TFAP2A mediates HIF-1α dependent activation of hypoxia-inducible genes via interaction with Ku70/80 complex in a SUMO-dependent manner
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1132937 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Hypoxia can be established under pathological conditions, such as cancer, due to the imbalance between oxygen supply and consumption. Hypoxia Inducible Transcription Factor HIF-1 mediates the physiological response to hypoxia but also regulates multiple steps of carcinogenesis. Despite its well-defined oxygen-dependent activation, many aspects of HIF-1 transcriptional activity as well as interaction with chromatin remain elusive. We have recently shown that TFAP2A physically interacts with HIF-1 and hypoxia-dependent deSUMOylation of TFAP2A positively affects HIF-1 activity. We now present ChIP-seq analysis showing that TFAP2A resides together with HIF-1α on the promoters of a subset of hypoxia-regulated genes, the mRNA expression of which is downregulated by silencing of TFAP2A. Interestingly, CRISPR-mediated knockdown of TFAP2A expression under hypoxia decreased the occupancy of HIF-1α on these promoters and affected chromatin accessibility. Mechanistically, we reveal that the Ku70/Ku80 protein complex interacts with TFAP2A in a SUMO-dependent manner under hypoxia and participates in HIF-dependent gene expression. Moreover, using stable expression of TFAP2A forms that either lack or constitutively carry a SUMO modification, we could show that SUMOylation affects binding of TFAP2A to chromatin. Overall, our data suggest that TFAP2A is an important co-regulator of the HIF-1-dependent transcriptional response to hypoxia and SUMOylation fine-tunes this regulation. As both TFAP2A and HIF-1 play critical roles in cancer progression, a detailed characterization of their crosstalk could lead to novel therapeutic strategies for targeting and killing cancer cells in hypoxic tumors
2025-08-27 | GSE271695 | GEO
Project description:Hypoxia can be established under pathological conditions, such as cancer, due to the imbalance between oxygen supply and consumption. Hypoxia Inducible Transcription Factor HIF-1 mediates the physiological response to hypoxia but also regulates multiple steps of carcinogenesis. Despite its well-defined oxygen-dependent activation, many aspects of HIF-1 transcriptional activity as well as interaction with chromatin remain elusive. We have recently shown that TFAP2A physically interacts with HIF-1 and hypoxia-dependent deSUMOylation of TFAP2A positively affects HIF-1 activity. We now present ChIP-seq analysis showing that TFAP2A resides together with HIF-1α on the promoters of a subset of hypoxia-regulated genes, the mRNA expression of which is downregulated by silencing of TFAP2A. Interestingly, CRISPR-mediated knockdown of TFAP2A expression under hypoxia decreased the occupancy of HIF-1α on these promoters and affected chromatin accessibility. Mechanistically, we reveal that the Ku70/Ku80 protein complex interacts with TFAP2A in a SUMO-dependent manner under hypoxia and participates in HIF-dependent gene expression. Moreover, using stable expression of TFAP2A forms that either lack or constitutively carry a SUMO modification, we could show that SUMOylation affects binding of TFAP2A to chromatin. Overall, our data suggest that TFAP2A is an important co-regulator of the HIF-1-dependent transcriptional response to hypoxia and SUMOylation fine-tunes this regulation. As both TFAP2A and HIF-1 play critical roles in cancer progression, a detailed characterization of their crosstalk could lead to novel therapeutic strategies for targeting and killing cancer cells in hypoxic tumors.
2025-05-23 | PXD053747 | Pride
Project description:To identify Ku70 phosphoS155-specific proximal protein interactors, we used the proximity-dependent biotin identification (BioID2) technique with wild-type Ku70, Ku70 S155A, and Ku70 S155D in the doxycycline-inducible T-REx 293 cell line and compared the different datasets.
2023-05-10 | PXD025581 | Pride
Project description: Not available
| S-EPMC12366265 | biostudies-literature
Project description:Mutations in the gene encoding transcription factor TFAP2A result in pigmentation anomalies in model organisms and premature hair graying in humans. However, the pleiotropic functions of TFAP2A and its redundantly-acting paralogs have made the precise contribution of TFAP2-type activity to melanocyte differentiation unclear. Defining this contribution may help to explain why TFAP2A expression is reduced in advanced-stage melanoma compared to benign nevi. To identify genes with TFAP2A-dependent expression in melanocytes, we profile zebrafish tissue and mouse melanocytes deficient in Tfap2a, and find that expression of a small subset of genes underlying pigmentation phenotypes is TFAP2A-dependent, including Dct, Mc1r, Mlph, and Pmel. We then conduct TFAP2A ChIP-seq in mouse and human melanocytes and find that a much larger subset of pigmentation genes is associated with active regulatory elements bound by TFAP2A. These elements are also frequently bound by MITF, which is considered the “master regulator” of melanocyte development. For example, the promoter of TRPM1 is bound by both TFAP2A and MITF, and we show that the activity of a minimal TRPM1 promoter is lost upon deletion of the TFAP2A binding sites. However, the expression of Trpm1 is not TFAP2A-dependent, implying that additional TFAP2 paralogs function redundantly to drive melanocyte differentiation, which is consistent with previous results from zebrafish. Paralogs Tfap2a and Tfap2b are both expressed in mouse melanocytes, and we show that mouse embryos with Wnt1-Cre-mediated deletion of Tfap2a and Tfap2b in the neural crest almost completely lack melanocytes but retain neural crest-derived sensory ganglia. These results suggest that TFAP2 paralogs, like MITF, are also necessary for induction of the melanocyte lineage. Finally, we observe a genetic interaction between tfap2a and mitfa in zebrafish, but find that artificially elevating expression of tfap2a does not increase levels of melanin in mitfa hypomorphic or loss-of-function mutants. Collectively, these results show that TFAP2 paralogs, operating alongside lineage-specific transcription factors such as MITF, directly regulate effectors of terminal differentiation in melanocytes. In addition, they suggest that TFAP2A activity, like MITF activity, has the potential to modulate the phenotype of melanoma cells.
2017-02-23 | GSE67555 | GEO
Project description:Damage to the gene regulatory network governing terminal differentiation of melanocytes leads to pigmentation phenotypes and increases the risk for melanoma. Microphthalmia-associated transcription factor (MITF) directly activates expression of melanocyte differentiation effectors, and levels of MITF have been proposed to govern the melanoma phenotype. Mutations in the gene encoding Transcription Factor Activator Protein 2 alpha (TFAP2A) cause reduced pigmentation in model organisms and premature hair graying in humans, and TFAP2A expression tends to be lower in advanced melanoma tumors than in benign nevi. However, the transcriptional targets of TFAP2A in melanocytes, and the epistatic relationship of TFAP2A and MITF, have been unclear. Using microarray-based analysis of zebrafish tfap2a mutant embryos, we generated a profile of genes whose expression is Tfap2a-dependent. We conducted anti-TFAP2A chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) in immortalized mouse melanocytes and human primary melanocytes, and discovered that TFAP2A peaks are present near the promoters of Tfap2a-dependent genes expressed in melanocytes, and also at the majority of enhancers active in melanocytes. Comparison of TFAP2A ChIP-seq data to published MITF ChIP-seq data showed that the set of genes with promoters bound by both MITF and TFAP2A is enriched for the gene ontology term “pigment cell differentiation.” Deletion analysis of one such co-bound promoter, for Transient Receptor Potential Melastatin-like 1(TRPM1), confirmed that its expression depends on the presence of MITF binding sites as previously shown, but also depends on the presence of TFAP2A binding sites. Finally, we find that mitfa and tfap2a interact genetically in zebrafish. Collectively, these results show that TFAP2A, operating in parallel with MITF, directly regulates effectors of terminal differentiation in melanocytes and melanoma.
2017-02-17 | GSE72953 | GEO
Project description:To explore the role of Ku70 in Treg cell biology, we isolated conventional T cells (Tconv) and regulatory T cells (Treg) from Foxp3Yfp-cre mice and performed Ku70 ChIP-seq. Furthermore, to investigate the impact of Ku70 deficiency on Foxp3 binding patterns, we sorted Treg cells from Foxp3Cre (WT) and Xrcc6fl/fl;Foxp3Cre (KO) mice and conducted Foxp3 ChIP-seq. These analyses revealed that Ku70 forms a complex with FOXP3, which supports FOXP3 transcriptional activity.
2024-11-04 | GSE278950 | GEO
Project description:To study the function of SUMOylation and SUMO-SIM interactions for particular substrates poses several challenges. SUMOylation occurs transiently and often in a small percentage of the total copy numbers of a given substrate. Modified proteins can be readily deSUMOylated and SUMO can be recycled and passed to other substrates. SUMO-SIM interactions are also difficult to analyze due to their weak affinity (Kd 1-100 µM). To overcome these technical issues, we developed SUMO-ID, a new strategy based on Split-TurboID to identify SUMO- interactors of specific substrates dependent on SUMO conjugation or interaction. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors. , the high sensitivity of SUMO-ID allowed to identify novel interactors of SUMOylated SALL1, a less characterized SUMO substrate.
2021-09-21 | PXD021923 | Pride
Project description:Proximity-dependent labelling (PL) methods are based on promiscuous labeling enzymes that produce reactive molecules that covalently bind neighbor proteins. Labeled proteins can be then purified and identified using affinity-purification coupled to mass spectrometry methods39. Proximity-dependent biotin identification (BioID)40 uses a promiscuously active Escherichia coli biotin ligase (BirA*) generated by a point mutation (R118G) to biotinylate lysines in nearby proteins within an estimated range of 10 nm41. By fusing BirA* to specific proteins, BioID efficiently identifies interactors at physiological levels in living cells42, 43. It has been extensively used in the Ub field, for instance, to identify substrates of E3 ligases44-46. Recently, a more efficient version of BioID, termed TurboID, has been developed47. TurboID can proximally biotinylate in 10 minutes to the same levels as BioID does in 18 hours, thus making it more suitable for transient protein-protein interaction (PPI) detection. Several studies have developed split-versions and applied “protein fragment complementation” (PCA) to BioID and TurboID, where proximal biotinylation is dependent on the proximity of the fusion partners, opening new opportunities for spatial and temporal identification of complex-dependent interactomes48-50. To study how SUMOylation and SUMO-SIM interactions can lead to other roles and fates for particular substrates poses particular challenges. SUMOylation occurs transiently and often in a small percentage of a given substrate. Modified proteins can be readily deSUMOylated and SUMO can be recycled and passed to other substrates. SUMO-SIM interactions are also difficult to analyze due to their weak affinity (Kd ranging from 1-100 µM). To overcome those technical issues, we developed SUMO-ID, a new strategy based on Split-TurboID to identify SUMO- interactors of specific substrates dependent on SUMO conjugation or interaction. Using PML as a model, we demonstrate that SUMO-ID can enrich for factors that depend on PML-SUMO interaction. Importantly, those are represented among proximal interactors of PML identified using full-length TurboID. We also applied SUMO-ID to a less-characterized SUMO substrate, Spalt like transcription factor 1 (SALL1), and identified both known and novel interactors that depend on intact SUMOylation sites in SALL1. SUMO-ID is thus a powerful tool to study transient and dynamic SUMO-dependent interaction events.
2021-09-20 | PXD021809 | Pride
Project description:Proximity-dependent labelling (PL) methods are based on promiscuous labeling enzymes that produce reactive molecules that covalently bind neighbor proteins. Labeled proteins can be then purified and identified using affinity-purification coupled to mass spectrometry methods39. Proximity-dependent biotin identification (BioID)40 uses a promiscuously active Escherichia coli biotin ligase (BirA*) generated by a point mutation (R118G) to biotinylate lysines in nearby proteins within an estimated range of 10 nm41. By fusing BirA* to specific proteins, BioID efficiently identifies interactors at physiological levels in living cells42, 43. It has been extensively used in the Ub field, for instance, to identify substrates of E3 ligases44-46. Recently, a more efficient version of BioID, termed TurboID, has been developed47. TurboID can proximally biotinylate in 10 minutes to the same levels as BioID does in 18 hours, thus making it more suitable for transient protein-protein interaction (PPI) detection. Several studies have developed split-versions and applied “protein fragment complementation” (PCA) to BioID and TurboID, where proximal biotinylation is dependent on the proximity of the fusion partners, opening new opportunities for spatial and temporal identification of complex-dependent interactomes48-50. To study how SUMOylation and SUMO-SIM interactions can lead to other roles and fates for particular substrates poses particular challenges. SUMOylation occurs transiently and often in a small percentage of a given substrate. Modified proteins can be readily deSUMOylated and SUMO can be recycled and passed to other substrates. SUMO-SIM interactions are also difficult to analyze due to their weak affinity (Kd ranging from 1-100 µM). To overcome those technical issues, we developed SUMO-ID, a new strategy based on Split-TurboID to identify SUMO- interactors of specific substrates dependent on SUMO conjugation or interaction. Using PML as a model, we demonstrate that SUMO-ID can enrich for factors that depend on PML-SUMO interaction. Importantly, those are represented among proximal interactors of PML identified using full-length TurboID. We also applied SUMO-ID to a less-characterized SUMO substrate, Spalt like transcription factor 1 (SALL1), and identified both known and novel interactors that depend on intact SUMOylation sites in SALL1. SUMO-ID is thus a powerful tool to study transient and dynamic SUMO-dependent interaction events.
2021-09-20 | PXD021770 | Pride