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:The ETO-family transcriptional corepressors, including ETO, ETO2 and MTGR1, are all involved in leukemia-causing chromosomal translocations. In every case, an ETO-family corepressor acquires a DNA-binding domain (DBD) to form a typical transcription factor – the DBD binds to target genes, while the ETO moiety contributes essentially to its transcriptional property. A direct comparative study of these “homologous” fusion transcription factors may clarify their similarities and differences in regulating transcription and leukemogenesis. Here, we performed a side-by-side comparison between AML1-ETO and ETO2-GLIS2, the most common fusion proteins in the M2 and M7 subtypes of acute myeloid leukemia, respectively, by inducible expression of them in U937 leukemia cells. We found that, although AML1-ETO and ETO2-GLIS2 can use their own DBDs (i.e., the Runt domain of AML1 and the zinc finger domain of GLIS2) to bind DNA, they actually share a large proportion of genome-wide binding regions dependent on other cooperative transcription factors such as ETS- and CEBP-family proteins. Functionally, AML1-ETO acts as either transcriptional repressor or activator, whereas ETO2-GLIS2 mainly acts as an activator. The repressor-versus-activator functions of AML1-ETO is determined by the abundance of cooperative transcription factors/cofactors on the target genes. Through these mechanisms, AML1-ETO and ETO2-GLIS2 differentially regulate several key transcription factors that are essential for myeloid differentiation. Indeed, AML1-ETO inhibits myeloid differentiation of U937 cells, whereas ETO2-GLIS2 facilitates it. Taken together, this study is the first direct comparative study between AML1-ETO and ETO2-GLIS2 in the same cellular context, and the results provide new insights into the context-dependent transcriptional regulatory mechanisms that may underlie how these seemingly “homologous” fusion transcription factors exert distinct functions to drive different subtypes of leukemia.

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The ProCisTranomic of human ETS family

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