The role of ORC4 in enucleation of Murine Erythroleukemia (MEL) cells is similar to that in oocyte polar body extrusion

ABSTRACT

The Origin Replication Complex subunit 4 (ORC4) is one in six subunits of the Origin Replication Complexes (ORCs) which is essential for initiating licensing at DNA replication origins and recruiting adaptor molecules necessary for various cellular processes. Previously, we reported that ORC4 also plays a vital role in polar body extrusion (PBE) during oogenesis in which half the chromosomes are extruded from the oocyte. We hypothesized that ORC4 might play a broader role in chromatin elimination. We tested its role in enucleation during the development of erythrocytes. Murine erythroleukemia (MEL) cells can be propagated in culture indefinitely and can be induced to enucleate their DNA by treatment with Vacuolin-1, thereby mimicking normal erythrocyte enucleation. We found that ORC4 appeared around the nuclei of the MEL cells with Vacuolin-1 treatment, gradually increasing in thickness before enucleation. We then tested whether ORC4 was required for MEL enucleation by down regulating ORC4 with siRNA-ORC4 during Vacuolin-1 treatment and found that this prevented MEL enucleation. These data are consistent with the model that ORC4 is required for erythroblast enucleation just as it is for oocyte PBE. They suggest a new model in which ORC4 expression is a marker for the initiation to the enucleation pathway.

KEYWORDS:

• ORC4
• polar body
• Murine Erythroleukemia cell (MEL)
• erythroblast
• enucleation
• DNA Replication
• extrusion

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Acknowledgments

The authors would like to thank Dr. John Crispino for important discussions and advice during this work and for the gift of the MEL cells.

Author’s contributions

Performed all the experiments and contributed to the design of the work: HN; contributed to most of the experiments: AU; designed the overall work and directed the experiments performed: WSW.

Disclosure statement

The authors report no conflicts of interest. This work was supported by NIH Grant Nos. R01 GM123048 and P30 GM131944 to WSW.

Data availability statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This work was supported by NIH Grants R01 GM123048, and by the Transgenic Mouse, ICSI and IVF Core supported by IBR-COBRE, NIH grant number P30 GM131944.