Histone variant H3.3 is an essential maternal factor for oocyte reprogramming

Abstract

Mature oocyte cytoplasm can reprogram somatic cell nuclei to the pluripotent state through a series of sequential events including protein exchange between the donor nucleus and ooplasm, chromatin remodeling, and pluripotency gene reactivation. Maternal factors that are responsible for this reprogramming process remain largely unidentified. Here, we demonstrate that knockdown of histone variant H3.3 in mouse oocytes results in compromised reprogramming and down-regulation of key pluripotency genes; and this compromised reprogramming for developmental potentials and transcription of pluripotency genes can be rescued by injecting exogenous H3.3 mRNA, but not H3.2 mRNA, into oocytes in somatic cell nuclear transfer embryos. We show that maternal H3.3, and not H3.3 in the donor nucleus, is essential for successful reprogramming of somatic cell nucleus into the pluripotent state. Furthermore, H3.3 is involved in this reprogramming process by remodeling the donor nuclear chromatin through replacement of donor nucleus-derived H3 with de novo synthesized maternal H3.3 protein. Our study shows that H3.3 is a crucial maternal factor for oocyte reprogramming and provides a practical model to directly dissect the oocyte for its reprogramming capacity. P ioneering nuclear transfer experiments in amphibians have revealed that the cytoplasm of the egg is able to reprogram a differentiated nucleus to the embryonic state (1, 2). The success of somatic cell nuclear transfer (SCNT) to produce cloned animals using enucleated metaphase II (MII) oocytes (3, 4), and, recently, the successful derivation of SCNT human embryonic stem cells (5), have demonstrated that maternal factors in the mature ooplasm are capable and sufficient to reprogram a differentiated cell nucleus to pluripotency. This process is known to involve a series of sequential events including protein exchange between donor nucleus and ooplasm, donor nuclear chromatin remodeling, and pluripotency gene reactivation (6-12). However, maternal factors responsible for this reprogramming process and the underlying mechanism(s) remain largely unknown.Thousands of different maternal proteins and mRNAs have been found in mouse mature oocytes (13,14), including variants of the core histone proteins that, along with DNA, constitute nucleosomes. Accumulating evidence suggests that histone variants play important roles in chromatin remodeling and epigenetic regulation orchestrating gene expression changes during reprogramming (12,15,16). In mammals, the histone variant H3.3 is encoded by two different genes (h3f3a and h3f3b), whose translation results in an identical protein product (17, 18). Unlike canonical H3 histones that are expressed and incorporated into chromatin during S phase, expression of H3.3 is not cell cycle-regulated, and the variant is expressed in quiescent cells, postmitotic cells, and proliferating cells throughout the whole cycle, enabling H3.3 deposition in a DNA synthesis-independent manner during and outside of S phase (19). It has been suggested that matern...

Histone Variant H3.3: A versatile H3 variant in health and in disease

“…It has been shown that exogenous H3.3 deposits at not only the regulatory regions of Oct4, but also other genomic regions, including the major satellite repeats and rDNA (Jullien et al, 2012). In addition, knockdown of maternal H3.3 results in compromised reprogramming and failure to reactivate key pluripotency genes, such as Oct4 in mouse embryos (Wen et al, 2014a). These results indicated that H3.3 is required for epigenetic reprogramming and embryonic development of SCNT embryos.…”

“…The function of H3.3 in early embryo development H3.3 takes part in various stages of development, such as gametogenesis, fertilization and differentiation (Bush et al, 2013;Lin et al, 2013;Tang et al, 2015;Wen et al, 2014a;Yuen et al, 2014) (Figure 2A). Knockout animal models for H3.3 or H3.3-specific chaperones have revealed the essential functions of H3.3 during development (Garrick et al, 2006;Michaelson et al, 1999;Roberts et al, 2002;Tang et al, 2015).…”

“…The cytoplasm of mature mouse oocytes contains abundant amounts of both H3f3a and H3f3b mRNAs; however, these maternal transcripts are largely degraded at the first embryonic cleavage (20 h after oocyte activation), whereas zygotic H3.3 mRNA increases after the two-cell stage (Wen et al, 2014a).…”

Chemical “Diversity” of Chromatin Through Histone Variants and Histone Modifications

“…The persistence of this epigenetic memory correlated with the presence of H3.3 at the lineage-specific genes, and alterations of the overall H3.3 levels affected the epigenetic memory of those genes. Since that study, the role of H3.3 in epigenetic reprogramming has been an area of great interest (reviewed in [26]). For example, studies in Xenopus showed that HIRA-dependent H3.3 deposition is required for the transition of a somatic cell-type transcription to an oocytetype transcription profile in SCNT [27].…”

siRNA-mediated depletion of stable proteins in mouse oocytes

“…This can be achieved by microinjection of short interfering RNA (siRNA) into oocytes. Many studies have indeed tried to deplete maternal proteins by injecting siRNA into metaphase II (MII)-stage oocytes (Okada et al, 2010;Wen et al, 2014;Wossidlo et al, 2011) or fully grown GV-stage oocytes (Inoue et al, 2012). However, oftentimes siRNA-injection at these stages cannot significantly deplete maternal proteins that have already been stored in oocytes.…”