Takashi Ito

Mediator complex subunit 19 (MED19), a component of the Mediator complex, has emerged as a potential regulator of cancer progression, although its downstream transcriptional programs remain poorly understood. Here, we investigated the role of MED19 across 20 human cancer cell lines. An siRNA-mediated knockdown (KD) screen identified A549 lung cancer and MCF-7 breast cancer cells as particularly sensitive to MED19 KD, exhibiting markedly reduced cell viability. Flow cytometric analysis revealed that MED19 KD induced G1-phase arrest, suggesting a role in G1/S-phase progression. RNA sequencing followed by differential expression, Gene Ontology (GO), and chromosomal mapping analyses identified amphiregulin (AREG) and cyclin-dependent kinase regulatory subunit 1B (CKS1B) as candidate downstream effectors in A549 and MCF-7 cells, respectively. Knockdown of AREG or CKS1B inhibited cell proliferation and recapitulated the G1-phase arrest induced by MED19 KD. Moreover, ectopic expression of AREG or CKS1B partially rescued the cell-cycle defects caused by MED19 KD. These findings identify MED19 as a context-dependent regulator of cancer cell proliferation and G1/S-phase progression through distinct downstream transcriptional programs in lung and breast cancer cells.

The Mediator complex is a central regulator of eukaryotic transcription, functioning as a dynamic molecular interface between gene-specific transcription factors and RNA polymerase II (Pol II). Although its overall architecture and general role in transcription have been extensively reviewed, accumulating genetic, genomic, and clinical evidence indicates that individual Mediator subunits make distinct and non-redundant contributions to human physiology and disease. In this review, we move beyond a generic description of Mediator function and present a subunit-resolved synthesis of Mediator biology with an emphasis on disease pathogenesis. A key feature of this review is a comprehensive table integrating disease associations and molecular functions of individual human Mediator subunits, enabling rapid assessment of functional specialization across the complex. We further discuss chromatin-based mechanisms of Mediator action, including cooperation with cohesin and architectural factors to regulate enhancer-promoter communication and higher-order genome organization. By organizing recent structural, mechanistic, and pathological findings into a unified framework, this review highlights how disruption of specific Mediator subunits contributes to cancer, developmental disorders, and metabolic disease, and outlines emerging opportunities for therapeutic intervention.

The Mediator complex is a central regulator of eukaryotic transcription, functioning as a dynamic molecular bridge between gene-specific transcription factors and RNA polymerase II (Pol II). Although decades of research have established its modular architecture and fundamental role in transcriptional control, recent advances have significantly expanded our understanding of its structural conformations, subunit-specific functions, and links to human disease. This review provides a comprehensive overview of the Mediator complex, highlighting key structural and functional discoveries from the past decade and synthesizing its diverse roles in transcriptional regulation. We further discuss emerging concepts and future directions for therapeutically targeting Mediator, particularly in cancer. Together, these insights position the Mediator complex as a highly conserved yet adaptable, signal-responsive regulatory hub with broad implications for both normal physiology and disease pathogenesis.

Usp21, a member of the ubiquitin protease family, plays a vital role in various biological functions. However, the effects of Usp21 dysfunction remain incompletely understood. In this study, we generated Usp21 knockout (KO) mice. Blood tests showed no impairment of liver function but did reveal elevated levels of total cholesterol (T-CHOL) and free fatty acid (FFA) in Usp21 KO mice compared to wild-type (WT) mice. Next, we performed RNA-sequencing (RNA-seq) to identify genes that Usp21 regulates. The results highlighted several candidate genes based on their biological relevance, and their expression levels were validated by RT-qPCR. The Usp21 KO mice exhibited significant elevations in the expression of the genes Fabp7, Nlrc5, and Ppargc1a, which play an important role in lipid metabolism, compared to WT. These data suggest that Usp21 may play roles in lipid metabolism in association with Fabp7, Nlrc5 and Ppargc1a. To clarify the involvement of USP21 in human hypercholesterolemia, we examined single-nucleotide polymorphisms (SNPs) around USP21 in non-hypercholesterolemic and hypercholesterolemic outpatients. We found that the rs11421 SNP downstream of USP21 was significantly associated with hypercholesterolemia. These data suggest that Usp21 plays a role in mice and human lipid metabolism and that its polymorphism may be a diagnostic marker for human hypercholesterolemia.

USP21, a member of the ubiquitin protease family, plays a vital role in various biological functions. However, the effects of USP21 dysfunction have yet to be fully understood. Here, we generated Usp21 knockout (KO) mice and conducted microarray to identify genes that Usp21 regulates. Its results raised several genes based on their biological characteristics, and RT-qPCR confirmed their expression. KO mice exhibited significant elevations in the expression of the genes Fabp7 and Nlrc5, which play a role in lipid metabolisms, compared to wild-type mice. Blood tests showed no impairment of liver function but did reveal elevated levels of total cholesterol and free fatty acids in KO mice compared to wild-type mice. These data suggest that Usp21 may play roles in lipid metabolism associated with Fabp7 and Nlrc5. To clarify the involvement of USP21 in human hypercholesterolemia, we examined single nucleotide polymorphisms (SNPs) around USP21 in non-hypercholesterolemic and hypercholesterolemic outpatients. We found that the rs11421 SNP downstream of USP21 was associated with hypercholesterolemia. These data suggest that USP21 plays a role in mice and human lipid metabolism and that its polymorphism may be a diagnostic marker for human hypercholesterolemia.

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide, and pulmonary epithelial cell apoptosis is regarded as one of the most important factors in its pathogenesis. Here we examined the molecular mechanisms of apoptosis caused by cigarette smoke (CS). In the normal bronchial epithelium cell line BEAS-2B, a CS extract markedly induced apoptosis together with transient early growth response 1 (EGR1) protein expression, which is activated over time via the aryl hydrocarbon receptor (AHR). The CS extract induced apoptosis and decreased cell count of BEAS-2B cells and was significantly reversed by knockdown of either EGR1 or AHR. In vivo, the CS extract caused alveolar wall destruction, mimicking COPD, 1 week after intrathoracic injection. Bronchoalveolar lavage fluid (BALF) from the CS extract-treated mice contained massive numbers of apoptotic epithelial cells. Furthermore, it was found that aminoanthracene induced EGR1 expression and cell apoptosis. By contrast, the AHR antagonist stemregenin 1 (SR1) restored apoptosis upon CS treatment. These results suggest that aryl hydrocarbons, such as aminoanthracene, induce EGR1 expression via the AHR, resulting in cell apoptosis and that this can be prevented by administration of an antagonist of AHR.

The five β-like globin genes (ε, Gγ, Aγ, δ and β) at the human β-globin gene locus are known to be expressed at specific developmental stages, although details of the underlying mechanism remain to be uncovered. Here we used an in vitro transcription assay to clarify the mechanisms that control this gene expression. We first tested nuclear RNA from HeLa cells using RT-qPCR and discovered a long noncoding RNAs (lncRNAs) within a 5.2-kb region beginning 4.4 kb downstream of the β-globin gene coding region. We investigated nuclear RNA from K562 cells using a primer-extension assay and determined the transcription start sites (TSSs) of these lncRNAs. To clarify their functional role, we performed knockdown (KD) of these lncRNAs in K562 cells. Hydroxyurea (HU), which induces differentiation of K562 cells, increased haemoglobin peptide production, and the effect was enhanced by KD of these lncRNAs, which also enhanced upregulation of the γ-globin expression induced by HU. To confirm these results, we performed an in vitro transcription assay. Noncoding single-stranded RNAs inhibited β-globin expression, which was upregulated by GATA1. Furthermore, lncRNAs interacted with GATA1 without sequence specificity and inhibited its binding to its target DNA response element in vitro. Our results suggest that lncRNAs downstream of the β-globin gene locus are key factors regulating globin gene expression.

Acetylation of histone H1 is generally considered to activate transcription, whereas deacetylation of H1 represses transcription. However, the precise mechanism of the acetylation is unknown. Here, using chromatography, we identified nucleosome assembly protein 1 (NAP-1) as having inhibitory activity against histone H1 acetylation by acetyltransferase p300. We found that native NAP-1 interacts with H1 in a Drosophila crude extract. We also found that it inhibits the deacetylation of histone H1 by histone deacetylase 1. The core histones in nucleosomes were acetylated in a GAL4–VP16 transcriptional activator-dependent manner in vitro. This acetylation was strongly repressed by hypoacetylated H1 but to a lesser extent by hyperacetylated H1. Consistent with these findings, a micrococcal nuclease assay indicated that hypoacetylated H1, which represses activator-dependent acetylation, was incorporated into chromatin, whereas hyperacetylated H1 was not. To determine the contribution of NAP-1 to transcriptional regulation in vivo, we compared NAP-1 knockdown (KD) with coactivator CREB-binding protein (CBP) KD using RNA sequencing in Drosophila Schneider 2 cells. Most genes were downregulated rather than upregulated by NAP-1 KD, and those downregulated genes were also downregulated by CBP KD. Our results suggest that NAP-1 plays a role in transcriptional regulation by fine-tuning the acetylation of histone H1.

The nucleolus is a membrane-less organelle sequestered from the nucleus by liquid droplet formation through a liquid–liquid phase separation (LLPS). It plays important roles in cell homoeostasis through its internal thermodynamic changes. Reversible nucleolar transitions between coalescence and dispersion are dependent on the concentrations, conformations and interactions of its molecular liquid droplet-forming components, including DNA, RNA and protein. The liquid droplet-like properties of the nucleolus enable its diverse dynamic roles. The liquid droplet formation mechanism, by which the nucleolus is sequestered from the nucleoplasm despite the absence of a membrane, explains a number of complex nucleolar functions.