Project description:Transcription factors (TFs) engage in protein-protein interactions throughout the process of transcriptional control. In this study, we have successfully identified the protein-protein interactions for more than 100 distinct human transcription factors (TFs) using the techniques of proximity-dependent biotinylation (BioID) and affinity purification mass spectrometry (AP-MS).
Project description:How transcription factors (TFs), the ultimate targets and executors of cellular signalling pathways, are regulated via protein-protein interactions remains elusive. To systematically investigate the regulations and functions of human transcription factors, we performed proteomics studies of soluble and chromatin-associated complexes of 56 TFs in HEK293T cells, using tandem-affinity-purification followed by mass spectrometry (TAP/MS). We performed 214 purifications and identified 2,156 high-confident protein-protein interactions.
Project description:The stoichiometries of TFIID and SAGA (TAF10-containing complexes) have been quantified in mouse and human erythroid cells. TAF10 immunoprecipitations (IPs) have been carried out in erythroid cells at different stages of differentiation and development followed by quantitative mass spectrometry (MS). Interactions of TFIID and SAGA with several transcription factors and specifically with GATA-1 have been identified. GATA-1 immunoprecipitation (IP) in MEL cells also identified the reverse interactions with subunits of the TFIID and SAGA after MS analysis.
Project description:Despite binding similarciselements in multiple locations, transcription factors often perform context-dependent functions at different loci. How factors integratecissequence and genomic context is still poorly understood and has implications for off-target effects in genetic engineering. TheDrosophilacontext-dependent transcription factor CLAMP targets similar GA-richciselements on the X-chromosome and at the histone gene locus but recruits very different, loci-specific factors. We discover that CLAMP leverages information from bothciselement and local sequence to perform context-specific functions. Our observations imply the importance of other cues, including protein-protein interactions and the presence of additional cofactors.
Project description:Transcription factors play a key role in the development of a number of cancers, and therapeutically targeting them has remained a challenge. In prostate cancer, the ETS transcription factor ERG is recurrently rearranged and likely plays a critical role in prostate oncogenesis. Here we identified a series of peptides from a phage-display library that interact specifically with the DNA binding domain of ERG. The interactive interface was mapped to 9-residues in the 3rd helix of the ETS domain that is critical for ERG transcriptional activity. The peptides were found to efficiently disrupt ERG-mediated protein-protein interactions, transcription, DNA damage, and cell invasion, as well as attenuate ERG recruitment to target gene loci. Furthermore, a retroinverso peptidomimetic version of the peptide sequence suppressed tumor growth, intravasation, and metastasis in vivo. Taken together, our results demonstrate that transcription factors have specific residues important for protein-protein interactions and disrupting those critical interactions may be an effective therapeutic strategy.
Project description:The nucleosome is a fundamental unit of chromatin in eukaryotes, and generally prevents the binding of transcription factors to genomic DNA. Pioneer transcription factors overcome the nucleosome barrier, and bind their target DNA sequences in chromatin. OCT4 is a representative pioneer transcription factor that plays a role in stem cell pluripotency. In the present study, we biochemically analyzed the nucleosome binding by OCT4. Crosslinking mass spectrometry showed that OCT4 binds the nucleosome.
Project description:The Nuclear Factor I (NFI) family of transcription factors (TFs) plays key roles in cellular differentiation, proliferation, and homeostasis. As such, NFI family members engage in large number of interactions with other proteins and the chromatin. However, despite their well-established significance, the NFIs interactomes, their dynamics, and their functions have not been comprehensively examined. Here, we employed complementary omics-level techniques, i.e. interactomics (affinity purification mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID)), and chromatin immunoprecipitation sequencing (ChIP-Seq), to obtain a comprehensive view of the NFI proteins and their interactions. Our analyses included all four main NFI family members, and a less studied short isoform of NFIB (NFIB4), which lacks the DNA binding domain. We observed that, despite exhibiting some redundancy, each family member had unique high-confidence interactors and target genes, highlighting distinct roles within the transcriptional regulatory networks. The study revealed that NFIs interact with a large number of other TFs to co-regulate a broad range of regulatory networks and cellular processes. Notably, time-dependent proximity-labeling unveiled a highly dynamic nature of NFI protein-protein interaction networks and hinted at temporal modulation of NFI interactions. Furthermore, gene ontology (GO) enrichment analysis of NFI interactome and targetome revealed the involvement of NFIs in transcriptional regulation, chromatin organization, and cellular signaling pathways, and pathways related with cancer. Additionally, we observed that NFIB4 engages with proteins associated with mRNA regulation, which suggests that NFIs have roles beyond traditional DNA binding and transcriptional modulation. We propose that NFIs serve as pioneering TFs, playing critical roles in regulating other TFs and influencing a wide range of cellular processes. These insights into NFI protein-protein interactions and their dynamic, context-dependent nature provide a deeper understanding of gene regulation mechanisms and hint at the role of NFIs as master regulators.
Project description:Programmable regulation of gene expression promises to be a powerful therapeutic venue for diseases caused by the misexpression of genes, haploinsufficiencies, or gain of function mutations. While dCas9-effector domain fusions have been used to alter expression levels, their in vivo application is limited by size and immunogenicity. Conversely, the Cys2His2 Zinc Finger domain offers ideal characteristics for in vivo use. However, their intricate engagement with the DNA has made the design of Zinc Finger arrays challenging. We here describe the screening of 49 billion protein-DNA interactions and development of the first deep learning model that solves Zinc Finger design for any genomic target. We demonstrate the versatility of designed Zinc Fingers as nucleases and as activators or repressors by seamlessly reprogramming human transcription factors.
Project description:Transcription factors play a key role in the development of a number of cancers, and therapeutically targeting them has remained a challenge. In prostate cancer, the ETS transcription factor ERG is recurrently rearranged and likely plays a critical role in prostate oncogenesis. Here we identified a series of peptides from a phage-display library that interact specifically with the DNA binding domain of ERG. The interactive interface was mapped to 9-residues in the 3rd -helix of the ETS domain that is critical for ERG transcriptional activity. The peptides were found to efficiently disrupt ERG-mediated protein-protein interactions, transcription, DNA damage, and cell invasion, as well as attenuate ERG recruitment to target gene loci. Furthermore, a retroinverso peptidomimetic version of the peptide sequence suppressed tumor growth, intravasation, and metastasis in vivo. Taken together, our results demonstrate that transcription factors have specific residues important for protein-protein interactions and disrupting those critical interactions may be an effective therapeutic strategy. Prostate cancer cell line VCaP were treated with 10µM of RI-EIP1 or RI-muEIP1 for 48 hr