Endoreduplication in Mouse Liver After Conditional Deletion of ORC2 and Combined Deletion of ORC1 and ORC2

Core Concepts

Mouse liver cells can undergo extensive endoreduplication and DNA synthesis even in the absence of two subunits of the Origin Recognition Complex (ORC), suggesting an alternative mechanism for loading the MCM2-7 helicase complex onto chromatin.

Abstract

The study investigates the role of the Origin Recognition Complex (ORC) in DNA replication and endoreduplication in mouse liver cells. The key findings are:

ORC2 is essential for embryonic development and proliferation of mouse embryo fibroblasts (MEFs), but its deletion in hepatocytes does not impair liver development and function. Hepatocytes in the ORC2-deficient livers undergo endoreduplication, leading to larger nuclei and cells.

Combined deletion of ORC1 and ORC2 in hepatocytes also allows extensive endoreduplication and DNA synthesis, suggesting an alternative mechanism for loading the MCM2-7 helicase complex onto chromatin in the absence of two ORC subunits.

Male mice tolerate the combined deletion of ORC1 and ORC2 better than female mice, with the male livers reaching near-normal size despite the genetic deletions.

The results indicate that while ORC is essential for normal mitotic DNA replication, liver cells can bypass the requirement for a complete ORC complex and still undergo endoreduplication, potentially through an alternative MCM2-7 loading mechanism.

Stats

Conditional deletion of Orc2 in mouse embryo fibroblasts (MEFs) leads to impaired proliferation.
Deletion of Orc2 in mouse hepatocytes results in fewer but larger hepatocytes with larger nuclei.
Hepatocytes from Orc2-deficient livers can incorporate EdU, indicating DNA synthesis, though the number of EdU-positive nuclei is decreased by 70% compared to wild-type.
Livers from mice with combined deletion of Orc1 and Orc2 in hepatocytes can regenerate to near-normal size after partial hepatectomy.
Nearly 100% of hepatocytes in the Orc1/Orc2 double knockout livers are EYFP-positive, indicating high penetrance of Cre recombinase activity.

Quotes

"Conditional deletion of Orc2 in mouse embryo fibroblasts (MEFs) leads to early embryonic lethality and impairs the proliferation of normal diploid MEFs in culture."
"Deletion of Orc2 in mouse hepatocytes results in fewer but larger hepatocytes with larger nuclei, suggesting that the liver cells can undergo endoreduplication in the absence of ORC2."
"Hepatocytes from Orc2-deficient livers can incorporate EdU, indicating DNA synthesis, though the number of EdU-positive nuclei is decreased by 70% compared to wild-type."
"Livers from mice with combined deletion of Orc1 and Orc2 in hepatocytes can regenerate to near-normal size after partial hepatectomy, suggesting that liver cells can undergo extensive endoreduplication even in the absence of two ORC subunits."

Key Insights Distilled From

Endo-reduplication in mouse liver after conditional mutation of ORC2 and combined mutation of ORC1 and ORC2

by Przanowska,R... at www.biorxiv.org 04-06-2024

https://www.biorxiv.org/content/10.1101/2024.04.04.588006v1

Deeper Inquiries

What are the potential alternative mechanisms by which mouse liver cells can load the MCM2-7 helicase complex onto chromatin and support DNA replication in the absence of a complete ORC complex?

In the context of the study, the observed ability of mouse liver cells to support DNA replication in the absence of a complete ORC complex, specifically ORC1 and ORC2, suggests the presence of alternative mechanisms for loading the MCM2-7 helicase complex onto chromatin. One potential mechanism could involve the compensation by other proteins or factors that can partially fulfill the role of ORC in initiating DNA replication. For example, it is possible that other ATPases or DNA-binding proteins may assist in recruiting MCM2-7 to replication origins in the absence of ORC subunits. Additionally, there could be redundant pathways or backup systems that can activate the licensing of replication origins and facilitate DNA synthesis in a manner independent of the canonical ORC complex. Further investigation into the specific proteins and pathways involved in this alternative mechanism is warranted to fully understand how DNA replication is sustained in ORC-deficient liver cells.

How do sex-specific differences in liver regeneration and tolerance to ORC1/ORC2 deletion arise, and what are the underlying molecular and physiological factors involved?

The study highlights sex-specific differences in liver regeneration and tolerance to ORC1/ORC2 deletion, with male mice showing higher tolerance and larger liver sizes compared to female mice. These differences could arise from the influence of sex hormones, particularly androgens, which are known to impact liver physiology and regeneration. Androgens may play a role in promoting cell proliferation and hypertrophy in male livers, contributing to their larger size and enhanced regeneration capacity. Additionally, sex-specific gene expression patterns and epigenetic regulation in the liver could also contribute to the observed differences in response to ORC1/ORC2 deletion. Understanding the molecular and physiological factors underlying these sex-specific differences may involve investigating the interplay between sex hormones, gene expression profiles, and epigenetic modifications that influence liver regeneration and function in male and female mice.

Could the endoreduplication and altered hepatocyte morphology observed in the ORC-deficient livers lead to any functional consequences for liver physiology and homeostasis?

The endoreduplication and altered hepatocyte morphology observed in ORC-deficient livers could have significant functional consequences for liver physiology and homeostasis. Endoreduplication, characterized by the replication of DNA without subsequent cell division, can lead to the generation of polyploid cells with enlarged nuclei. This altered cellular morphology may impact the functional capacity of hepatocytes, affecting processes such as metabolism, detoxification, and protein synthesis. The presence of polyploid cells in the liver could also influence the regenerative capacity of the organ and its response to injury or stress. Additionally, the changes in hepatocyte morphology and DNA replication patterns may disrupt normal liver function, potentially leading to impaired metabolic processes and liver dysfunction. Further studies are needed to elucidate the specific consequences of endoreduplication and altered hepatocyte morphology on liver physiology and homeostasis in ORC-deficient conditions.