Peter W. Lewis

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.

Isolation of the Cdc45/Mcm2–7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase

The protein Cdc45 plays a critical but poorly understood role in the initiation and elongation stages of eukaryotic DNA replication. To study Cdc45s function in DNA replication, we purified Cdc45 protein from Drosophila embryo extracts by a combination of traditional and immunoaffinity chromatography steps and found that the protein exists in a stable, high-molecular-weight complex with the Mcm2–7 hexamer and the GINS tetramer. The purified Cdc45/Mcm2–7/GINS complex is associated with an active ATP-dependent DNA helicase function. RNA interference knock-down experiments targeting the GINS and Cdc45 components establish that the proteins are required for the S phase transition in Drosophila cells. The data suggest that this complex forms the core helicase machinery for eukaryotic DNA replication.

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.

Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres

The histone variant H3.3 is implicated in the formation and maintenance of specialized chromatin structure in metazoan cells. H3.3-containing nucleosomes are assembled in a replication-independent manner by means of dedicated chaperone proteins. We previously identified the death domain associated protein (Daxx) and the α-thalassemia X-linked mental retardation protein (ATRX) as H3.3-associated proteins. Here, we report that the highly conserved N terminus of Daxx interacts directly with variant-specific residues in the H3.3 core. Recombinant Daxx assembles H3.3/H4 tetramers on DNA templates, and the ATRX–Daxx complex catalyzes the deposition and remodeling of H3.3-containing nucleosomes. We find that the ATRX–Daxx complex is bound to telomeric chromatin, and that both components of this complex are required for H3.3 deposition at telomeres in murine embryonic stem cells (ESCs). These data demonstrate that Daxx functions as an H3.3-specific chaperone and facilitates the deposition of H3.3 at heterochromatin loci in the context of the ATRX–Daxx complex.

Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.

ATRX ADD domain links an atypical histone methylation recognition mechanism to human mental-retardation syndrome

ATR-X (alpha-thalassemia/mental retardation, X-linked) syndrome is a human congenital disorder that causes severe intellectual disabilities. Mutations in the ATRX gene, which encodes an ATP-dependent chromatin-remodeler, are responsible for the syndrome. Approximately 50% of the missense mutations in affected persons are clustered in a cysteine-rich domain termed ADD (ATRX-DNMT3-DNMT3L, ADD(ATRX)), whose function has remained elusive. Here we identify ADD(ATRX) as a previously unknown histone H3-binding module, whose binding is promoted by lysine 9 trimethylation (H3K9me3) but inhibited by lysine 4 trimethylation (H3K4me3). The cocrystal structure of ADD(ATRX) bound to H3(1-15)K9me3 peptide reveals an atypical composite H3K9me3-binding pocket, which is distinct from the conventional trimethyllysine-binding aromatic cage. Notably, H3K9me3-pocket mutants and ATR-X syndrome mutants are defective in both H3K9me3 binding and localization at pericentromeric heterochromatin; thus, we have discovered a unique histone-recognition mechanism underlying the ATR-X etiology.ATR-X (alpha-thalassemia/mental retardation, X-linked) syndrome is a human congenital disorder that causes severe intellectual disabilities. Mutations in the ATRX gene, which encodes an ATP-dependent chromatin-remodeler, are responsible for the syndrome. Approximately 50% of the missense mutations in affected persons are clustered in a cysteine-rich domain termed ADD (ATRX-DNMT3-DNMT3L, ADDATRX), whose function has remained elusive. Here we identify ADDATRX as a previously unknown histone H3–binding module, whose binding is promoted by lysine 9 trimethylation (H3K9me3) but inhibited by lysine 4 trimethylation (H3K4me3). The cocrystal structure of ADDATRX bound to H31–15K9me3 peptide reveals an atypical composite H3K9me3-binding pocket, which is distinct from the conventional trimethyllysine-binding aromatic cage. Notably, H3K9me3-pocket mutants and ATR-X syndrome mutants are defective in both H3K9me3 binding and localization at pericentromeric heterochromatin; thus, we have discovered a unique histone-recognition mechanism underlying the ATR-X etiology.

DAXX envelops a histone H3.3-H4 dimer for H3.3-specific recognition

Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. DAXX is a metazoan histone chaperone specific to the evolutionarily conserved histone variant H3.3. Here we report the crystal structures of the DAXX histone-binding domain with a histone H3.3–H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies that elucidate the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3–H4 dimer, with complex formation accompanied by structural transitions in the H3.3–H4 histone fold. DAXX uses an extended α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, and functional studies address the contributions of Gly 90 in H3.3 and Glu 225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.

Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation

Over 70% of diffuse intrinsic pediatric gliomas, an aggressive brainstem tumor, harbor heterozygous mutations that create a K27M amino acid substitution (methionine replaces lysine 27) in the tail of histone H3.3. The role of the H3.3K27M mutation in tumorigenesis is not fully understood. Here, we use a human embryonic stem cell system to model this tumor. We show that H3.3K27M expression synergizes with p53 loss and PDGFRA activation in neural progenitor cells derived from human embryonic stem cells, resulting in neoplastic transformation. Genome-wide analyses indicate a resetting of the transformed precursors to a developmentally more primitive stem cell state, with evidence of major modifications of histone marks at several master regulator genes. Drug screening assays identified a compound targeting the protein menin as an inhibitor of tumor cell growth in vitro and in mice. A stem cell model of a lethal brain tumor in children shows how a recurrent histone mutation leads to cancer. [Also see Perspective by Becher and Wechsler-Reya] Modeling brain cancer from stem to stern Diffuse intrinsic pontine gliomas (DIPGs) are aggressive brain tumors primarily affecting children. Because the tumors arise in the brainstem, which controls many vital functions, they cannot be surgically excised and are often fatal. To study the pathogenesis of DIPGs—in particular, the role of a histone H3.3 mutation that occurs in 70% of cases—Funato et al. developed a new tumor model (see the Perspective by Becher and Wechsler-Reya). They first directed the differentiation of embryonic stem cells into neural progenitor cells. They then introduced a specific combination of genes, including the mutant histone gene, and found that this caused the progenitor cells to acquire features characteristic of cancer cells. During this oncogenic transformation, the cells reverted to a more primitive differentiation state and displayed altered histone marks at several key regulatory genes. Science, this issue p. 1529; see also p. 1458

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.

KDM4A Lysine Demethylase Induces Site-Specific Copy Gain and Rereplication of Regions Amplified in Tumors

Acquired chromosomal instability and copy number alterations are hallmarks of cancer. Enzymes capable of promoting site-specific copy number changes have yet to be identified. Here, we demonstrate that H3K9/36me3 lysine demethylase KDM4A/JMJD2A overexpression leads to localized copy gain of 1q12, 1q21, and Xq13.1 without global chromosome instability. KDM4A-amplified tumors have increased copy gains for these same regions. 1q12h copy gain occurs within a single cell cycle, requires S phase, and is not stable but is regenerated each cell division. Sites with increased copy number are rereplicated and have increased KDM4A, MCM, and DNA polymerase occupancy. Suv39h1/KMT1A or HP1γ overexpression suppresses the copy gain, whereas H3K9/K36 methylation interference promotes gain. Our results demonstrate that overexpression of a chromatin modifier results in site-specific copy gains. This begins to establish how copy number changes could originate during tumorigenesis and demonstrates that transient overexpression of specific chromatin modulators could promote these events.