SUMMARY Polycomb repressive complex 2 (PRC2) regulates gene expression during lineage specification through trimethylation of lysine 27 on histone H3 (H3K27me3). In Drosophila, polycomb binding sites are dynamic chromatin regions enriched with the histone variant H3.3. Here we show that in mouse embryonic stem cells (ESCs) H3.3 is required for proper establishment of H3K27me3 at the promoters of developmentally regulated genes. Upon H3.3 depletion, these promoters show reduced nucleosome turnover measured by deposition of de novo synthesized histones, and reduced PRC2 occupancy. Further, we show H3.3-dependent interaction of PRC2 with the histone chaperone, Hira, and that Hira localization to chromatin requires H3.3. Our data demonstrate the importance of H3.3 in maintaining a chromatin landscape in ESCs that is important for proper gene regulation during differentiation. Moreover, our findings support the emerging notion that H3.3 has multiple functions in distinct genomic locations that are not always correlated with an “active” chromatin state.

*Brdt is a testis-specific member of the distinctive BET sub-family of bromodomain motif-containing proteins, a motif that binds acetylated lysines and is implicated in chromatin remodeling. Its expression is restricted to the germ line, specifically to pachytene and diplotene spermatocytes and early spermatids. Targeted mutagenesis was used to generate mice carrying a mutant allele of Brdt, Brdt ⌬BD1, which lacks only the first of the two bromodomains that uniquely characterize BET proteins. Homozygous Brdt ⌬BD1/⌬BD1 mice were viable but males were sterile, producing fewer and morphologically abnormal sperm. Aberrant morphogenesis was first detected in step 9 elongating spermatids, and those elongated spermatids that were formed lacked the distinctive foci of heterochromatin at the peri-nuclear envelope. Quantitative reverse transcription (RT)-PCR showed threefold increased levels of histone H1t (Hist1h1t) in Brdt ⌬BD1/⌬BD1 testes and chromatin immunoprecipitation revealed that Brdt protein, but not Brdt ⌬BD1 protein, was associated with the promoter of H1t. Intracytoplasmic sperm injection suggested that the DNA in the Brdt ⌬BD1 mutant sperm could support early embryonic development and yield functional embryonic stem cells. This is the first demonstration that deletion of just one of the two bromodomains in members of the BET sub-family of bromodomain-containing proteins has profound effects on in vivo differentiation.

The BET subfamily of bromodomain-containing genes is characterized by the presence of two bromodomains and a unique ET domain at their carboxyl termini. Here, we show that the founding member of this subfamily, Brd2, is an essential gene by generating a mutant mouse line lacking Brd2 function. Homozygous Brd2 mutants are embryonic lethal, with most Brd2 ؊/؊ embryos dying by embryonic day 11.5. Before death, the homozygous embryos were notably smaller and exhibited abnormalities in the neural tube where the gene is highly expressed. Brd2-deficient embryonic fibroblast cells were observed to proliferate more slowly than controls. Experiments to explore whether placental insufficiency could be a cause of the embryonic lethality showed that injecting diploid mutant embryonic stem cells into tetraploid wild-type blastocysts did not rescue the lethality; that is Brd2-deficient embryos could not be rescued by wild-type extraembryonic tissues. Furthermore, there were enhanced levels of cell death in Brd2-deficient embryos. Developmental Dynamics 238:908 -917, 2009.

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...