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Catalytic-subunit-selective transcriptional regulation of the proteasome by E4F1

This study identifies the transcription factor E4F1 as a selective regulator of the proteasome catalytic β1 subunit (PSMB6), revealing a novel layer of proteasome homeostasis where individual catalytic subunits are controlled by distinct transcription factors.

Original authors: Shigeo Murata, Ayaka Kido, Maki Inami, Akihiro Kawabe, yan Wang, Yi Wang, Sota Hashimoto, Ayako Watanabe, Hiroki Sugishita, Yukiko Gotoh, Jun Hamazaki

Published 2026-09-15

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CC BY 4.0

Original authors: Shigeo Murata, Ayaka Kido, Maki Inami, Akihiro Kawabe, yan Wang, Yi Wang, Sota Hashimoto, Ayako Watanabe, Hiroki Sugishita, Yukiko Gotoh, Jun Hamazaki

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

Inside every living cell, a relentless cleanup crew works to maintain order. This system, known as the ubiquitin-proteasome system, acts as the cell's primary waste disposal unit, identifying and destroying damaged or unnecessary proteins to keep the internal environment stable. Without this constant recycling, toxic clumps of protein would accumulate, leading to cell death and contributing to diseases like Alzheimer's and cancer. The machinery responsible for this destruction is the proteasome, a massive, barrel-shaped complex that functions like a molecular shredder. For years, scientists believed that the cell built this shredder by turning on a single master switch that simultaneously increased the production of all its parts. This coordinated approach seemed logical, ensuring that the machine was assembled with the right balance of components. However, the idea that every piece of this complex is regulated in exactly the same way has recently been challenged by a new discovery that reveals a more nuanced and selective control mechanism.

Researchers at the University of Tokyo, led by Shigeo Murata, set out to find the specific genetic switches that control the construction of the proteasome. They suspected that while a general master switch exists, there might be other regulators that fine-tune specific parts of the machine. To find these regulators, the team used a powerful screening method called CRISPR interference. This technique allowed them to systematically turn down the activity of nearly every gene in human cells, one by one, while watching a fluorescent light that indicated how well the proteasome was working. When the proteasome functioned poorly, the fluorescent light glowed brighter. By sorting through millions of cells, the team identified a transcription factor, a protein that controls gene activity, called E4F1. When they reduced the levels of E4F1, the fluorescent light surged, signaling a breakdown in the proteasome's ability to clean up cellular waste.

The investigation into why E4F1 was so critical revealed a surprising level of specificity. The researchers found that removing E4F1 did not shut down the production of all proteasome parts equally. Instead, it selectively silenced the gene for a single component: the beta-1 subunit. This specific part is one of the three catalytic blades inside the proteasome barrel that actually cut the protein chains. Without enough of this single blade, the entire shredding machine could not assemble correctly. The cell accumulated incomplete, half-built versions of the proteasome, and the mature, working machines disappeared. Consequently, the cell could not degrade its waste, leading to a buildup of damaged proteins. The team confirmed that this was not a side effect of general cell stress but a direct result of E4F1 failing to activate the gene for the beta-1 subunit. They showed that E4F1 physically binds to the DNA of this specific gene to keep it active, acting as a dedicated manager for just this one piece of the puzzle.

This finding forces a rethinking of how cells manage their most essential machinery. The study demonstrates that the cell does not rely solely on a broad, coordinated command to build the proteasome. Instead, it employs a strategy of selective regulation, where different transcription factors control individual catalytic parts independently. The researchers noted that this mirrors a previous discovery where another factor, THAP1, was found to specifically regulate a different blade, the beta-5 subunit. Together, these results suggest that the cell can adjust the capacity of its waste disposal system by tweaking specific components rather than just turning the whole system up or down. This selective control might allow cells to adapt the proteasome's function to the unique needs of different tissues or changing environmental conditions. Furthermore, the study linked E4F1 to the regulation of mitochondrial genes, the power plants of the cell, suggesting that this transcription factor coordinates the cell's energy production with its waste management. While the full implications for human disease are still being explored, the work establishes that the assembly of the proteasome is governed by a sophisticated network of specialized regulators, each responsible for a distinct part of the whole.

Technical Summary: Catalytic-subunit-selective transcriptional regulation of the proteasome by E4F1

Problem Statement
The ubiquitin-proteasome system (UPS) is essential for cellular proteostasis, yet the transcriptional mechanisms governing proteasome biogenesis are not fully elucidated. While the transcription factor Nrf1 is well-established as a coordinator that induces proteasome subunit genes in response to proteasome impairment, the prevailing view suggests that proteasome gene expression occurs primarily through coordinated transcriptional programs. However, recent findings identifying THAP1 as a selective regulator of the catalytic $\beta$ 5 subunit gene (PSMB5) independent of Nrf1 suggest that coordinated transcription may not be the sole mechanism. The specific mechanisms regulating other individual catalytic subunits, and whether distinct transcription factors can selectively target specific catalytic subunits, remained unknown.

Methodology
The authors employed a multi-faceted approach to identify and characterize novel regulators of proteasome biogenesis:

Genome-wide CRISPRi Screening: A fluorescent proteasome activity reporter (mCherry-P2A-ZsGreen-mODC) was adapted to a genome-wide CRISPR interference (CRISPRi) platform in HEK293T cells. Cells exhibiting increased ZsGreen fluorescence (indicating proteasome dysfunction) were sorted and subjected to next-generation sequencing to identify candidate regulatory genes.
Validation and Functional Assays: Candidate hits were validated using independent sgRNAs for knockdown in HEK293T, U2OS, and HCT116 cells. Proteasome function was assessed via flow cytometry of the reporter, chymotrypsin-like peptidase activity assays (with and without SDS to distinguish 26S and 20S activity), and immunoblotting for ubiquitinated proteins and proteasome subunits.
Assembly Analysis: Glycerol gradient centrifugation was used to fractionate cell lysates and analyze proteasome assembly intermediates, specifically monitoring the incorporation of $\beta$ subunits and the accumulation of precursors.
Transcriptomics: RT-qPCR and RNA-seq were performed at early time points post-knockout to distinguish primary transcriptional targets from secondary effects of proteasome dysfunction.
Rescue Experiments: HEK293T cells stably expressing individual FLAG-tagged $\beta$ 1– $\beta$ 7 subunits were used to determine which specific subunit restoration could rescue the defects caused by E4F1 depletion.
Mechanistic Studies: Publicly available ChIP-seq datasets were analyzed, followed by luciferase reporter assays with promoter deletions and Chromatin Immunoprecipitation (ChIP)-qPCR to verify direct binding of E4F1 to the PSMB6 promoter.
Comparative Analysis: The transcriptional profiles of E4F1-deficient cells were compared with previously published data from THAP1-deficient cells to determine overlapping or distinct target gene sets, including mitochondrial genes.

Key Results

Identification of E4F1: The CRISPRi screen identified the transcription factor E4F1 as a strong hit. Depletion of E4F1 led to a marked accumulation of the proteasome reporter, reduced peptidase activity, and accumulation of ubiquitinated proteins.
Selective Regulation of PSMB6: Among all proteasome subunit genes, E4F1 depletion selectively reduced the mRNA and protein levels of the catalytic $\beta$ 1 subunit (PSMB6). Other $\beta$ subunits, $\alpha$ subunits, regulatory particle subunits, and assembly chaperones remained largely unchanged.
Impact on Assembly: E4F1 depletion impaired 20S core particle (CP) assembly. Glycerol gradient analysis revealed an accumulation of assembly intermediates containing precursor forms of $\beta$ 2 and $\beta$ 5, while mature $\beta$ 1 was absent. This indicated a stall in assembly after $\beta$ 5 incorporation but before completion.
Causality via $\beta$ 1 Restoration: Re-expression of the $\beta$ 1 subunit in E4F1-deficient cells was sufficient to rescue proteasome activity, restore mature CP levels, resolve assembly intermediates, and reduce ubiquitinated protein accumulation. This confirmed that the assembly defect was primarily driven by the loss of PSMB6 expression.
Direct Transcriptional Regulation: E4F1 was found to bind directly to the PSMB6 promoter region. Deletion of putative E4F1-binding motifs significantly reduced promoter activity, and ChIP-qPCR confirmed that wild-type E4F1, but not a DNA-binding domain mutant, associates with the PSMB6 promoter.
Distinct Transcriptional Programs: While both E4F1 and THAP1 regulate specific catalytic subunits (PSMB6 and PSMB5, respectively) and mitochondrial gene sets, their transcriptional targets are largely non-overlapping. Restoration of $\beta$ 1 or $\beta$ 5 did not rescue the mitochondrial gene expression defects caused by E4F1 or THAP1 depletion, respectively, indicating that these transcription factors regulate proteasome subunits and mitochondrial genes through independent mechanisms.
Conservation: The selective regulation of PSMB6 and associated mitochondrial genes by E4F1 was observed in multiple cell types (SH-SY5Y, U2OS, MEFs) and in vivo models (mouse spermatogonia), as well as in Thap1 mutant mice for the THAP1 pathway.

Significance and Claims
The paper establishes catalytic-subunit-selective transcriptional regulation as a previously unrecognized layer of proteasome gene regulation. The authors claim that individual catalytic proteasome subunits are not regulated exclusively through coordinated, proteasome-wide transcriptional programs (such as the Nrf1-mediated response) but can also be regulated independently by distinct transcription factors.

Specifically, the study identifies E4F1 as a direct transcriptional regulator of PSMB6 ( $\beta$ 1) and, together with their previous work on THAP1 and PSMB5 ( $\beta$ 5), proposes a model where distinct transcription factors fine-tune specific catalytic subunits. This mechanism may allow cells to adapt proteasome capacity to specific physiological demands or environmental conditions. Furthermore, the findings suggest that pathogenic variants in transcription factors like E4F1 and THAP1 may contribute to human diseases (such as mitochondrial disorders and dystonia) not only through their known targets but also via the dysregulation of specific proteasome catalytic subunits. The work expands the conceptual framework of proteasome transcriptional regulation beyond the prevailing model of coordinated induction.

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