ORC4 Surrounds Extruded Chromatin in Female Meiosis

Abstract

Six proteins, ORC1-6, make up the origin recognition complex (ORC) that initiates licensing of DNA replication origins. We have previously reported that subunit ORC2 is localized between the separating maternal chromosomes at anaphase II just after fertilization and is present in zygotic pronuclei at G1. Here, we found that ORC1, 3, and 5 all localize between the chromosomes at anaphase II, but could not be detected in zygotic G1. ORC6 localized to the periphery of the nucleoli at all zygotic stages. We identified an unexpected potential role for ORC4 in polar body formation. We found that in both female meiotic divisions, ORC4 surrounds the set of chromosomes, as a sphere-like structure, that will eventually be discarded in the polar bodies, but not the chromosomes that segregate into the oocyte. None of the other five ORC proteins are involved in this structure. In Zygotic G1, ORC4 surrounds the nuclei of the polar bodies, but was not detectable in the pronuclei. When the zygote entered mitosis ORC4 was only detected in the polar body. However, ORC4 appeared on both sets of separating chromosomes at telophase. At this point, the ORC4 that was in the polar body also migrated into the nuclei, suggesting that ORC4 or an associated protein is modified during the first embryonic cell cycle to allow it to bind DNA. Our results suggest that ORC4 may help identify the chromosomes that are destined to be expelled in the polar body, and may play a role in polar body extrusion. extrusion. ORC4 surrounds the chromatin that will be extruded in the polar body in both female meiotic divisions, then makes a transition from the cytoplasm to the chromosomes at zygotic anaphase, suggesting multiple roles for this replication licensing protein.

ORC proteins in the mammalian zygote

“…1D). This spindle-like formation and behavior observed by ORC2 is mirrored by ORC1, ORC3, and ORC5 (Nguyen et al 2015). ORC2 also localizes at the spindles in metaphase I (M-I) during oocyte meiosis, but it is not yet known whether ORC1, ORC3 and ORC5 are similarly present at the M-I spindles (Fig.…”

“…In D. melanogaster studies, ORC6 associates with Pnut in forming a septin-collar in dividing embryos and it has been argued that ORC6 may therefore be directly involved in cytokinesis (Chesnokov et al 2003). In HeLa cells, ORC6 localizes to the area between the separating chromosomes (Prasanth et al 2002), just as ORC1-3 and ORC5 were found to do during A-II of the mouse zygote (Nguyen et al 2015). Disruption of ORC6 in human cells results in cytokinetic defects further supporting the argument for a role of ORC6 in this process (Prasanth et al 2002).…”

“…In the mouse, as the sperm decondenses it donates centrosome proteins (Goto et al 2010) and RNA (Krawetz et al 2011) to the zygote during protamine-histone exchange. Concurrently, ORC4 can be transiently visualized during decondensation prior to pronuclear formation (Figure 1 B) (Nguyen et al 2015). However, after pronuclear formation only ORC2 and ORC6 are detectable by ICC with ORC2 binding throughout the PN while ORC6 localizes around the nucleoli.…”

“…MCM7 is present on the M-II chromatin which implies partial licensing (Ortega et al 2012). It is possible that transient ORC association in the maturing oocyte prior to M-II might explain the observed helicase deposit (Nguyen et al 2015). If true, ORC might recruit MCM to the origins in late meiosis-I, then leave the chromatin before M-II.…”

“…ORC4 forms an ovoid structure surrounding the extruding A-II chromosomes during both meiotic divisions (Fig. 1C and E) (Nguyen et al 2015). During zygotic anaphase, ORC4 behaves in a way that is more consistent with its role as a DNA replication licensing protein by more clearly associating with both chromosome sets (Fig.…”