereas Type II genes appear to have increased following whole genome duplication events. An exception to the latter rule occurs in the FLC and SVP/AGL24 Type II subfamilies, in which species-specific tandem duplicates have been retained in a number of perennial species. These subfamilies comprise part of a genetic toolkit that regulates endodormancy transitions, but phylogenetic and expression data suggest that individual orthologs may not function identically across all species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0436-2) contains supplementary material, which is available to authorized users.

Targeting DNA Repair Gene, RAD52, Induces Exhaustion of the Proliferating CML-CP Leukemia Stem Cells Carrying Oxidative DNA Damage

Abstract 447BCR-ABL1 transforms hematopoietic stem cells (HSCs) into leukemia stem cells (LSCs) to induce chronic myeloid leukemia in chronic phase (CML-CP). We detected that the most primitive LSCs display elevated levels of reactive oxygen species (ROS) and accumulate excessive numbers of potentially lethal DNA double-strand breaks (DSBs). We also reported that BCR-ABL1-transformed cells exhibit enhanced RAD51-mediated homologous recombination repair (HRR) activity occurring in S and G2/M cell cycle phases. In normal cells initiation of RAD51-mediated HRR is directed either by BRCA1– or RAD52–dependent mechanisms. Since BCR-ABL1 kinase downregulated BRCA1, LSCs containing high number of DSBs should depend more on RAD52 to promote HRR to repair lethal DSBs.We found that in vivo leukemogenic potential of BCR-ABL1 –positive RAD52−/− hematopoietic cells is abrogated in comparison to their BCR-ABL1 -positive RAD52+/+ counterparts. The absence of RAD52 in BCR-ABL1 –positive cells reduced the percentage of Lin−Kit+Sca1+ cells by >2-fold and inhibited their clonogenic potential and proliferation by >10-fold. In addition RAD52 knockout caused approximately 2-fold reduction of Lin−Kit+Sca1+CD34−Flt3− long-term LSCs (LT-LSCs) and Lin−Kit+Sca1+CD34+Flt3− short-term LSCs (ST-LSCs). Conversely, 4-fold accumulation of BCR-ABL1 –positive RAD52−/− Lin−Kit+Sca1+eFluor670max quiescent cells was detected in comparison to BCR-ABL1 –positive RAD52+/+ counterparts. These effects were accompanied by 2-fold reduction of the percentage of BCR-ABL1 –positive RAD52−/− cells in S and G2/M and 7-fold increase of these cells in sub-G1 when compared to BCR-ABL1 –positive RAD52+/+ counterparts. BCR-ABL1-positive RAD52−/− Lin−Kit+Sca1+ cells accumulated more DSBs than BCR-ABL1 –positive RAD52+/+ cells. These differences were not observed between non-transformed RAD52−/− and RAD52+/+ cells. Expression of the wild-type RAD52 reduced the accumulation of lethal DSBs and rescued the clonogenic potential and proliferation of BCR-ABL1-positive RAD52−/− Lin−Kit+Sca1+ cells. Downregulation of ROS with antioxidants vitamin E (VE) and N-acetyl-cysteine (NAC) exerted similar effect as restored expression of RAD52. Thus it appears that RAD52 is necessary to repair the extensive ROS-induced DSBs in LSC-enriched Lin−Kit+Sca1+ cells. BCR-ABL1 kinase does not affect the expression of RAD52 protein, but phosphorylates RAD52 on Y104. However, expression of RAD52(Y104F) phosphorylation-less mutant reduced the number of DSBs and rescued the clonogenic potential of BCR-ABL1-positive RAD52−/− Lin−Kit+Sca1+ cells in a similar way to the wild-type RAD52. Accordingly, RAD52-mediated DSB repair activity in CML-CP cells should not be affected by imatinib treatment. RAD52 mediates the annealing of complementarry DNA strands during DSB repair. To exert this function RAD52 has two DNA binding domains. Expression of RAD52(F79A) and RAD52(K102A) DNA binding-deficient mutants (each amino acid substitution inactivated different DNA binding domain) failed to prevent the accumulation of DSBs and did not rescue the clonogenic and proliferative potential of BCR-ABL1-positive RAD52−/− cells. In addition, RAD52(F79A), but not RAD52(Y104F) inhibited DSB repair by HRR. Therefore DNA binding capability of RAD52 appears essential for BCR-ABL1 –mediated leukemogenesis, but it is dispensable in normal hematopoietic cells. The “addiction” of BCR-ABL1 leukemia cells to RAD52 was confirmed by demonstration that RAD52(F79A) mutant inhibited clonogenic potential of CD34+ CML-CP cells, but not normal counterparts. Furthermore, to determine if RAD52 DNA binding domains could be targeted to selectively inhibit CML-CP, peptide aptamers containing RAD52 DNA binding domain amino acids sequence surrounding F79 were employed as potential decoys for RAD52 DNA binding. Aptamer containing F79, but not the A79 substitution, diminished the number of RAD52 foci and reduced the clonogenic potential and proliferation of CD34+ cells from CML-CP, but not from normal donors. In conclusion, we postulate that RAD52 is essential for BCR-ABL1 –mediated leukemogenesis and that DNA binding domains of RAD52 may be targeted for selective elimination of the proliferating CML-CP LSCs. Disclosures:No relevant conflicts of interest to declare.

Identification of a Novel Mutation inFOXL2Gene That Leads to Blepharophimosis Ptosis Epicanthus Inversus and Telecanthus Syndrome in a Tunisian Consanguineous Family

Mutations in FOXL2 gene are responsible for blepharophimosis ptosis epicanthus inversus and telecanthus syndrome (BPES). The BPES syndrome is a rare autosomal dominant genetic disease characterized by eyelid malformations associated with premature ovarian failure (BPES type I) or not (BPES type II). The human FOXL2 protein (376 aa) contains a 100 amino-acid DNA-binding forkhead domain (residues 52-152) and a polyalanine tract (residues 221-234). In the present study, we report the molecular investigation of four affected members with BPES syndrome in a Tunisian consanguineous family. To identify the causative mutation, we performed a direct sequencing of the FOXL2 gene. The sequence analysis of the coding exon revealed a novel frameshift mutation g.1113 dup C, c.876 dup C, p.P292 Fs. The mutation is located downstream of the polyalanine tract and causes the protein extension to 532 aa. This study reports for the first time a novel frameshift mutation in two-generation consanguineous Tunisian family with BPES. Our results expand the spectrum of FOXL2 mutations.

DNA Binding Domain in the Replication Checkpoint Protein Mrc1 of Schizosaccharomyces pombe

The replication checkpoint is activated when replication forks are obstructed by DNA lesions or protein complexes bound to DNA or when DNA synthesis is restrained by the limited availability of deoxyribonucleotides. This checkpoint preserves genome integrity by stabilizing stalled forks and delaying the onset of mitosis. In the fission yeast Schizosaccharomyces pombe, Mrc1 is a replication checkpoint adaptor protein that allows the sensor kinase Rad3-Rad26 to activate the effector kinase Cds1. In Saccharomyces cerevisiae, Mrc1 associates with replication forks and co-precipitates with the DNA replication protein Cdc45. Whether or not Mrc1 interacts directly with DNA is unknown. Here we define a approximately 150 amino acid DNA binding domain (DBD) in the N-terminal region of S. pombe Mrc1. The DBD interacts preferentially with branched DNA structures in vitro. Deletion of the DBD or point mutations that diminish its DNA binding activity render cells sensitive to the replication inhibitor hydroxyurea. These mutations also impair the replication checkpoint arrest. The DBD has a helix-loop-helix motif that is predicted to bind DNA. This motif is conserved in the recently identified N-terminal DBD of human Claspin, a presumptive homolog of yeast Mrc1 proteins.

The role of Tbx2 and Tbx3 in mammary development and tumorigenesis.

TBX2 and TBX3 are members of a family of genes encoding developmental transcription factors, characterized by a 200 amino acid DNA binding domain (T-box). Tbx2 and Tbx3 are closely related T-box proteins that have been implicated in development of a number of different tissues including the mammary gland. TBX3 is required for normal mammary development in mouse models and in patients with ulnar-mammary syndrome (UMS). In addition to a role in development, TBX2 and TBX3 have been implicated in tumor development through downregulation of the alternative reading frame (ARF) tumor suppressor and an associated bypass of senescence. Here we review the current information on the roles of Tbx2 and Tbx3 in mammary gland development and tumorigenesis.

Basic Residues within the Kruppel Zinc Finger DNA Binding Domains Are the Critical Nuclear Localization Determinants of EKLF/KLF-1

EKLF/KLF-1 is an erythroid-restricted transcription factor essential for expression of the adult beta-globin gene. EKLF/KLF-1 is a 358-amino acid nuclear protein with an amino-terminal proline-rich domain and a carboxyl-terminal DNA binding domain. The nuclear localization signal (NLS) of EKLF/KLF-1 has not been empirically determined. We generated a series of epitope-tagged deletion and point mutants and assessed their subcellular localization. Our results delimit the NLS to the 83-amino acid (amino acids 276-358) DNA binding domain that consists of three Kruppel zinc fingers. All three zinc fingers are necessary for efficient nuclear localization; deletion of any individual finger results in cytoplasmic accumulation. Fusion of the three zinc fingers to green fluorescent protein (GFP) targeted GFP to the nucleus, demonstrating that the zinc finger domain is sufficient for nuclear localization. EKLF/KLF-1 containing histidine to alanine mutations that disrupt the structure of all three fingers retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization. We demonstrate that basic residues within the fingers are the critical determinants for nuclear localization; mutations of these basic residues to alanine resulted in cytoplasmic mislocalization. The basic residues of all mammalian Kruppel zinc fingers are highly conserved; therefore we propose that these basic residues are a common NLS shared by all Kruppel family members.

DNA binding domains in diverse nuclear receptors function as nuclear export signals

Background: The nuclear receptor superfamily of transcription factors directs gene expression through DNA sequence-specific interactions with target genes. Nuclear import of these receptors involves recognition of a nuclear localization signal (NLS) by importins, which mediate translocation into the nucleus. Nuclear receptors lack a leucine-rich nuclear export signal (NES), and export is insensitive to leptomycin B, indicating that nuclear export is not mediated by Crm1.Results: We set out to define the NES in the glucocorticoid receptor (GR) and to characterize the export pathway. We found that the 69 amino acid DNA binding domain (DBD) of GR, which is unrelated to any known NES, is necessary and sufficient for export. Mutational analysis revealed that a 15 amino acid sequence between the two zinc binding loops in the GR-DBD confers nuclear export to a GFP reporter protein, and alanine-scanning mutagenesis was used to identify the residues within this sequence that are critical for export. The DBD is highly related (41%–88% identity) in steroid, nonsteroid, and orphan nuclear receptors, and we found that the DBDs from ten different nuclear receptors all function as export signals. DBD-dependent nuclear export is saturable, and prolonged nuclear localization of the GR increases its transcriptional activity.Conclusions: Multiple members of the nuclear receptor superfamily use a common pathway to exit the nucleus. We propose that NLS-mediated import and DBD-mediated export define a shuttling cycle that integrates the compartmentalization and activity of nuclear receptors.

SOX7 transcription factor: sequence, chromosomal localisation, expression, transactivation and interference with Wnt signalling.

The Sox gene family consists of several genes related by encoding a 79 amino acid DNA-binding domain known as the HMG box. This box shares strong sequence similarity to that of the testis determining protein SRY. SOX proteins are transcription factors having critical roles in the regulation of diverse developmental processes in the animal kingdom. We have characterised the human SOX7 gene and compared it to its mouse orthologue. Chromosomal mapping analyses localised mouse Sox7 on band D of mouse chromosome 14, and assigned human SOX7 in a region of shared synteny on human chromosome 8 (8p22). A detailed expression analysis was performed in both species. Sox7 mRNA was detected during embryonic development in many tissues, most abundantly in brain, heart, lung, kidney, prostate, colon and spleen, suggesting a role in their respective differentiation and development. In addition, mouse Sox7 expression was shown to parallel mouse Sox18 mRNA localisation in diverse situations. Our studies also demonstrate the presence of a functional transactivation domain in SOX7 protein C-terminus, as well as the ability of SOX7 protein to significantly reduce Wnt/beta-catenin-stimulated transcription. In view of these and other findings, we suggest different modes of action for SOX7 inside the cell including repression of Wnt signalling.

Mutations in the Yeast Nhp6 Protein Can Differentially Affect Its in Vivo Functions

Nhp6A and Nhp6B from S. cerevisiae are required for viability at 38°C because they are involved in transcription of SNR6 by RNA polymerase III. Nhp6A also represses transcription of NHP6B by RNA polymerase II. Nhp6 belongs to the HMG1 family, defined by an 80 amino acid DNA binding domain, which includes six highly conserved residues. These amino acids were mutated in Nhp6A and their affects on Nhp6 function were assessed in vivo. Surprisingly, most of the changes allowed Nhp6A to function normally in supporting growth at 38°C. However, six mutants had differential effects on in vivo function. Finally, two of the mutant proteins that did not restore Nhp6A function in vivo were shown to bind and bend DNA in vitro as well as wild type. Together, these results suggest that Nhp6 interacts with another protein(s) to carry out some of its biological functions and that this interaction might differ at promoters transcribed by RNA polymerase II versus RNA polymerase III.

Ets target genes: past, present and future.

Ets is a family of transcription factors present in species ranging from sponges to human. All family members contain an approximately 85 amino acid DNA binding domain, designated the Ets domain. Ets proteins bind to specific purine-rich DNA sequences with a core motif of GGAA/T, and transcriptionally regulate a number of viral and cellular genes. Thus, Ets proteins are an important family of transcription factors that control the expression of genes that are critical for several biological processes, including cellular proliferation, differentiation, development, transformation, and apoptosis. Here, we tabulate genes that are regulated by Ets factors and describe past, present and future strategies for the identification and validation of Ets target genes. Through definition of authentic target genes, we will begin to understand the mechanisms by which Ets factors control normal and abnormal cellular processes.

Molecular Dynamics Simulation in Solvent of the Bacteriophage 434 cI Repressor Protein DNA Binding Domain Amino Acids (R1–69) in Complex with its Cognate Operator (OR1) DNA Sequence

We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstom water layer model of the 434 cl Repressor protein DNA binding domain (DBD) amino acids (R1–69) and DNA of operator (OR1) and its flanks consisting of 28 nucleotide base pairs. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the 434 cl repressor DNA recognition helix 3 formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the ORl DNA major groove halfsites and flanking regions. In addition, hydrophilic amino acids within the loop between helix 3 and helix 4 have strong electrostatic attraction to codon-anticodon nucleotides located within the central nucleotides of the minor groove between the ORI major groove halfsites These interactions together induced significant structural changes in the operator DNA manifested by overtwisting of the central nucleotide base pairs and narrowing of the minor groove between the DNA major groove halfsites. Finally, these findings offer a code for site specific DNA recognition by the 434 cl repressor protein.

Brachyury and the T-box genes

Brachyury, or T, is the founder member of a family of transcription factors that share the so-called T-box — a 200 amino acid DNA-binding domain. Recent work has addressed the regulation of Brachyury expression and its function in the embryo. New T-box family members have been found in vertebrate and invertebrate embryos and the importance of this gene family is illustrated by the discovery that mutations in human TBX5 are responsible for Holt-Oram syndrome, which is characterised by abnormalities in heart and forelimb development.

Correction of the NMR structure of the ETS1/DNA complex.

The ETS family of transcription factors consists of a group of proteins that share a highly conserved 85 amino acid DNA-binding domain (DBD). This family recognizes a consensus sequence rich in purine bases with a central GGAA motif. A comparison of the published three-dimensional structures of the DBD/DNA complexes of ETS1 by NMR [Werner et al. (1995) Cell, 83, 761-771] and the related Pu.1 by X-ray crystallography [Kodandapani et al. (1996) Nature, 380, 456-460] reveals an apparent discrepancy in which the protein domains bind with opposite polarity to their target sequences. This surprising and highly unlikely result prompted us to reexamine our NMR structure. Additional NMR experiments now reveal an error in the original interpretation of the spectra defining the orientation of the ETS1-DBD on DNA. It was originally reported that the ETS1-DBD bound to DNA with a bipartite motif involving major groove recognition via a helix-turn-helix element and minor groove recognition via protein side-chain intercalation. The presence of intercalation was deduced on the basis of numerous NOEs between several amino acids in the protein and a resonance at 12.33 ppm originally assigned to a DNA imino proton. New NMR experiments now conclusively demonstrate that this resonance, which is located within the DNA imino proton region of the spectrum, arises from the hydroxyl proton of Tyr86. Realization of this error necessitated reanalysis of the intermolecular NOEs. This revealed that the orientation of the ETS1-DBD in the complex is opposite to that originally reported and that a tryptophan residue does not intercalate into the DNA. The calculation of a new ensemble of structures based on the corrected data indicates that the structure of the ETS1-DBD/DNA complex is indeed similar to the X-ray structure of the Pu.1-DBD/DNA complex.

Clustered arrangement of winged helix genes fkh-6 and MFH-1: possible implications for mesoderm development.

The 'winged helix' or 'forkhead' transcription factor gene family is defined by a common 100 amino acid DNA binding domain which is a variant of the helix-turn-helix motif. Here we describe the structure and expression of the mouse fkh-6 and MFH-1 genes. Both genes are expressed in embryonic mesoderm from the headfold stage onward. Transcripts for both genes are localised mainly to mesenchymal tissues, fkh-6 mRNA is enriched in the mesenchyme of the gut, lung, tongue and head, whereas MFH-1 is expressed in somitic mesoderm, in the endocardium and blood vessels as well as the condensing mesenchyme of the bones and kidney and in head mesenchyme. Both genes are located within a 10 kb region (in mouse chromosome 8 at 5.26 +/- 2.56 cM telomeric to Actsk1. The close physical linkage of these two winged helix genes is conserved in man, where the two genes map to chromosome 16q22-24. This tandem arrangement suggests the common use of regulatory mechanisms. The fkh-6/MFH-1 locus maps close to the mouse mutation amputated, which is characterised by abnormal development of somitic and facial mesoderm. Based on the expression patterns we suggest that a mutation in MFH-1, not fkh-6 is the possible cause for the amputated phenotype.

A FORKHEAD TRANSCRIPTION FACTOR EXPRESSED IN DEVELOPING HUMAN LUNG, KIDNEY AND CENTRAL NERVOUS SYSTEM. • 380

Forkhead transcription factors appear to play important roles in cell fate determination and cell-specific gene expression during development. To study the role of these transcription factors in human development and disease, a human forkhead transcription factor HFH-4 was isolated by screening a human fetal lung cDNA library with a rat HFH-4 probe. Sequence analysis of isolated clones revealed an open reading frame of 421 amino acids with a conserved 100 amino acid DNA binding domain. Comparison of the human amino acid sequence to the murine and rat proteins revealed greater than 90% amino acid identity overall and 100% identity within the DNA binding domain. Comparison to previously identified human forkhead DNA binding domains revealed less than 50% amino acid homology. The human cDNA is encoded by a single Hfh-4 gene localized to chromosome 17q21-25 by Southern blot analysis of human/hamster somatic cell hybrid DNA. Examination of adult human tissues by RNA blot analysis revealed expression of a 2.5 kb HFH-4 transcript in male and female reproductive tissues, lung, and brain. In human fetal tissue at 22 to 24 weeks gestation a 2.5 kb transcript was present in lung, kidney and brain. By in situ hybridization, expression of HFH-4 in the human fetal lung was restricted to the developing bronchiolar epithelium. Expression of HFH-4 in developing human organs suggests a role for this transcription factor in regulating human developmental processes.