Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates the RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure–function analyses, we show that human TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1—the largest protein in the complex—as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.

Core promoters are sites where transcriptional regulatory inputs of a gene are integrated to direct the assembly of the preinitiation complex (PIC) and RNA polymerase II (Pol II) transcription output. Until now, core promoter functions have been investigated by distinct methods, including Pol II transcription initiation site mappings and structural characterization of PICs on distinct promoters. Here, we bring together these previously unconnected observations and hypothesize how, on metazoan TATA promoters, the precisely structured building up of transcription factor (TF) IID-based PICs results in sharp transcription start site (TSS) selection; or, in contrast, how the less strictly controlled positioning of the TATA-less promoter DNA relative to TFIID-core PIC components results in alternative broad TSS selections by Pol II.

Most factors that regulate gene transcription in eukaryotic cells are multimeric, often large, protein complexes. The understanding of the biogenesis pathways of such large and heterogeneous protein assemblies, as well as the dimerization partner choice among transcription factors, is crucial to interpret and control gene expression programs and consequent cell fate decisions. Co-translational assembly (Co-TA) is thought to play key roles in the biogenesis of protein complexes by directing complex formation during protein synthesis. In this review we discuss the principles of Co-TA with a special focus for the assembly of transcription regulatory complexes. We outline the expected molecular advantages of establishing co-translational interactions, pointing at the available, or missing, evidence for each of them. We hypothesize different molecular mechanisms based on Co-TA to explain the allocation "dilemma" of paralog proteins and subunits shared by different transcription complexes. By taking as a paradigm the different assembly pathways employed by three related transcription regulatory complexes (TFIID, SAGA and ATAC), we discuss alternative Co-TA strategies for nuclear multiprotein complexes and the widespread - yet specific - use of Co-TA for the formation of nuclear complexes involved in gene transcription. Ultimately, we outlined a series of open questions which demand well-defined lines of research to investigate the principles of gene regulation that rely on the coordinated assembly of protein complexes.

NF-Y is a transcription factor (TF) comprising three subunits (NF-YA, NF-YB, NF-YC) that binds with high specificity to the CCAAT sequence, a widespread regulatory element in gene promoters of prosurvival, cell-cycle-promoting, and metabolic genes. Tumor cells undergo “metabolic rewiring” through overexpression of genes involved in such pathways, many of which are under NF-Y control. In addition, NF-YA appears to be overexpressed in many tumor types. Thus, limiting NF-Y activity may represent a desirable anti-cancer strategy, which is an ongoing field of research. With virtual-screening docking simulations on a library of pharmacologically active compounds, we identified suramin as a potential NF-Y inhibitor. We focused on suramin given its high water-solubility that is an important factor for in vitro testing, since NF-Y is sensitive to DMSO. By electrophoretic mobility shift assays (EMSA), isothermal titration calorimetry (ITC), STD NMR, X-ray crystallography, and molecular dynamics (MD) simulations, we showed that suramin binds to the histone fold domains (HFDs) of NF-Y, preventing DNA-binding. Our analyses, provide atomic-level detail on the interaction between suramin and NF-Y and reveal a region of the protein, nearby the suramin-binding site and poorly conserved in other HFD-containing TFs, that may represent a promising starting point for rational design of more specific and potent inhibitors with potential therapeutic applications.

NF-YA, the regulatory subunit of the trimeric transcription factor (TF) NF-Y, is regulated by alternative splicing (AS) generating two major isoforms, “long” (NF-YAl) and “short” (NF-YAs). Muscle cells express NF-YAl. We ablated exon 3 in mouse C2C12 cells by a four-guide CRISPR/Cas9n strategy, obtaining clones expressing exclusively NF-YAs (C2-YAl-KO). C2-YAl-KO cells grow normally, but are unable to differentiate. Myogenin and—to a lesser extent, MyoD— levels are substantially lower in C2-YAl-KO, before and after differentiation. Expression of the fusogenic Myomaker and Myomixer genes, crucial for the early phases of the process, is not induced. Myomaker and Myomixer promoters are bound by MyoD and Myogenin, and Myogenin overexpression induces their expression in C2-YAl-KO. NF-Y inactivation reduces MyoD and Myogenin, but not directly: the Myogenin promoter is CCAAT-less, and the canonical CCAAT of the MyoD promoter is not bound by NF-Y in vivo. We propose that NF-YAl, but not NF-YAs, maintains muscle commitment by indirectly regulating Myogenin and MyoD expression in C2C12 cells. These experiments are the first genetic evidence that the two NF-YA isoforms have functionally distinct roles.