# The Generation and Characterization of a Mouse Embryonic Stem Cell Line with Psmb9 Immunoproteasome Gene Knockout

## TL;DR

This study creates a mouse embryonic stem cell line with a knockout of the Psmb9 gene, revealing how immunoproteasomes adapt to maintain cellular function during differentiation.

## Contribution

The novel contribution is the generation and characterization of a Psmb9 knockout mouse ESC line, revealing compensatory mechanisms in immunoproteasome assembly.

## Key findings

- Psmb9KO cells retained pluripotency and normal proliferation but showed altered immunoproteasome assembly during differentiation.
- Differentiation induced accumulation of Mecl-1 precursors and increased caspase-like activity in Psmb9KO cells.
- Compensatory integration of constitutive β1-subunit into immunoproteasomes was observed in the absence of Lmp2.

## Abstract

Immunoproteasomes, a unique type of proteasome complex, play a critical role in
antigen presentation and cellular homeostasis. Unlike the constitutive 20S
proteasome, the catalytic subunits β1, β2, and β5 in the
immunoproteasome are replaced by inducible isoforms: β1i (LMP2), β2i
(MECL-1), and β5i (LMP7). The expression of the genes encoding these
subunits (Psmb9, Psmb10, and Psmb8) is activated by cytokines, primarily
interferon-γ (IFNγ). Although it has been demonstrated more and more
convincingly that immunoproteasomes are expressed in embryonic stem cells
(ESCs), their involvement in maintaining pluripotency, promoting self-renewal,
and regulating differentiation processes remains unexplored. This study
implemented CRISPR/Cas9 technology to generate a Psmb9 gene
knockout (Psmb9KO) mouse ESC line. The resulting cells exhibited a normal
karyotype and morphology, maintained normal proliferation rates, and retained
the capacity to form teratomas containing derivatives of all three germ layers.
However, the differentiation induced by retinoic acid (RA) and IFNγ caused
an accumulation of Mecl-1 precursors in Psmb9KO cells, suggesting modifications
in immunoproteasome assembly. Furthermore, an increase in the caspase-like
activity of immunoproteasomes was detected, suggesting the integration of a
constitutive β1-subunit into the complex in place of Lmp2. The findings
underscore the adaptability of the ubiquitin-proteasome system in maintaining
cellular proteostasis by compensatory mechanisms that counteract the lack of
Lmp2. The Psmb9KO line can serve as a valuable model for examining the function
of immunoproteasomes in proteostasis regulation during early mammalian
embryogenesis differentiation.