The maternal nucleolus is required for proper activation of the embryonic genome (EGA) and early embryonic development. Nucleologenesis is characterized by the transformation of a nucleolar precursor body (NPB) to a mature nucleolus during preimplantation development. However, the function of NPBs and the involved molecular factors are unknown. We uncover a novel role for the pluripotency factor LIN28, the biological significance of which was previously demonstrated in the reprogramming of human somatic cells to induced pluripotent stem (iPS) cells. Here, we show that LIN28 accumulates at the NPB and the mature nucleolus in mouse preimplantation embryos and embryonic stem cells (ESCs), where it colocalizes with the nucleolar marker B23 (nucleophosmin 1). LIN28 has nucleolar localization in non-human primate (NHP) preimplantation embryos, but is cytoplasmic in NHP ESCs. Lin28 transcripts show a striking decline before mouse EGA, whereas LIN28 protein localizes to NPBs at the time of EGA. Following knockdown with a Lin28 morpholino, the majority of embryos arrest between the 2- and 4-cell stages and never develop to morula or blastocyst. Lin28 morpholino-injected embryos arrested at the 2-cell stage were not enriched with nucleophosmin at presumptive NPB sites, indicating that functional NPBs were not assembled. Based on these results, we propose that LIN28 is an essential factor of nucleologenesis during early embryonic development.

Centromeres are the fragments of DNA that are responsible for proper chromosome segregation. They consist of centromeric chromatin surrounded by blocks of pericentric heterochromatin, playing an important role in centromere function. In somatic cells, the pericentric domains have a specific pattern of epigenetic modifications of core histones and contain specific pericentric proteins. These features are probably more important for the centromere function than the sequence of the centromeric DNA itself. In somatic cells, the HP1alpha and HP1beta proteins are indispensable for constitutive heterochromatin formation and maintenance. We have analyzed the localization of these proteins in the primordial, growing, fully-grown, and maturing mouse oocytes. Additionally, we have analyzed post-translational modifications of histone H3, which can influence HP1alpha and HP1beta association with the heterochromatin. We showed that the regions of constitutive heterochromatin have a distinct pattern of histone H3 acetylation and di-, and trimethylation of its lysine 9. We demonstrated that HP1beta protein was present in pericentric chromatin domains in primordial oocytes, growing (transcriptionally active) oocytes, and in fully-grown oocytes, and was released to the cytoplasm after germinal vesicle breakdown. In contrast, the HP1alpha was never detected in primordial oocytes, was first detected in pericentric heterochromatin in growing oocytes, dissociated from pericentric heterochromatin in fully-grown oocytes, and it was never detected in maturing oocytes. The presence of HP1alpha and HP1beta proteins on the heterochromatin of transcriptionally active oocytes and their absence in transcriptionally silent oocytes suggest that they are necessary for the repression of RNA synthesis in heterochromatin domains of transcribing oocytes.

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Pericentric constitutive heterochromatin surrounds centromeric regions and is important for centromere function and chromatid cohesion. HP1 (heterochromatin protein 1), a homolog of yeast Swi6, has been shown to be indispensible for proper heterochromatin structure and function. In mammalian somatic cells, two HP1 isoforms, HP1α and HP1β, are constitutively present in pericentric heterochromatin until late G 2, when they dissociate from heterochromatin. Subsequently, they re-associate with heterochromatin at late anaphase. In one-cell mouse embryos, pericentric heterochromatin has a unique configuration and features. It does not form heterochromatin clusters observed in somatic cells and known as chromocenters. Instead, in both pronuclei, it surrounds nucleolar precursor bodies (NBPs), forming ring-like structures. These regions contain HP1β but lack HP1α in both pronuclei. In subsequent interphases, HP1β is constitutively found in heterochromatin until the blastocyst stage. It is not known when HP1α appears and what is its function in early mouse embryos. Here, we show that HP1α appears for the first time at late S phase of two-cell stage, at the time when pericentric heterochromatin is replicated. Its appearance is regulated at the level of translation. In two-cell embryos, the amount of HP1α that can bind to these regions is regulated by phosphorylation of serine 10 of histone H3 (H3S10Ph). Elimination of HP1α by siRNA interfered with centromere relocation from heterochromatin surrounding NPBs to pro-chromocenters at the two-cell stage but did not affect preimplantation develoment to the blastocyst stage.