scholarly journals The structural basis for forkhead box family specificity revealed by the crystal structure of human FOXN1 in complex with DNA

2018 ◽  
Author(s):  
Joseph A. Newman ◽  
Hazel Aitkenhead ◽  
Angeline Gravard ◽  
Ioanna A. Rota ◽  
Adam E. Handel ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Large Family ◽  
Structural Basis ◽  
Thymic Epithelial Cell ◽  
Forkhead Box ◽  
Recognition Helix ◽  
Dna Specificity

SignificanceFOXN1 is a transcription factor that is essential for the development of the thymus and the production of T-lymphocytes. It is a member of a large family of transcription factors which recognize DNA sequences through the conserved Forkhead (FH) domain. FOXN1 recognizes a DNA sequence that is different from the common consensus binding sequence of FH domains, although key binding residues are identical. We present crystal structures of the FH domain of FOXN1, free and DNA-bound, which shed light on the different binding specificities; the structure also revelas the basis of the immunocompromised nude mutation, as well as a preferential binding to non-methylated CpG motifs.AbstractFOXN1 is a member of the forkhead box (FOX) family of transcription factors, and plays an important role in thymic epithelial cell differentiation and function. FOXN1 mutations in humans and mice give rise to the "nude" phenotype which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognize an alternate consensus sequence (GACGC), which is different from the more widely-recognized canonical sequence consensus RYAAAYA. Here, we present the structure of FOXN1 in complex with DNA at 1.6 Å resolution, in which the DNA sequence is recognised by a mixture of direct and water-mediated contacts provided by residues in an a-helix inserted in the DNA major groove (the recognition helix). Comparisons with other FOX family structures reveal that the canonical and alternate DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identify a single alternate rotamer within the recognition helix itself as an important determinant of DNA specificity, and indicate sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally we demonstrate that FOXN1 has a significantly reduced affinity for DNA containing 5-methylcytosine, which may have implications for the role of FOXN1 in thymic senescence.

scholarly journals The crystal structure of human forkhead box N1 in complex with DNA reveals the structural basis for forkhead box family specificity

2019 ◽  
Vol 295 (10) ◽  
pp. 2948-2958 ◽  
Author(s):  
Joseph A. Newman ◽  
Hazel Aitkenhead ◽  
Angeline E. Gavard ◽  
Ioanna A. Rota ◽  
Adam E. Handel ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Consensus Sequence ◽  
Family Members ◽  
Structural Basis ◽  
Thymic Epithelial Cell ◽  
Thymic Involution ◽  
Forkhead Box ◽  
Recognition Helix ◽  
Dna Specificity ◽  
Α Helix

Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the “nude” phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC) that is different from the more widely recognized forkhead (FKH) sequence RYAAAYA (where R is purine, and Y is pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5′-methylcytosine, which may have implications for the role of FOXN1 in thymic involution.

Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells

1985 ◽  
Vol 5 (4) ◽  
pp. 619-627
Author(s):  
M Montoya-Zavala ◽  
J L Hamlin
Keyword(s):  
Dna Sequence ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chinese Hamster Cells ◽  
Reductase Gene ◽  
Reductase Domain

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.

scholarly journals Comparative structure analysis of the ETSi domain of ERG3 and its complex with the E74 promoter DNA sequence

2018 ◽  
Vol 74 (10) ◽  
pp. 656-663 ◽  
Author(s):  
Ruby Sharma ◽  
Shanti P. Gangwar ◽  
Ajay K. Saxena
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Dna Sequence ◽  
Structure Analysis ◽  
Dna Sequences ◽  
Space Group ◽  
Class 1 ◽  
Comparative Structure ◽  
Ets Domain ◽  
Promoter Dna

ERG3 (ETS-related gene) is a member of the ETS (erythroblast transformation-specific) family of transcription factors, which contain a highly conserved DNA-binding domain. The ETS family of transcription factors differ in their binding to promoter DNA sequences, and the mechanism of their DNA-sequence discrimination is little known. In the current study, crystals of the ETSi domain (the ETS domain of ERG3 containing a CID motif) in space group P41212 and of its complex with the E74 DNA sequence (DNA9) in space group C2221 were obtained and their structures were determined. Comparative structure analysis of the ETSi domain and its complex with DNA9 with previously determined structures of the ERGi domain (the ETS domain of ERG containing inhibitory motifs) in space group P65212 and of the ERGi–DNA12 complex in space group P41212 were performed. The ETSi domain is observed as a homodimer in solution as well as in the crystallographic asymmetric unit. Superposition of the structure of the ETSi domain on that of the ERGi domain showed a major conformational change at the C-terminal DNA-binding autoinhibitory (CID) motif, while minor changes are observed in the loop regions of the ETSi-domain structure. The ETSi–DNA9 complex in space group C2221 forms a structure that is quite similar to that of the ERG–DNA12 complex in space group P41212. Upon superposition of the complexes, major conformational changes are observed at the 5′ and 3′ ends of DNA9, while the conformation of the core GGA nucleotides was quite conserved. Comparison of the ETSi–DNA9 structure with known structures of ETS class 1 protein–DNA complexes shows the similarities and differences in the promoter DNA binding and specificity of the class 1 ETS proteins.

scholarly journals DNA Sequence and Functional Analysis of Homologous ARS Elements of Saccharomyces cerevisiae and S. carlsbergensis

Genetics ◽  
1999 ◽  
Vol 152 (3) ◽  
pp. 943-952
Author(s):  
James F Theis ◽  
Chen Yang ◽  
Christopher B Schaefer ◽  
Carol S Newlon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Chromosomal Dna ◽  
Functional Elements ◽  
Sequence Comparisons ◽  
Cis Acting ◽  
Homologous Sequences ◽  
Ars Elements

Abstract ARS elements of Saccharomyces cerevisiae are the cis-acting sequences required for the initiation of chromosomal DNA replication. Comparisons of the DNA sequences of unrelated ARS elements from different regions of the genome have revealed no significant DNA sequence conservation. We have compared the sequences of seven pairs of homologous ARS elements from two Saccharomyces species, S. cerevisiae and S. carlsbergensis. In all but one case, the ARS308-ARS308carl pair, significant blocks of homology were detected. In the cases of ARS305, ARS307, and ARS309, previously identified functional elements were found to be conserved in their S. carlsbergensis homologs. Mutation of the conserved sequences in the S. carlsbergensis ARS elements revealed that the homologous sequences are required for function. These observations suggested that the sequences important for ARS function would be conserved in other ARS elements. Sequence comparisons aided in the identification of the essential matches to the ARS consensus sequence (ACS) of ARS304, ARS306, and ARS310carl, though not of ARS310.

scholarly journals Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells.

1985 ◽  
Vol 5 (4) ◽  
pp. 619-627 ◽  
Author(s):  
M Montoya-Zavala ◽  
J L Hamlin
Keyword(s):  
Dna Sequence ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Chinese Hamster Cells ◽  
Reductase Gene ◽  
Reductase Domain

We have isolated overlapping recombinant cosmids that represent 150 kilobases of contiguous DNA sequence from the amplified dihydrofolate reductase domain of a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400). This sequence includes the 25-kilobase dihydrofolate reductase gene and an origin of DNA synthesis. Eight cosmids that span this domain have been utilized as radioactive hybridization probes to analyze the similarities among the dihydrofolate reductase amplicons in four independently derived methotrexate-resistant Chinese hamster cell lines. We have observed no significant differences among the four cell lines within the 150-kilobase DNA sequence that we have examined, except for polymorphisms that result from the amplification of one or the other of two possible alleles of the dihydrofolate reductase domain. We also show that the restriction patterns of the amplicons in these four resistant cell lines are virtually identical to that of the corresponding, unamplified sequence in drug-susceptible parental cells. Furthermore, measurements of the relative copy numbers of fragments from widely separated regions of the amplicon suggest that all fragments in this 150-kilobase region may be amplified in unison. Our data show that in methotrexate-resistant Chinese hamster cells, the amplified unit is large relative to the dihydrofolate reductase gene itself. Furthermore, within the 150-kilobase amplified consensus sequence that we have examined, significant rearrangements do not seem to occur during the amplification process.

An AT-rich satellite DNA sequence, E180, in alfalfa (Medicago sativa)

Genome ◽  
1993 ◽  
Vol 36 (3) ◽  
pp. 427-432 ◽  
Author(s):  
X. Xia ◽  
L. Erickson
Keyword(s):  
Medicago Sativa ◽  
Dna Sequence ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Satellite Dna ◽  
Nuclear Dna ◽  
Consensus Sequence ◽  
Repeated Dna Sequence ◽  
Dna Fragment ◽  
High Degree

A DNA fragment of ~750 bp was cloned from EcoRI-digested nuclear DNA of alfalfa (Medicago sativa). Southern blot and sequence analysis showed that the cloned DNA fragment represents a tetramer of a highly tandemly repeated DNA sequence of 185–188 bp (E180). The consensus sequence deduced from the four repeating units is 189 bp in length with an AT content of 67%. The copy number of the satellite DNA was estimated to be ~1.8 × 105 per genome and constitutes about 1% of the alfalfa genome. Sequence comparison revealed no identity to any repetitive DNA sequences that have been published to date. Digestion with HpaII and MspI indicated a high degree of methylation at the internal C of the restriction site CCGG within E180.Key words: alfalfa, Medicago, repetitive DNA sequence, satellite DNA.

DNA sequence requirements for transcriptional initiator activity in mammalian cells

1994 ◽  
Vol 14 (1) ◽  
pp. 116-127
Author(s):  
R Javahery ◽  
A Khachi ◽  
K Lo ◽  
B Zenzie-Gregory ◽  
S T Smale
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Tata Box ◽  
Start Site ◽  
Sequence Element ◽  
Synthetic Promoters ◽  
Sequence Requirements

A transcriptional initiator (Inr) for mammalian RNA polymerase II can be defined as a DNA sequence element that overlaps a transcription start site and is sufficient for (i) determining the start site location in a promoter that lacks a TATA box and (ii) enhancing the strength of a promoter that contains a TATA box. We have prepared synthetic promoters containing random nucleotides downstream of Sp1 binding sites to determine the range of DNA sequences that convey Inr activity. Numerous sequences behaved as functional Inrs in an in vitro transcription assay, but the Inr activities varied dramatically. An examination of the functional elements revealed loose but consistent sequence requirements, with the approximate consensus sequence Py Py A+1 N T/A Py Py. Most importantly, almost every functional Inr that has been described fits into the consensus sequence that we have defined. Although several proteins have been reported to bind to specific Inrs, manipulation of those elements failed to correlate protein binding with Inr activity. The simplest model to explain these results is that all or most Inrs are recognized by a universal binding protein, similar to the functional recognition of all TATA sequences by the same TATA-binding protein. The previously reported proteins that bind near specific Inr elements may augment the strength of an Inr or may impart transcriptional regulation through an Inr.

scholarly journals General and Genomic DNA-Binding Specificity for the Thermus thermophilus HB8 Transcription Factor TTHB023

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 94 ◽  
Author(s):  
James Shell Cox ◽  
Michael W. Van Dyke
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Sequences ◽  
Fatty Acid Biosynthesis ◽  
Thermus Thermophilus ◽  
Consensus Sequence ◽  
Specific Gene ◽  
Thermus Thermophilus Hb8 ◽  
Dna Binding Specificity ◽  
Primary Means

Transcription factors are proteins that recognize specific DNA sequences and affect local transcriptional processes. They are the primary means by which all organisms control specific gene expression. Understanding which DNA sequences a particular transcription factor recognizes provides important clues into the set of genes that they regulate and, through this, their potential biological functions. Insights may be gained through homology searches and genetic means. However, these approaches can be misleading, especially when comparing distantly related organisms or in cases of complicated transcriptional regulation. In this work, we used a biochemistry-based approach to determine the spectrum of DNA sequences specifically bound by the Thermus thermophilus HB8 TetR-family transcription factor TTHB023. The consensus sequence 5′–(a/c)Y(g/t)A(A/C)YGryCR(g/t)T(c/a)R(g/t)–3′ was found to have a nanomolar binding affinity with TTHB023. Analyzing the T. thermophilus HB8 genome, several TTHB023 consensus binding sites were mapped to the promoters of genes involved in fatty acid biosynthesis. Notably, some of these were not identified previously through genetic approaches, ostensibly given their potential co-regulation by the Thermus thermophilus HB8 TetR-family transcriptional repressor TTHA0167. Our investigation provides additional evidence supporting the usefulness of a biochemistry-based approach for characterizing putative transcription factors, especially in the case of cooperative regulation.

scholarly journals Half-Site DNA Sequence and Spacing Length Contributions to PrrA Binding to PrrA Site 2 of RSP3361 in Rhodobacter sphaeroides 2.4.1

2009 ◽  
Vol 191 (13) ◽  
pp. 4353-4364 ◽  
Author(s):  
Jesus M. Eraso ◽  
Samuel Kaplan
Keyword(s):  
Dna Binding ◽  
Dna Sequence ◽  
Rhodobacter Sphaeroides ◽  
Dna Sequences ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Binding Affinities ◽  
Transcriptional Fusions

ABSTRACT The consensus DNA binding sequence for PrrA, a global regulator in Rhodobacter sphaeroides 2.4.1, is poorly defined. We have performed mutational analysis of PrrA site 2, of the RSP3361 gene, to which PrrA binds in vitro (J. M. Eraso and S. Kaplan, J. Bacteriol. 191:4341-4352, 2009), to further define the consensus sequence for DNA binding. Two half-sites of equal length, containing 6 nucleotides each, were required for PrrA binding to this DNA sequence. Systematic nucleotide substitutions in both inverted half-sites led to a decrease in binding affinity of phosphorylated PrrA in vitro, the level of which was dependent on the substitution. The reduced binding affinities were confirmed by competition experiments and led to proportional decreases in the expression of lacZ transcriptional fusions to the RSP3361 gene in vivo. The 5-nucleotide spacer region between the half-sites was found to be optimal for PrrA binding to the wild-type half-sites, as shown by decreased PrrA DNA binding affinities to synthetic DNA sequences without spacer regions or with spacer regions ranging from 1 to 10 nucleotides. The synthetic spacer region alleles also showed decreased gene expression in vivo when analyzed using lacZ transcriptional fusions. We have studied three additional DNA sequences to which PrrA binds in vitro. They are located in the regulatory regions of genes positively regulated by PrrA and contain spacer regions with 5 or 8 nucleotides. We demonstrate that PrrA can bind in vitro to DNA sequences with different lengths in the spacer regions between the half-sites.

scholarly journals Cryo-EM structure of the nucleosome containing the ALB1 enhancer DNA sequence

Open Biology ◽  
2018 ◽  
Vol 8 (3) ◽  
pp. 170255 ◽  
Author(s):  
Yoshimasa Takizawa ◽  
Hiroki Tanaka ◽  
Shinichi Machida ◽  
Masako Koyama ◽  
Kazumitsu Maehara ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Precursor Cells ◽  
Dna Accessibility ◽  
Nucleosome Structure ◽  
Gene Enhancer ◽  
Pioneer Transcription Factor

Pioneer transcription factors specifically target their recognition DNA sequences within nucleosomes. FoxA is the pioneer transcription factor that binds to the ALB1 gene enhancer in liver precursor cells, and is required for liver differentiation in embryos. The ALB1 enhancer DNA sequence is reportedly incorporated into nucleosomes in cells, although the nucleosome structure containing the targeting sites for FoxA has not been clarified yet. In this study, we determined the nucleosome structure containing the ALB1 enhancer (N1) sequence, by cryogenic electron microscopy at 4.0 Å resolution. The nucleosome structure with the ALB1 enhancer DNA is not significantly different from the previously reported nucleosome structure with the Widom 601 DNA. Interestingly, in the nucleosomes, the ALB1 enhancer DNA contains local flexible regions, as compared to the Widom 601 DNA. Consistently, DNaseI treatments revealed that, in the nucleosome, the ALB1 enhancer (N1) DNA is more accessible than the Widom 601 sequence. The histones also associated less strongly with the ALB1 enhancer (N1) DNA than the Widom 601 DNA in the nucleosome. Therefore, the local histone–DNA contacts may be responsible for the enhanced DNA accessibility in the nucleosome with the ALB1 enhancer DNA.

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