The REL domain of the NFATc2 protein binding to four types of DNA using protein binding microarrays
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ABSTRACT:
PROVIDER: PRJNA413374 | ENA |
REPOSITORIES: ENA
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Project description:NFATc2 is a sequence specific DNA binding protein in the Rel family that binds a 5-mer motif CGGAA better when both cytosines in the CG dinucleotide are methylated. Using protein binding microarrays (PBMs) we examined the DNA binding of NFATc2 to three additional types of DNA: (1) single-stranded DNA (ssDNA), (2) double stranded DNA (dsDNA) with 5-methylcytosine (5mC, M) in one strand and cytosine in the second strand (dsDNA(5mC|C)) and (3) DNA with 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand (dsDNA(5hmC|C)). NFATc2 binding to ssDNA is strongest for the 7-mer ATTTCCA. While NFAT can bind ssDNA, the average binding to the 40,000 DNA features of the PBM was twice as high for ssDNA compared to dsDNA, with maximal binding observed to dsDNA, suggesting that NFAT binding to dsDNA is more specific. dsDNA sequences containing the 5-mer MGGAA with 5mC in one DNA strand are better bound than CGGAA, with methylation of a single cytosine being sufficient for the stronger binding seen when both cytosines in the CG dinucleotide are methylated. Several DNA sequences containing HGGAA are also better bound than those containing cytosine, however the effect of 5hmC is not as dramatic as that of 5mC. Analysis of available NFATc2:DNA complexes rationalizes our PBM data.
2018-12-31 | GSE104631 | GEO
Project description:NFATc2 is a DNA binding protein in the Rel family transcription factors, which binds a CGGAA motif better when both cytosines in the CG dinucleotide are methylated. Using protein binding microarrays (PBMs), we examined the DNA binding of NFATc2 to three additional types of DNA: single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with either 5-methylcytosine (5mC, M) or 5-hydroxymethylcytosine (5hmC, H) in one strand and a cytosine in the second strand. ATTTCCAC, the complement of the core GGAA motif, is better bound as ssDNA compared to dsDNA. dsDNA containing the 5-mer CGGAA with either 5mC or 5hmC in one DNA strand is bound stronger than CGGAA. In contrast, the reverse complement TTCCG is bound weaker when it contains 5mC. Analysis of the available NFATc2:dsDNA complexes rationalizes these PBM data.
| S-EPMC7906578 | biostudies-literature
Project description:Based on previous work on the KDM histone demethylase family in AML, the transcription factor NFATC2 was identified as a regulatory target of KDM4A in MLL-AF9-rearrangement THP-1 cells. Our existing work examined the elements of the transcriptome regulated by NFATC2 in THP-1 cells and so, to further this work, we aimed to characterise the DNA-binding targets of NFATc2 in these cells. In this study, we used NFATc2 immunoprecipitation for ChIP-seq in untreated THP-1 cells, in order to determine these.
2024-09-01 | GSE241260 | GEO
Project description:Rel and RelA proteins were stably expressed in the chicken DT40 pre-B cell line and analyzed by transcription profiling to identify transformation-impacting genes regulated by NF-kB’s oncogenic v-Rel and c-Rel proteins. This analysis uncovered both common and differential gene expression profiles in cells expressing Rel vs. RelA proteins, and revealed that Rel protein expression can lead to gene-specific transcriptional repression, as seen for key B-cell receptor (BCR) components and signaling molecules like B-cell linker (BLNK), the B-cell adaptor for PI3K (BCAP) and Igλ. These were also downregulated in cells expressing a transformation-competent chimeric RelA/v-Rel protein, suggesting a correlation with the capacity of Rel proteins to transform lymphocytes. DNA binding, ChIP and transformation assays indicate that downregulation of BLNK and BCAP is an important contributing factor to the malignant transformation of lymphocytes by Rel and suggest that gene repression may be as important as transcriptional activation for the transforming activity of Rel proteins. Keywords: Comparative transcriptional profiling
2008-02-14 | GSE9544 | GEO
Project description:Adaptive immunity and the five vertebrate NF-kB/Rel family members first appeared in cartilaginous fish, suggesting that NF-kB family expansion allowed the acquisition of new functions to regulate adaptive immune responses. Transcriptome profiling revealed that, even in macrophages, the NF-kB family member, c-Rel, most potently regulates a cytokine gene linked to adaptive immunity, Il12b, with limiting roles at key regulators of innate immunity. Neofunctionalization of c-Rel to regulate Il12b depends on its unique DNA-binding properties, which we examined using structural, biochemical, functional, and genomic approaches. Among our findings was functional c-Rel homodimer binding to motifs with little resemblance to canonical NF-kB motifs. To determine whether c-Rel’s unique binding properties drove c-Rel-RelA divergence, we compared binding properties in various vertebrate species. c-Rel-RelA binding properties diverged in mammals and amphibians but were comparable in earlier vertebrates, suggesting that divergent DNA binding emerged relatively late during vertebrate evolution to support increasing complexity of adaptive immune regulation.
2023-11-29 | GSE243010 | GEO
Project description:Adaptive immunity and the five vertebrate NF-kB/Rel family members first appeared in cartilaginous fish, suggesting that NF-kB family expansion allowed the acquisition of new functions to regulate adaptive immune responses. Transcriptome profiling revealed that, even in macrophages, the NF-kB family member, c-Rel, most potently regulates a cytokine gene linked to adaptive immunity, Il12b, with limiting roles at key regulators of innate immunity. Neofunctionalization of c-Rel to regulate Il12b depends on its unique DNA-binding properties, which we examined using structural, biochemical, functional, and genomic approaches. Among our findings was functional c-Rel homodimer binding to motifs with little resemblance to canonical NF-kB motifs. To determine whether c-Rel’s unique binding properties drove c-Rel-RelA divergence, we compared binding properties in various vertebrate species. c-Rel-RelA binding properties diverged in mammals and amphibians but were comparable in earlier vertebrates, suggesting that divergent DNA binding emerged relatively late during vertebrate evolution to support increasing complexity of adaptive immune regulation.
2024-03-11 | GSE243011 | GEO
Project description:The transcription factor Nfatc2 is a potent β-cell mitogen in mouse and human islets, however, the direct genomic targets that mediate the mitogenic effects have not been identified. We expressed a constitutively active form of Nfatc2, and the closely related Nfatc1, in human islets and identified ~5,600 differentially expressed genes. Nfatc2 binding sites in human islets were identified via ChIP-seq, yielding ~8,600 high-confidence peaks. By integrating gene expression changes induced by Nfatc2 with genomic binding sites for Nfatc2, we identified ~2,200 direct transcriptional targets of Nfatc2 in human islets. Direct targets that were induced by Nfatc2 were enriched for transcripts that regulate the cell cycle, and for motifs associated with the transcription factor FoxP1. We showed that islets from an endocrine-specific FoxP1, FoxP2 and FoxP4 triple-knockout mouse are less responsive to Nfatc2-induced β-cell proliferation, suggesting the FoxP family may work in concert with Nfatc2 to regulate β-cell proliferation. Nfatc2 induced β-cell proliferation in both mouse and human islets, whereas Nfatc1 does so only in human islets. Exploiting this species difference in Nfatc1-responsiveness, we identified ~250 direct transcriptional targets with similar transcriptional profiles. This gene set strongly enriches for cell cycle-associated transcripts, and includes the nuclear hormone receptor, Nr4a1. We show that deletion of Nr4a1 greatly reduces the capacity of Nfatc2 to induce β-cell proliferation, suggesting that much of the effect on Nfatc2 occurs through its induction of Nr4a1. Integration of ncRNA profiling, chromatin accessibility and Nfatc2 binding sites enabled us to identify key Nfatc2-dependent enhancer loci that mediate enhanced β-cell proliferation.
2021-09-15 | GSE158498 | GEO
Project description:The transcription factor Nfatc2 is a potent β-cell mitogen in mouse and human islets, however, the direct genomic targets that mediate the mitogenic effects have not been identified. We expressed a constitutively active form of Nfatc2, and the closely related Nfatc1, in human islets and identified ~5,600 differentially expressed genes. Nfatc2 binding sites in human islets were identified via ChIP-seq, yielding ~8,600 high-confidence peaks. By integrating gene expression changes induced by Nfatc2 with genomic binding sites for Nfatc2, we identified ~2,200 direct transcriptional targets of Nfatc2 in human islets. Direct targets that were induced by Nfatc2 were enriched for transcripts that regulate the cell cycle, and for motifs associated with the transcription factor FoxP1. We showed that islets from an endocrine-specific FoxP1, FoxP2 and FoxP4 triple-knockout mouse are less responsive to Nfatc2-induced β-cell proliferation, suggesting the FoxP family may work in concert with Nfatc2 to regulate β-cell proliferation. Nfatc2 induced β-cell proliferation in both mouse and human islets, whereas Nfatc1 does so only in human islets. Exploiting this species difference in Nfatc1-responsiveness, we identified ~250 direct transcriptional targets with similar transcriptional profiles. This gene set strongly enriches for cell cycle-associated transcripts, and includes the nuclear hormone receptor, Nr4a1. We show that deletion of Nr4a1 greatly reduces the capacity of Nfatc2 to induce β-cell proliferation, suggesting that much of the effect on Nfatc2 occurs through its induction of Nr4a1. Integration of ncRNA profiling, chromatin accessibility and Nfatc2 binding sites enabled us to identify key Nfatc2-dependent enhancer loci that mediate enhanced β-cell proliferation.
2021-09-15 | GSE158497 | GEO
Project description:The transcription factor Nfatc2 is a potent β-cell mitogen in mouse and human islets, however, the direct genomic targets that mediate the mitogenic effects have not been identified. We expressed a constitutively active form of Nfatc2, and the closely related Nfatc1, in human islets and identified ~5,600 differentially expressed genes. Nfatc2 binding sites in human islets were identified via ChIP-seq, yielding ~8,600 high-confidence peaks. By integrating gene expression changes induced by Nfatc2 with genomic binding sites for Nfatc2, we identified ~2,200 direct transcriptional targets of Nfatc2 in human islets. Direct targets that were induced by Nfatc2 were enriched for transcripts that regulate the cell cycle, and for motifs associated with the transcription factor FoxP1. We showed that islets from an endocrine-specific FoxP1, FoxP2 and FoxP4 triple-knockout mouse are less responsive to Nfatc2-induced β-cell proliferation, suggesting the FoxP family may work in concert with Nfatc2 to regulate β-cell proliferation. Nfatc2 induced β-cell proliferation in both mouse and human islets, whereas Nfatc1 does so only in human islets. Exploiting this species difference in Nfatc1-responsiveness, we identified ~250 direct transcriptional targets with similar transcriptional profiles. This gene set strongly enriches for cell cycle-associated transcripts, and includes the nuclear hormone receptor, Nr4a1. We show that deletion of Nr4a1 greatly reduces the capacity of Nfatc2 to induce β-cell proliferation, suggesting that much of the effect on Nfatc2 occurs through its induction of Nr4a1. Integration of ncRNA profiling, chromatin accessibility and Nfatc2 binding sites enabled us to identify key Nfatc2-dependent enhancer loci that mediate enhanced β-cell proliferation.
2021-09-15 | GSE158496 | GEO
Project description:The transcription factor Nfatc2 is a potent β-cell mitogen in mouse and human islets, however, the direct genomic targets that mediate the mitogenic effects have not been identified. We expressed a constitutively active form of Nfatc2, and the closely related Nfatc1, in human islets and identified ~5,600 differentially expressed genes. Nfatc2 binding sites in human islets were identified via ChIP-seq, yielding ~8,600 high-confidence peaks. By integrating gene expression changes induced by Nfatc2 with genomic binding sites for Nfatc2, we identified ~2,200 direct transcriptional targets of Nfatc2 in human islets. Direct targets that were induced by Nfatc2 were enriched for transcripts that regulate the cell cycle, and for motifs associated with the transcription factor FoxP1. We showed that islets from an endocrine-specific FoxP1, FoxP2 and FoxP4 triple-knockout mouse are less responsive to Nfatc2-induced β-cell proliferation, suggesting the FoxP family may work in concert with Nfatc2 to regulate β-cell proliferation. Nfatc2 induced β-cell proliferation in both mouse and human islets, whereas Nfatc1 does so only in human islets. Exploiting this species difference in Nfatc1-responsiveness, we identified ~250 direct transcriptional targets with similar transcriptional profiles. This gene set strongly enriches for cell cycle-associated transcripts, and includes the nuclear hormone receptor, Nr4a1. We show that deletion of Nr4a1 greatly reduces the capacity of Nfatc2 to induce β-cell proliferation, suggesting that much of the effect on Nfatc2 occurs through its induction of Nr4a1. Integration of ncRNA profiling, chromatin accessibility and Nfatc2 binding sites enabled us to identify key Nfatc2-dependent enhancer loci that mediate enhanced β-cell proliferation.
2021-09-15 | GSE158495 | GEO