Replication-independent Variant Research Articles

Loss of histone H3.3 results in DNA replication defects and altered origin dynamics in C. elegans.

Histone H3.3 is a replication-independent variant of histone H3 with important roles in development, differentiation, and fertility. Here, we show that loss of H3.3 results in replication defects in Caenorhabditis elegans embryos at elevated temperatures. To characterize these defects, we adapt methods to determine replication timing, map replication origins, and examine replication fork progression. Our analysis of the spatiotemporal regulation of DNA replication shows that despite the very rapid embryonic cell cycle, the genome is replicated from early and late firing origins and is partitioned into domains of early and late replication. We find that under temperature stress conditions, additional replication origins become activated. Moreover, loss of H3.3 results in altered replication fork progression around origins, which is particularly evident at stress-activated origins. These replication defects are accompanied by replication checkpoint activation, a delayed cell cycle, and increased lethality in checkpoint-compromised embryos. Our comprehensive analysis of DNA replication in C. elegans reveals the genomic location of replication origins and the dynamics of their firing, and uncovers a role of H3.3 in the regulation of replication origins under stress conditions.

Differential Expression of Histone H3.3 Genes and Their Role in Modulating Temperature Stress Response in Caenorhabditis elegans.

Replication-independent variant histones replace canonical histones in nucleosomes and act as important regulators of chromatin function. H3.3 is a major variant of histone H3 that is remarkably conserved across taxa and is distinguished from canonical H3 by just four key amino acids. Most genomes contain two or more genes expressing H3.3, and complete loss of the protein usually causes sterility or embryonic lethality. Here, we investigate the developmental expression patterns of the five Caenorhabditis elegans H3.3 homologs and identify two previously uncharacterized homologs to be restricted to the germ line. Despite these specific expression patterns, we find that neither loss of individual H3.3 homologs nor the knockout of all five H3.3-coding genes causes sterility or lethality. However, we demonstrate an essential role for the conserved histone chaperone HIRA in the nucleosomal loading of all H3.3 variants. This requirement can be bypassed by mutation of the H3.3-specific residues to those found in H3. While even removal of all H3.3 homologs does not result in lethality, it leads to reduced fertility and viability in response to high-temperature stress. Thus, our results show that H3.3 is nonessential in C. elegans but is critical for ensuring adequate response to stress.

Abstract 3767: Identification of a novel and a shared H3.3K27M mutation derived neoantigen epitope and H3.3K27M specific TCR engineered T cell therapy for glioma

Abstract Brain cancers are the leading cause of cancer related mortality in children and young adults with median overall survival of 9-10 months and hence represents a significant unmet medical need. Genome-wide sequencing efforts of pediatric gliomas have identified a recurrent and shared missense mutation in the gene encoding the replication-independent variant of histone 3, H3.3. Approximately 70% of diffuse intrinsic pontine gliomas (DIPG) and 50% of thalamic and other midline gliomas harbor the amino acid substitution from lysine (K) to methionine (M) at the position 27 of H3.3 gene. Tumor specific missense mutations are not subjected to self-tolerance and can be suitable targets (neoantigens) for cancer immunotherapy. Herein, we evaluated whether the H3.3K27M mutation can induce specific cytotoxic T lymphocyte (CTL) response in HLA-A2+ T cells. In vitro stimulation of HLA-A2+ donor derived CD8+ T cells with a synthetic peptide encompassing the H3.3K27M mutation (H3.3K27M epitope) induced CTL lines which recognized not only T2 cells loaded with the synthetic H3.3K27M epitope peptide but also lysed the HLA-A2+ DIPG cells which endogenously harbor the H3.3K27M mutation. On the other hand, the CTL lines did not react to either HLA-A2+ but H3.3K27M- negative DIPG cell lines or H3.3K27M positive but HLA-A2 negative DIPG cells. The H3.3K27M epitope peptide but not the non-mutant counterpart indicated an excellent binding affinity (Kd 151nM) to HLA-A2 based on competitive binding inhibition assay. From CTL clones with high and specific affinities to HLA-A2-H3.3K27M-tetramer, cDNAs for T cell receptor (TCR) alpha and beta chains were cloned into a retroviral vector. Human HLA-A2+ T cells transduced with the TCR demonstrated antigen specific reactivity as well as anti-glioma responses in vitro. Peptide titration assays suggested that the H3.3K27M specific TCR had the half-maximal reactivity for peptide recognition of around 100nM. Furthermore, critically important for safety of clinical application, alanine scanning demonstrated that the key amino acid sequence motif in the epitope of the TCR reactivity is not shared by any known human protein. Finally, intravenous administration of T cells transduced with H3.3K27M specific TCR significantly inhibited the growth of intracranial HLA-A2+ H3.3K27M positive glioma xenografts in immune deficient NSG mice. These data provide us with a strong basis for developing peptide based vaccines as well as adoptive transfer therapy using autologous T cells transduced with the H3.3K27M specific TCR. Acknowledgements: This study is supported by the NIH/NINDS (1RO1NS096954), V Foundation and Parker Institution for Cancer Immunotherapy. Citation Format: Zinal Chheda, Gary Kohanbash, John Sidney, Kaori Okada, Naznin Jahan, Diego Carrera, Payal Watchmaker, Kira Downey, Shuming Liu, Shruti Shrivastav, Sabine Mueller, Ian F. Pollack, Angel M. Carcaboso, Alessandro Sette, Yafei Hou, Hideho Okada. Identification of a novel and a shared H3.3K27M mutation derived neoantigen epitope and H3.3K27M specific TCR engineered T cell therapy for glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3767. doi:10.1158/1538-7445.AM2017-3767

OS09.4 Identification of a novel H3.3.K27M mutation-derived neoantigen epitope and cloning of H3.3.K27M-specific T-cell receptor for T-cell therapy in gliomas

Brain cancers are the leading cause of cancer related mortality and morbidity in children and young adults. Children with diffuse intrinsic brainstem malignant gliomas have one-year progression-free survival rates below 25% and median overall survival of 9 to 10 months with current treatment options and hence represent a significant unmet medical need. Genome-wide sequencing efforts of pediatric gliomas have identified a recurrent and shared missense mutation in the gene encoding the replication-independent variant of histone 3, H3.3. Approximately 70% of diffuse intrinsic pontine gliomas (DIPG) and 50% of thalamic and other midline gliomas harbor the amino-acid substitution from lysine (K) to methionine (M) at the position 27 of H3.3 gene (H3.3.K27M mutation). Tumor-specific missense mutations are not subjected to self-tolerance and can be suitable targets (i.e. neoantigen) for cancer immunotherapy. We evaluated whether the H3.3.K27M mutation can induce specific cytotoxic T lymphocyte (CTL) response in HLA-A2+ human T cells. In vitro stimulation of HLA-A2+ donor-derived CD8+ T-cells with a synthetic peptide encompassing the H3.3.K27M mutation (H3.3.K27M epitope) induced CTL lines which recognized not only T2 cells loaded with the synthetic H3.3.K27M epitope peptide but also lysed HLA-A2+ DIPG cell lines which endogenously harbor the H3.3.K27M mutation. On the other hand, the CTL lines did not react to either HLA-A2+ H3.3.K27M -negative DIPG cell lines or H3.3K27M-positive but HLA-A2-negative cells. The H3.3.K27M epitope peptide, but not the non-mutant counterpart, indicated an excellent affinity (Kd 151nM) to HLA-A2 based on competitive binding inhibition assay. From CTL clones with high and specific affinities to HLA-A2-H3.3.K27M-tetramer, cDNAs for T cell receptor (TCR) α- and β-chains were cloned into a retroviral vector. Human HLA-A2+ T-cells transduced with the TCR demonstrated antigen-specific reactivity as well as anti-glioma responses in vitro. Peptide titration assay suggested that the H3.3.K27M-specific TCR had the half-maximal reactivity for peptide recognition of around 100nM. Furthermore, critically important for safety of clinical application, alanine scanning demonstrated that the key amino-acid sequence motif in the epitope for the TCR reactivity is not shared by any known human protein. Finally, intravenous administration of T-cells transduced with the H3.3.K27M-specific TCR significantly inhibited the growth of intracranial HLA-A2+ H3.3.K27M-positive glioma xenografts in immune-deficient mice. These data provide us with a strong basis for developing peptide-based vaccines as well as adoptive transfer therapy using autologous T-cells transduced with the H3.3.K27M-specific TCR.

Histone variants on the move: substrates for chromatin dynamics.

Most histones are assembled into nucleosomes behind the replication fork to package newly synthesized DNA. By contrast, histone variants, which are encoded by separate genes, are typically incorporated throughout the cell cycle. Histone variants can profoundly change chromatin properties, which in turn affect DNA replication and repair, transcription, and chromosome packaging and segregation. Recent advances in the study of histone replacement have elucidated the dynamic processes by which particular histone variants become substrates of histone chaperones, ATP-dependent chromatin remodellers and histone-modifying enzymes. Here, we review histone variant dynamics and the effects of replacing DNA synthesis-coupled histones with their replication-independent variants on the chromatin landscape.

Contribution of the Two Genes Encoding Histone Variant H3.3 to Viability and Fertility in Mice

Histones package DNA and regulate epigenetic states. For the latter, probably the most important histone is H3. Mammals have three near-identical H3 isoforms: canonical H3.1 and H3.2, and the replication-independent variant H3.3. This variant can accumulate in slowly dividing somatic cells, replacing canonical H3. Some replication-independent histones, through their ability to incorporate outside S-phase, are functionally important in the very slowly dividing mammalian germ line. Much remains to be learned of H3.3 functions in germ cell development.Histone H3.3 presents a unique genetic paradigm in that two conventional intron-containing genes encode the identical protein. Here, we present a comprehensive analysis of the developmental effects of null mutations in each of these genes. H3f3a mutants were viable to adulthood. Females were fertile, while males were subfertile with dysmorphic spermatozoa. H3f3b mutants were growth-deficient, dying at birth. H3f3b heterozygotes were also growth-deficient, with males being sterile because of arrest of round spermatids. This sterility was not accompanied by abnormalities in sex chromosome inactivation in meiosis I. Conditional ablation of H3f3b at the beginning of folliculogenesis resulted in zygote cleavage failure, establishing H3f3b as a maternal-effect gene, and revealing a requirement for H3.3 in the first mitosis. Simultaneous ablation of H3f3a and H3f3b in folliculogenesis resulted in early primary oocyte death, demonstrating a crucial role for H3.3 in oogenesis.These findings reveal a heavy reliance on H3.3 for growth, gametogenesis, and fertilization, identifying developmental processes that are particularly susceptible to H3.3 deficiency. They also reveal partial redundancy in function of H3f3a and H3f3b, with the latter gene being generally the most important.

Nucleosome Assembly Dynamics Involve Spontaneous Fluctuations in the Handedness of Tetrasomes

Nucleosomes play crucial roles in eukaryotic cells as they form the basic compaction unit of DNA, and impact the accessibility of the DNA for transcription and replication. How the assembly, stability, and disassembly of nucleosomes affect the interplay between compaction and accessibility forms a question of key interest. Here we use freely-orbiting magnetic tweezers (FOMT), which can measure in real time the changes in length and twist of individually tethered DNA molecules. We monitor how Nucleosome Assembly Protein-1 (NAP1) loads either histone tetramers (an important intermediate in dis/assembly or remodeling) or complete histone octamers onto DNA, thus forming nucleosomes. Remarkably, tetrasomes are observed to exhibit a spontaneous flipping between DNA states with linking number ΔLk=-0.73 and ΔLk=+1.0, without concomitant changes in DNA end-to-end length. Such behavior is absent in the case of nucleosomes. The statistics of tetrasome flipping shows that the preferentially occupied left-handed state has a probability p=0.9. The difference in free energy between these two states is thus 2.3kBT. We also demonstrate how controlled application of positive torque can drive tetrasome occupancy from left-handed to right-handed states. To exclude effects of the assembly protein NAP1, we repeated these experiments with tetrasomes assembled in the conventional way using salt dialysis. These results show that the spontaneous fluctuations in the handedness of tetrasomes are not NAP1 induced. Since we expect the H3-H3 interface to play an important role in the handedness fluctuations, we also studied tetrasomes in which the replication-coupled canonical histone H3 was replaced by the replication-independent variant H3.3, which show very similar behavior. Our findings thus reveal unexpected dynamical rearrangements of the nucleosomal structure that suggest that chromatin can serve as a ‘twist reservoir’, offering a mechanistic explanation for the regulation of DNA supercoiling in chromatin.

Identification of a Replication-independent Replacement Histone H3 in the Basidiomycete Ustilago maydis

Ustilago maydis is a haploid basidiomycete with single genes for two distinct histone H3 variants. The solitary U1 gene codes for H3.1, predicted to be a replication-independent replacement histone. The U2 gene is paired with histone H4 and produces a putative replication-coupled H3.2 variant. These predictions were evaluated experimentally. U2 was confirmed to be highly expressed in the S phase and had reduced expression in hydroxyurea, and H3.2 protein was not incorporated into transcribed chromatin of stationary phase cells. Constitutive expression of U1 during growth produced ~25% of H3 as H3.1 protein, more highly acetylated than H3.2. The level of H3.1 increased when cell proliferation slowed, a hallmark of replacement histones. Half of new H3.1 incorporated into highly acetylated chromatin was lost with a half-life of 2.5 h, the fastest rate of replacement H3 turnover reported to date. This response reflects the characteristic incorporation of replacement H3 into transcribed chromatin, subject to continued nucleosome displacement and a loss of H3 as in animals and plants. Although the two H3 variants are functionally distinct, neither appears to be essential for vegetative growth. KO gene disruption transformants of the U1 and U2 loci produced viable cell lines. The structural and functional similarities of the Ustilago replication-coupled and replication-independent H3 variants with those in animals, in plants, and in ciliates are remarkable because these distinct histone H3 pairs of variants arose independently in each of these clades and in basidiomycetes.

Modifications of H3 and H4 during Chromatin Replication, Nucleosome Assembly, and Histone Exchange

Histone posttranslational modifications that accompany DNA replication, nucleosome assembly, and H2A/H2B exchange were examined in human tissue culture cells. Through microsequencing analysis and chromatin immunoprecipitation, it was found that a subset of newly synthesized H3.2/H3.3 is modified by acetylation and methylation at sites that correlate with transcriptional competence. Immunoprecipitation experiments suggest that cytosolic predeposition complexes purified from cells expressing FLAG-H4 contain H3/H4 dimers, not tetramers. Studies of the deposition of newly synthesized H2A/H2B onto replicating and nonreplicating chromatin demonstrated that H2A/H2B exchange takes place in chromatin regions that contain acetylated H4; however, there is no single pattern of H4 acetylation that accompanies exchange. H2A/H2B exchange is also largely independent of the deposition of replacement histone variant, H3.3. Finally, immunoprecipitation of nucleosomes replicated in the absence of de novo nucleosome assembly showed that histone modifications do not prevent the transfer of parental histones to newly replicated DNA and thus have the potential to serve as means of epigenetic inheritance. Our experiments provide an in-depth analysis of the "histone code" associated with chromatin replication and dynamic histone exchange in human cells.

Role for a YY1-binding element in replication-dependent mouse histone gene expression.

Expression of the highly conserved replication-dependent histone gene family increases dramatically as a cell enters the S phase of the eukaryotic cell cycle. Requirements for normal histone gene expression in vivo include an element, designated alpha, located within the protein-encoding sequence of nucleosomal histone genes. Mutation of 5 of 7 nucleotides of the mouse H3.2 alpha element to yield the sequence found in an H3.3 replication-independent variant abolishes the DNA-protein interaction in vitro and reduces expression fourfold in vivo. A yeast one-hybrid screen of a HeLa cell cDNA library identified the protein responsible for recognition of the histone H3.2 alpha sequence as the transcription factor Yin Yang 1 (YY1). YY1 is a ubiquitous and highly conserved transcription factor reported to be involved in both activation and repression of gene expression. Here we report that the in vitro histone alpha DNA-protein interaction depends on YY1 and that mutation of the nucleotides required for the in vitro histone alpha DNA-YY1 interaction alters the cell cycle phase-specific up-regulation of the mouse H3.2 gene in vivo. Because all mutations or deletions of the histone alpha sequence both abolish interactions in vitro and cause an in vivo decrease in histone gene expression, the recognition of the histone alpha element by YY1 is implicated in the correct temporal regulation of replication-dependent histone gene expression in vivo.

On the origins of genetic variants

Two contrasting mechanisms responsible for the creation of genetic variants are described: one is the manifestation of the limited accuracy of the cellular machinery for DNA replication, the other results from the ability of cells to repair damaged DNA. Replication-dependent variants and those caused by episodical DNA damage enhance the probability that a small fraction of a cell population may survive a sudden (physical or biological) change of environmental conditions. Replication-independent variants arise during persistent but not immediately lethal stress (e.g. starvation) of a non-dividing population. The variants observed under these conditions are of selective advantage because they are able to cope with the particular stress situation. The molecular basis of their creation is a matter of intensive debate.