Exploring the DNA-Recognition Potential of Homeodomains
Ontology highlight
ABSTRACT:
ORGANISM(S): synthetic construct
PROVIDER: GSE35806 | GEO | 2012/02/15
SECONDARY ACCESSION(S): PRJNA152555
REPOSITORIES: GEO
Similar Datasets
Project description:We utilized a bacterial selection system to isolate Engrailed variants from a randomized library that are compatible with each of the 64 possible 3’ triplet sites (i.e. TAANNN). The DNA-binding specificity of 151 representative HD variants from these populations was subsequently characterized. Many of these variants contain novel combinations of specificity determinants that are uncommon or absent in extant HDs. These novel determinants, when grafted into different HD backbones, produce a corresponding alteration in their specificity. The identified determinates expand our understanding of HD recognition. Homeodomains where selected against each of the 64 possible 3' triplet sites (ie: TAANNN). The DNA-binding specificity of 151 representative HD variants from these populations was subsequently characterized. 7 novel determinants, when grafted into 3 different HD backbones. 5' speicifity was changed for 3 novel determinants.
2012-02-15 | E-GEOD-35806 | biostudies-arrayexpress
Project description:Homeodomains (HDs) are the second largest class of DNA binding domains (DBDs) in eukaryotic sequence-specific transcription factors (TFs) and are the TF structural class with the largest number of disease mutations in the Human Gene Mutation Database (HGMD). Despite numerous structural studies and large-scale analyses of HD DNA binding specificity, HD-DNA recognition is still not fully understood. Here, we analyzed 92 human HD mutants, including disease-associated variants and variants of unknown significance (VUS), for their effects on DNA binding activity. Many of the variants altered DNA binding affinity and/or specificity. Structural analysis identified 14 novel specificity-determining positions, 5 of which do not contact DNA. The same missense substitution at analogous positions within different HDs exhibited different effects on DNA binding activity. Variant effect prediction tools perform moderately well in distinguishing variants with altered DNA binding affinity, but poorly in identifying those with altered binding specificity. Our results highlight the need for biochemical assays of TF coding variants and promote dozens of variants for further investigations into their pathogenicity and the development of clinical diagnostics and precision therapies.
2023-12-05 | GSE233827 | GEO
Project description:A subfamily of Drosophila homeodomain (HD) transcription factors (TFs) controls the identities of individual muscle founder cells (FCs). However, the molecular mechanisms by which these TFs generate unique FC genetic programs remain unknown. To investigate this problem, we first applied genome-wide mRNA expression profiling to identify genes that are activated or repressed by the muscle HD TFs Slouch (Slou) and Muscle segment homeobox (Msh). Next, we used protein binding microarrays to define the sequences that are bound by Slou, Msh and other HD TFs having mesodermal expression. These studies revealed that a large class of HDs, including Slou and Msh, predominantly recognize TAAT core sequences but that each HD also binds to unique sites that deviate from this canonical motif. To better understand the regulatory specificity of an individual FC identity HD, we evaluated the functions of atypical binding sites that are preferentially bound by Slou relative to other HDs within muscle enhancers that are either activated or repressed by this TF. These studies showed that Slou regulates the activities of particular myoblast enhancers through Slou-preferred sequences, whereas swapping these sequences for sites that are capable of binding to multiple HD family members does not support the normal regulatory functions of Slou. Moreover, atypical Slou binding sites are overrepresented in putative enhancers associated with additional Slou-responsive FC genes. Collectively, these studies provide new insights into the roles of individual HD TFs in determining cellular identity, and suggest that the diversity of HD binding preferences can confer regulatory specificity.
2012-01-28 | GSE35380 | GEO
Project description:Homeodomain (HD) proteins comprise a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, yet they paradoxically recognize very similar DNA sequences. To investigate how HDs control cell-specific gene expression patterns, we determined the DNA binding specificities of a broad range of HDs critical for Drosophila embryonic mesoderm development. These studies revealed particular sequences that are bound by one HD and not by others. Such HD-preferred binding sites are overrepresented in the noncoding regions of genes that are regulated by the corresponding HD. Moreover, we show at single-cell resolution in intact embryos that the HD Slouch (Slou) controls myoblast gene expression through unique DNA sequences that are preferentially bound by Slou. These findings demonstrate that the sequence of a HD-binding site dictates which HD family member binds to and regulates a particular enhancer. This represents a novel mechanism for how cell type-specific TFs induce the distinct genetic programs of individual embryonic cells.
2012-02-08 | GSE27163 | GEO
Project description:Homeodomain (HD) proteins comprise a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, yet they paradoxically recognize very similar DNA sequences. To investigate how HDs control cell-specific gene expression patterns, we determined the DNA binding specificities of a broad range of HDs critical for Drosophila embryonic mesoderm development. These studies revealed particular sequences that are bound by one HD and not by others. Such HD-preferred binding sites are overrepresented in the noncoding regions of genes that are regulated by the corresponding HD. Moreover, we show at single-cell resolution in intact embryos that the HD Slouch (Slou) controls myoblast gene expression through unique DNA sequences that are preferentially bound by Slou. These findings demonstrate that the sequence of a HD-binding site dictates which HD family member binds to and regulates a particular enhancer. This represents a novel mechanism for how cell type-specific TFs induce the distinct genetic programs of individual embryonic cells. Mesodermal cells were profiled from wild type, slou gain-of-function and msh gain-of-function backgrounds.
2012-02-08 | E-GEOD-27163 | biostudies-arrayexpress
Project description:Profiling TF sequence variants to map their specificity determinants
2023-05-03 | GSE209631 | GEO
Project description:A subfamily of Drosophila homeodomain (HD) transcription factors (TFs) controls the identities of individual muscle founder cells (FCs). However, the molecular mechanisms by which these TFs generate unique FC genetic programs remain unknown. To investigate this problem, we first applied genome-wide mRNA expression profiling to identify genes that are activated or repressed by the muscle HD TFs Slouch (Slou) and Muscle segment homeobox (Msh). Next, we used protein binding microarrays to define the sequences that are bound by Slou, Msh and other HD TFs having mesodermal expression. These studies revealed that a large class of HDs, including Slou and Msh, predominantly recognize TAAT core sequences but that each HD also binds to unique sites that deviate from this canonical motif. To better understand the regulatory specificity of an individual FC identity HD, we evaluated the functions of atypical binding sites that are preferentially bound by Slou relative to other HDs within muscle enhancers that are either activated or repressed by this TF. These studies showed that Slou regulates the activities of particular myoblast enhancers through Slou-preferred sequences, whereas swapping these sequences for sites that are capable of binding to multiple HD family members does not support the normal regulatory functions of Slou. Moreover, atypical Slou binding sites are overrepresented in putative enhancers associated with additional Slou-responsive FC genes. Collectively, these studies provide new insights into the roles of individual HD TFs in determining cellular identity, and suggest that the diversity of HD binding preferences can confer regulatory specificity. 10 Protein binding microarray (PBM) experiments of Drosophila transcription factors were performed. Briefly, the PBMs involved binding GST-tagged fly transcription factors to double-stranded 44K Agilent microarrays in order to determine their sequence preferences. The method is described in Berger et al., Nature Biotechnology 2006 (PMID: 16998473). A key feature is that the microarrays are composed of de Bruijn sequences that contain each 10-base sequence once and only once, providing an evenly balanced sequence distribution. Individual de Bruijn sequences have different properties, including representation of gapped patterns. The array probe sequences on the custom array design used in this study were reported previously in Berger et al., Cell 2008 (PMID: 18585359) and are available via an academic research use license. Here we provide the data transformed into median signal intensities (after normalization and detrending of the original array data) for all 32,896 8-base sequences, Z-scores for these intensities, and E-scores. E-scores are a modified version of AUC, and describe how well each 8-mer ranks the intensities of the spots. 'Keep fraction' (kf) parameter setting of 0.9 was used to calculate E-scores. In general the E-scores are slightly more reproducible than Z-scores, but contain less information about relative binding affinity. Additional experimental details are found in Berger et al., Nature Biotechnology 2006 (PMID: 16998473), and the accompanying Supplementary information.
2012-01-28 | E-GEOD-35380 | biostudies-arrayexpress
Project description:Th17 cells are rapidly depleted during HIV infection and their frequencies and functions are not fully restored by antiretroviral therapy (ART). In addition, Th17 cells contribute to viral persistence during ART. However, molecular mechanisms underlying these observations remain understudied. MemCCR6+ cells from STs vs. HDs showed alterations in the NOTCH and TGF-β1/smad2-3 pathways, indicative of impairments in their differentiation and stability. Consistently, the RORγt/C2 repressor NR1D1 was upregulated, while Semaphorin 4D, a RORγt/C2 inducer, was decreased in MemCCR6+ cells from STs vs. HDs. Frequencies of Th17 and Th1/Th17 cells within total MemCCR6+ cells were lower in STs and ECs compared to HD individuals, and expressed higher levels of activation and senescence/exhaustion markers. As a result, MemCCR6+ cells from STs produced less IL-17A/F, IL-22, and IFN-γ in vitro compared to HDs. Downregulation of HIV restriction factors (SERINC3, KLF3, and RNF125) and HIV inhibitors (tetraspanins), along with increased expression of MRE11 in MemCCR6+ cells from ST individuals supported higher susceptibility/permissiveness to HIV infection. MemCCR6+ cells from STs and ECs had lower TFAP4 expression and increased expression of EED and TP53BP1 vs. HDs, which is associated to HIV persistence. Markers of DNA damage/modification were elevated in STs and ECs compared to HDs, including NASP, RPA2, H2AX, PARP, and DNA methylases DNMT1/DNMT3A. Similar to the ST group, MemCCR6+ cells in ECs showed decreased expression of Semaphorin 4D and Notch1, along with higher expression of NR1D1 compared to HD donors. Despite successful ART, the transcriptional signature of MemCCR6+ cells revealed impaired differentiation, stability, functions, DNA damage/modifications, and higher susceptibility to HIV infection and viral persistence.
2024-06-30 | GSE252771 | GEO
Project description:Antigen-antibody interactions play a key role in the immune response post vaccination and the mechanism of action of antibody-based biopharmaceuticals. 4CMenB is a multicomponent vaccine against Neisseria meningitidis serogroup B in which factor H binding protein (fHbp) is one of the key antigens. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to identify epitopes in fHbp recognized by polyclonal antibodies (pAb) from two human donors (HDs) vaccinated with 4CMenB. Our HDX-MS data reveal several epitopes recognized by the complex mixture of human pAb. Furthermore, we show that the pAb from the two HDs recognize the same epitope regions. Epitope mapping of total pAb and purified fHbp-specific pAb from the same HD reveals that the two antibody samples recognize the same main epitopes, showing that HDX-MS based epitope mapping can, in this case at least, be performed directly using total IgG pAb samples that have not undergone Ab-selective purification. Two monoclonal antibodies (mAb) were previously produced from B-cell repertoire sequences from one of the HDs and used for epitope mapping of fHbp with HDX-MS. The epitopes identified for the pAb from the same HD in this study, overlap with the epitopes recognized by the two individual mAbs. Overall, HDX-MS epitope mapping appears highly suitable for simultaneous identification of epitopes recognized by pAb from human donors and to thus both guide vaccine development and study basic human immunity to pathogens, including viruses.
2024-05-22 | PXD047712 | Pride