Laura A. Banaszynski

Distinct Factors Control Histone Variant H3.3 Localization at Specific Genomic Regions

The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc-finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.

A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules

Rapid and reversible methods for perturbing the function of specific proteins are desirable tools for probing complex biological systems. We have developed a general technique to regulate the stability of specific proteins in mammalian cells using cell-permeable, synthetic molecules. We engineered mutants of the human FKBP12 protein that are rapidly and constitutively degraded when expressed in mammalian cells, and this instability is conferred to other proteins fused to these destabilizing domains. Addition of a synthetic ligand that binds to the destabilizing domains shields them from degradation, allowing fused proteins to perform their cellular functions. Genetic fusion of the destabilizing domain to a gene of interest ensures specificity, and the attendant small-molecule control confers speed, reversibility, and dose-dependence to this method. This general strategy for regulating protein stability should enable conditional perturbation of specific proteins with unprecedented control in a variety of experimental settings.

Inhibition of PRC2 Activity by a Gain-of-Function H3 Mutation Found in Pediatric Glioblastoma

EZ Inhibition Missense mutations in the core constituents of the genome packaging material, chromatin, have been implicated in several of human cancers. Nucleosomes are made up of histones, and a mutation of lysine 27 (K27) to methionine in the N-terminal tail of histone variants H3.3 and H3.1 has been identified in various pediatric gliomas. Lewis et al. (p. 857, published online 28 March; see the Perspective by Morgan and Shilatifard) show that the polycomb enzyme complex, which can epigenetically modify K27 by addition of a methyl group—and which is often a silencing signal—is itself potently inhibited by replacement of the H3.3/3.1 K27 by methionine. The inhibition of the EZH2 subunit causes an overall reduction of K27 methylation. Methionine mutants of other methylated lysine residues in histone H3 cause similar reductions in methylation levels of the cognate lysine, altering the epigenetic profiles of such cancer cells. Mutations of histones in some cancers result in inhibition of enzymes that lay down epigenetic marks on chromatin. [Also see Perspective by Morgan and Shilatifard] Sequencing of pediatric gliomas has identified missense mutations Lys27Met (K27M) and Gly34Arg/Val (G34R/V) in genes encoding histone H3.3 (H3F3A) and H3.1 (HIST3H1B). We report that human diffuse intrinsic pontine gliomas (DIPGs) containing the K27M mutation display significantly lower overall amounts of H3 with trimethylated lysine 27 (H3K27me3) and that histone H3K27M transgenes are sufficient to reduce the amounts of H3K27me3 in vitro and in vivo. We find that H3K27M inhibits the enzymatic activity of the Polycomb repressive complex 2 through interaction with the EZH2 subunit. In addition, transgenes containing lysine-to-methionine substitutions at other known methylated lysines (H3K9 and H3K36) are sufficient to cause specific reduction in methylation through inhibition of SET-domain enzymes. We propose that K-to-M substitutions may represent a mechanism to alter epigenetic states in a variety of pathologies.

Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells

Transposable elements comprise roughly 40% of mammalian genomes. They have an active role in genetic variation, adaptation and evolution through the duplication or deletion of genes or their regulatory elements, and transposable elements themselves can act as alternative promoters for nearby genes, resulting in non-canonical regulation of transcription. However, transposable element activity can lead to detrimental genome instability, and hosts have evolved mechanisms to silence transposable element mobility appropriately. Recent studies have demonstrated that a subset of transposable elements, endogenous retroviral elements (ERVs) containing long terminal repeats (LTRs), are silenced through trimethylation of histone H3 on lysine 9 (H3K9me3) by ESET (also known as SETDB1 or KMT1E) and a co-repressor complex containing KRAB-associated protein 1 (KAP1; also known as TRIM28) in mouse embryonic stem cells. Here we show that the replacement histone variant H3.3 is enriched at class I and class II ERVs, notably those of the early transposon (ETn)/MusD family and intracisternal A-type particles (IAPs). Deposition at a subset of these elements is dependent upon the H3.3 chaperone complex containing α-thalassaemia/mental retardation syndrome X-linked (ATRX) and death-domain-associated protein (DAXX). We demonstrate that recruitment of DAXX, H3.3 and KAP1 to ERVs is co-dependent and occurs upstream of ESET, linking H3.3 to ERV-associated H3K9me3. Importantly, H3K9me3 is reduced at ERVs upon H3.3 deletion, resulting in derepression and dysregulation of adjacent, endogenous genes, along with increased retrotransposition of IAPs. Our study identifies a unique heterochromatin state marked by the presence of both H3.3 and H3K9me3, and establishes an important role for H3.3 in control of ERV retrotransposition in embryonic stem cells.

Histone Variants in Metazoan Development

Embryonic development is regulated by both genetic and epigenetic mechanisms, with nearly all DNA-templated processes influenced by chromatin architecture. Sequence variations in histone proteins, core components of chromatin, provide a means to generate diversity in the chromatin structure, resulting in distinct and profound biological outcomes in the developing embryo. Emerging literature suggests that epigenetic contributions from histone variants play key roles in a number of developmental processes such as the initiation and maintenance of pericentric heterochromatin, X-inactivation, and germ cell differentiation. Here, we review the role of histone variants in the embryo with particular emphasis on early mammalian development.

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

Significance A differentiated cell nucleus can be reprogrammed into the pluripotent state by maternal factors in ooplasm; the factors that are responsible for this reprogramming process have not yet been identified. In this paper, we show that histone variant H3.3 is one of the essential maternal factors involved in somatic nuclear reprogramming. Maternal H3.3, not H3.3 in the donor chromatin, is required for development and the reactivation of many key pluripotency genes in somatic cell nuclear transfer (SCNT) embryos. H3.3 facilitates reprogramming by remodeling the donor nuclear chromatin through replacement of donor H3 in chromatin with de novo synthesized maternal H3.3 at the beginning of reprogramming in SCNT embryos. 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.

H3.3 replacement facilitates epigenetic reprogramming of donor nuclei in somatic cell nuclear transfer embryos

Transfer of a somatic nucleus into an enucleated oocyte is the most efficient approach for somatic cell reprogramming. While this process is known to involve extensive chromatin remodeling of the donor nucleus, the maternal factors responsible and the underlying chromatin-based mechanisms remain largely unknown. Here we discuss our recent findings demonstrating that the histone variant H3.3 plays an essential role in reprogramming and is required for reactivation of key pluripotency genes in somatic cell nuclear transfer (SCNT) embryos. Maternal-derived H3.3 replaces H3 in the donor nucleus shortly after oocyte activation, with the amount of replacement directly related to the differentiation status of the donor nucleus in SCNT embryos. We provide additional evidence to suggest that de novo synthesized H3.3 replaces histone H3 carrying repressive modifications in the donor nuclei of SCNT embryos, and hypothesize that replacement may occur at specific loci that must be reprogrammed for gene reactivation.

Histone variant H3.3-mediated chromatin remodeling is essential for paternal genome activation in mouse preimplantation embryos

Derepression of chromatin-mediated transcriptional repression of paternal and maternal genomes is considered the first major step that initiates zygotic gene expression after fertilization. The histone variant H3.3 is present in both male and female gametes and is thought to be important for remodeling the paternal and maternal genomes for activation during both fertilization and embryogenesis. However, the underlying mechanisms remain poorly understood. Using our H3.3B-HA–tagged mouse model, engineered to report H3.3 expression in live animals and to distinguish different sources of H3.3 protein in embryos, we show here that sperm-derived H3.3 (sH3.3) protein is removed from the sperm genome shortly after fertilization and extruded from the zygotes via the second polar bodies (PBII) during embryogenesis. We also found that the maternal H3.3 (mH3.3) protein is incorporated into the paternal genome as early as 2 h postfertilization and is detectable in the paternal genome until the morula stage. Knockdown of maternal H3.3 resulted in compromised embryonic development both of fertilized embryos and of androgenetic haploid embryos. Furthermore, we report that mH3.3 depletion in oocytes impairs both activation of the Oct4 pluripotency marker gene and global de novo transcription from the paternal genome important for early embryonic development. Our results suggest that H3.3-mediated paternal chromatin remodeling is essential for the development of preimplantation embryos and the activation of the paternal genome during embryogenesis.

Transcription pausing regulates mouse embryonic stem cell differentiation

The pluripotency of embryonic stem cells (ESCs) relies on appropriate responsiveness to developmental cues. Promoter-proximal pausing of RNA polymerase II (Pol II) has been suggested to play a role in keeping genes poised for future activation. To identify the role of Pol II pausing in regulating ESC pluripotency, we have generated mouse ESCs carrying a mutation in the pause-inducing factor SPT5. Genomic studies reveal genome-wide reduction of paused Pol II caused by mutant SPT5 and further identify a tight correlation between pausing-mediated transcription effect and local chromatin environment. Functionally, this pausing-deficient SPT5 disrupts ESC differentiation upon removal of self-renewal signals. Thus, our study uncovers an important role of Pol II pausing in regulating ESC differentiation and suggests a model that Pol II pausing coordinates with epigenetic modification to influence transcription during mESC differentiation.

Elsässer et al. reply

Epigenetic silencing of endogenous retroviruses (ERVs) is initiated by KRAB-ZFP–KAP1–SETDB1 repression complexes during early mammalian development. On the basis of biochemical evidence from histone H3.3-knockout embryonic stem (ES) cells, Elsässer et al.1 reported that histone H3.3 is deposited at KAP1– SETDB1-targeted ERVs by the chaperone DAXX–ATRX complex and that this deposition is required to repress ERV transcription and retrotransposition. However, our re-analysis of the published data revealed little evidence of genome-wide ERV upregulation in H3.3-knockout ES cells, and, more importantly, that the ES cells used for the analysis include polymorphic ERV insertions, which probably reflect a mixed genetic background and compromises their use for ERV expression and reintegration analysis. Thus, despite the strong evidence for H3.3 deposition at KAP1–SETDB1targeted ERV elements, it remains to be determined whether this deposition plays a major role in preventing ERV reactivation. There is a Reply to this Comment by Elsässer, S. J. et al. Nature 548, http://dx.doi.org/10.1038/nature23278 (2017). Elsässer et al.1 reported that H3.3 is deposited at ERVs by the H3.3 histone chaperone DAXX in mouse ES cells1. While we were able to confirm DAXX-dependent H3.3 deposition at ERVs in a newly generated conditional Daxx-knockout ES cell line (Extended Data Fig. 1a, b), we have concerns about the authors’ conclusion that H3.3 knockout leads to increased ERV expression1. Intracisternal A-type particle (IAP) ERVs were upregulated less than 1.5-fold in the two tested H3.3-knockout ES cell lines compared with a single wild-type line, and ERVK10C ERVs showed a modest upregulation (approximately 2-fold) in only one of these lines1. By contrast, IAPs and ERVK10C are upregulated 14and 99-fold in Setdb1-knockout ES cells, respectively2, with comparable levels of ERV reactivation observed in Kap1-knockout ES cells3. As Elsässer et al.1 presented expression data of H3.3-knockout ES cells for only a small selection of ERVs1, we re-analysed the published H3.3-knockout RNAsequencing (RNA-seq) datasets to obtain an expanded picture of ERV reactivation. Notably, we found that the number of annotated ERV families that are upregulated in H3.3-knockout ES cells approximately matches the number of ERV families that are downregulated in these cells (Extended Data Fig. 2a). Furthermore, comparing ERV expression in the two H3.3-knockout ES cell lines reveals that an up to 1.5fold difference in expression is within the variation ‘noise’ between two ES cell lines of the same genotype (Extended Data Fig. 2b). If H3.3 directly represses ERVs, ERV upregulation in H3.3-knockout ES cells and H3.3 enrichment at these elements in wild-type ES cells should be positively correlated. However, in contrast to H3K9me3 enrichment and ERV upregulation in Setdb1-knockout ES cells, our re-analysis does not support such a correlation for H3.3 (Fig. 1a, b). Moreover, the vast majority of ERVs that are upregulated in Setdb1knockout ES cells are not convincingly upregulated in H3.3-knockout ES cells (Fig. 1c and Extended Data Fig. 3), indicating that this histone variant is generally dispensable for KAP1–SETDB1-mediated ERV silencing. Curiously, the only robustly re-activated ERVs in H3.3knockout ES cells, namely class III MERVL elements, are not enriched in H3K9me3 or H3.3 in wild-type ES cells (Fig. 1a, b), suggesting that the strongest effect of H3.3 knockout on ERV expression is indirect. Although even a subtle difference in ERV expression could be biologically important, it is possible that secondary effects of H3.3 knockout or clonal variation are responsible for the observed weak phenotype in H3.3-knockout ES cells. More importantly, the genomic copy number of active ERVs, including IAPs, differs widely in inbred mouse strains (described in detail below) and their expression is influenced by genetic background. Given the low level of upregulation reported previously1, and the fact that mice of mixed genetic background (C57BL/6 and 129) were used to generate the single wild-type and two knockout ES cell clones used in the study1, we conclude that the data as originally provided do not convincingly support a requirement for H3.3 in transcriptional silencing of ERVs. The modest level of IAP derepression in H3.3-depleted ES cells1 was inconsistent with their observation of frequent de novo IAP insertions in this line. Notably, IAP elements are highly polymorphic