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PubMed2025

Two CENH3 paralogs in the green alga Chlamydomonas reinhardtii have a redundantly essential function and associate with ZeppL-LINE1 elements.

Centromeres in eukaryotes are defined by the presence of histone H3 variant CENP-A/CENH3. Chlamydomonas encodes two predicted CENH3 paralogs, CENH3.1 and CENH3.2, that have not been previously characterized. We generated peptide antibodies to unique N-terminal epitopes for each of the two predicted Chlamydomonas CENH3 paralogs as well as an antibody against a shared CENH3 epitope. All three CENH3 antibodies recognized proteins of the expected size on immunoblots and had punctate nuclear immunofluorescence staining patterns. These results are consistent with both paralogs being expressed and localized to centromeres. CRISPR-Cas9-mediated insertional mutagenesis was used to generate predicted null mutations in either CENH3.1 or CENH3.2. Single mutants were viable but cenh3.1 cenh3.2 double mutants were not recovered, confirming that the function of CENH3 is essential. We sequenced and assembled two chromosome-scale Chlamydomonas genomes from strains CC-400 and UL-1690 (a derivative of CC-1690) with complete centromere sequences for 17/17 and 14/17 chromosomes respectively, enabling us to compare centromere evolution across four isolates with near complete assemblies. These data revealed significant changes across isolates between homologous centromeres including mobility and degeneration of ZeppL-LINE1 (ZeppL) transposons that comprise the major centromere repeat sequence in Chlamydomonas. We used cleavage under targets and tagmentation (CUT&Tag) to purify and map CENH3-bound genomic sequences and found enrichment of CENH3-binding almost exclusively at predicted centromere regions. An interesting exception was chromosome 2 in UL-1690, which had enrichment at its genetically mapped centromere repeat region as well as a second, distal location, centered around a single recently acquired ZeppL insertion. The CENH3-bound regions of the 17 Chlamydomonas centromeres ranged from 63.5 kb (average lower estimate) to 175 kb (average upper estimate). The relatively small size of its centromeres suggests that Chlamydomonas may be a useful organism for testing and deploying artificial chromosome technologies.

Chlamydomonas reinhardtii↗
PubMed2025

The dynamic centromere.

Centromeres are fundamental chromosomal structures that ensure accurate chromosome segregation during cell division. Despite their conserved and essential role in maintaining genomic stability, centromeres are subject to rapid evolutionary change. At the heart of centromere identity is the histone H3 variant CENP-A, an epigenetic mark that defines and propagates active centromeres and is essential for their function. Recent evidence supports a rapid evolution of centromere DNA sequences but also suggests a certain degree of flexibility in CENP-A deposition and propagation. The phenomenon of centromere drift, recently observed in humans, highlights how the dynamic repositioning of CENP-A and associated epigenetic environment over time maintains a regulated equilibrium, ensuring centromere function despite positional variation. Understanding these processes is crucial for unraveling centromere dynamics and their broader implications for genome stability and evolution.

Centromere↗
PubMed2025

Preserving centromere identity: right amounts of CENP-A at the right place and time.

Four decades ago, the discovery of centromere protein-A (CENP-A) marked a pivotal breakthrough in chromosome biology, revealing the epigenetic foundation of centromere identity. CENP-A, a histone H3 variant, directs the formation of the microtubule-binding kinetochore complex, designating the chromosomal site for its assembly and underpins the accurate partitioning of genetic material during cell division. Errors in cell division can give rise to DNA instability and aneuploidy, implicated in human diseases such as cancer. Therefore, discovering the underlying pathways and mechanisms responsible for the formation, regulation and maintenance of the centromere is important to our understanding of genome stability, epigenetic inheritance, and in providing the knowledge to help generate possible treatments and therapeutics. Here, we review various molecular pathways and mechanisms implicated in maintaining centromere identity and highlight some of the key outstanding questions with a focus on the human centromere.

Humans↗
PubMed2026

Neocentromeres fail to maintain DNA methylation boundaries, driving CENP-A drift, instability, and chromosome missegregation.

Centromere identity is specified by CENP-A, a histone H3 variant that epigenetically defines centromere position. How CENP-A is maintained at one location in rapidly evolving centromeric DNA is unknown. Using single-cell-derived clones of human cell lines, we demonstrate heterogeneity in CENP-A position within cell populations at neocentromeres and a native centromere. CENP-A heterogeneity is accompanied by heterogeneous DNA methylation patterns, with DNA methylation shifting according to CENP-A position. We demonstrate centromere epigenetic plasticity over extended proliferation, with native centromeres maintaining stable DNA methylation boundaries, but neocentromeres exhibiting DNA methylation instability, boundary loss, and increased missegregation. Finally, we show that neocentromeres are more sensitive to DNA methylation inhibition than native centromeres, and that this inhibition is accompanied by expanded CENP-A-enriched domains and increased missegregation. This study supports a role for DNA methylation boundaries in maintaining centromere position, stability, and function and highlights the intrinsic instability of DNA methylation at neocentromeres.

CENP-A↗
PubMed2025

Quantitative Profiling of Histone Variants and Posttranslational Modifications by Tandem Mass Spectrometry in Arabidopsis.

Histone dynamics constitute an important layer of gene regulations associated with development and growth in multicellular eukaryotes. They also stand as key determinants of plant responses to environmental changes. Histone dynamics include the exchange of histone variants as well as post-translational modifications of their amino acid residues (such as acetylation and mono/di/trimethylation), commonly referred to as histone marks. Investigating histone dynamics with a focus on combinatorial changes occurring at their residues will greatly help unravel how plants achieve phenotypic plasticity.Mass spectrometry (MS) analysis offers unequaled resolution of the abundance of histone variants and of their marks. Indeed, relative to other techniques such as western blot or genome-wide profiling, this powerful technique allows quantifying the relative abundances of histone forms, as well as revealing coexisting marks on the same histone molecule. Yet, while MS-based histone analysis has proven efficient in several animals and other model organisms, this method stands out as more challenging in plants. One major challenge is the isolation of sufficient amounts of pure, high-quality histones, likely rendered difficult by the presence of the cell wall, for sufficiently deep and resolutive identification of histone species.In this chapter, we describe a straightforward MS-based proteomic method, implemented to characterize histone marks from Arabidopsis thaliana seedling tissues and cell culture suspensions. After acid extraction of histones, in vitro propionylation of free lysine residues, and digestion with trypsin, a treatment at highly basic pH allows obtaining sharp spectral signals of biologically relevant histone peptide forms.The method workflow described here shall be used to measure changes in histone marks between Arabidopsis thaliana genotypes, along developmental time-courses, or upon various stresses and treatments.

Histones↗
PubMed1978

Species-specific suppression of histone H1 and H2B production in human/mouse hybrids.

Ten human/mouse hybrid cell lines that segregate either human or mouse chromosomes were examined for the expression of human- and mouse-specific histones H1 and H2B. Results of this study indicate that the human and mouse chromosomes in hybrid cells that segregate human chromosomes (M greater than H hybrids) contain only mouse histone H1 and H2B. Chromosomes in hybrid cells that segregate mouse chromosomes (H greater than M hybrids) contain only human H1 and H2B histones. Loss of the ability to produce either human or mouse histones does not seem to be due to the loss of specific human or mouse chromosomes because M greater than H hybrids retaining at least one copy of each human chromosome contain only mouse H1 and H2B and H greater than M hybrids retaining at least one copy of each mouse chromosome contain only human H1 and H2B histones. These results, together with those concerning histone H4 acetylation levels and ratios of variants of histones H3 and H2A that are like those in the dominant parent cell type, indicate that the control mechanisms affecting H1 and H2B expression in H greater than M and in M greater than H hybrid cells affect expression of histones H2A, H3, and H4 genes as well. The present data thus support the hypothesis that none of the histone genes that are active in the recessive parent cell type is expressed in hybrid lines that segregate recessive cell chromosomes.

Cell Line↗
PubMed1979

Isolation and characterization of the histone variants in chicken erythrocytes.

Chicken erythrocyte histones 2A, 2B, and 3 can be resolved into nonallelic primary structure variants by polyacrylamide gel electrophoresis in the presence of Triton X-100. These variants were isolated and characterized by analysis of their tryptic and thermolytic peptides. The major variants of chicken H2A and H2B differ from the analogous component of calf thymus by a small number of conservative amino acid substitutions in the basic terminal regions, which interact with DNA. This moderate rate of allelic evolution of the slightly lysine-rich histones contrasts with the complete conservatism found in the arginine-rich histones. Chicken H4 and both chicken H3 variants are identical with their corresponding components in mammals. The amino acid substitutions distinguishing histone variants are located within the highly conserved hydrophobic regions, which are involved in histone--histone interactions.

Amino Acids↗
PubMed2026

A novel iPSC model of Bryant-Li-Bhoj neurodevelopmental/neurodegenerative syndrome demonstrates the role of histone H3.3 in chromatin dynamics, neuronal differentiation, and maturation.

BACKGROUND: Bryant-Li-Bhoj neurodevelopmental syndrome (BLBS) is neurogenetic disorder caused by variants in H3-3A and H3-3B, the two genes that encode histone H3.3. Ninety-nine percent of individuals with BLBS show developmental delay/intellectual disability, but the mechanism by which variants in H3.3 result in these phenotypes is not yet understood, limiting the therapeutic interventions available to individuals living with BLBS. METHODS: Here, we investigate how one BLBS-causative variant, H3-3B p.Leu48Arg (L48R), affects neurodevelopment using an induced pluripotent stem cell model differentiated to 2D neural progenitor cells (NPCs), 2D forebrain neurons (FBNs), and 3D dorsal forebrain organoids (DFBOs). We employ a multi-omic approach in the 2D models to quantify the resulting changes in gene expression and chromatin accessibility. We used immunofluorescence (IF) staining to define the identities of cells in the 3D DFBOs and whole-cell patch clamp to investigate the electrophysiological properties of neurons in DFBOs. RESULTS: In the 2D systems, we found dysregulated gene expression and chromatin accessibility affecting neuronal fate, adhesion, neurotransmission, and excitatory/inhibitory balance. Immunofluorescence of DFBOs corroborated altered proportions of radial glia and mature neuronal populations. Patch clamp recordings revealed decreased electrical activity in neurons from L48R DFBOs compared to control DFBOs. CONCLUSIONS: These data provide the first mechanistic insights into the pathogenesis of BLBS from a human-derived model of neurodevelopment, which suggest that H3.3 L48R increases H3-3B expression, resulting in the hyper-deposition of H3.3 into the nucleosome, which underlies changes in gene expression and chromatin accessibility. Functionally, this causes dysregulation of cell adhesion, neurotransmission, and the balance between excitatory and inhibitory signaling. These results are a crucial step towards preclinical development and testing of targeted therapies for this and related disorders.

Histones↗
PubMed1978

[Geometric complementarity of the primary structures of histone molecules H2A, H2B, H3, H4 and several possible consequences of this phenomenon].

The paper treats of the correlations between the position geometry of the extended successions of the residue of basic amino acids and those nonbearing any positive charge along the polypeptide chains of the histone H2A, H2B, H3, H4 molecules forming nucleosomes protein frame. The number of histone packing variants in the tetramere conforming to the conditions of the revealed complementarity reached the value of 10(4), and in respect to histone octamere--10(8). A hypothesis was put forward on structural heterogeneity of nucleosomes (of functional significance) the choice of whose protein frame packing variant could be realized by the mechanisms of allosteric regulation of the primary structure of DNA molecule section serving as the nucleosome element.

Chromosomes↗
PubMed2026

Developmental genetic determinants of the human cerebrospinal fluid-ventricular system.

Primary enlargement of the cerebrospinal fluid (CSF)-filled brain ventricles, known as congenital cerebral ventriculomegaly (CCV), is a hallmark of congenital hydrocephalus. CCV is also enigmatically but frequently associated with autism and other neurodevelopmental disorders. To gain insight into the developmental genetic regulation of the human CSF-ventricular system, we conducted an integrated, multiomic study of about 2700 trio-based exomes from patients with primary CCV. We found that about 25% of cases were associated with rare, damaging de novo variants in mutation-intolerant genes, many of which are linked to other dominant Mendelian disorders. Thirty-five exome-wide significant CCV genes and dozens of other high-confidence CCV genes converged on pathways involved in ATP-dependent Brahma-related gene 1/Brahma-associated factor chromatin remodeling, histone H3 lysine 4 methylation, and phosphoinositide 3-kinase signaling. Knockout of selected CCV genes in mouse models supported that de novo variants in CCV genes caused ventriculomegaly by impairing both CSF dynamics and cortical cytoarchitecture through dysregulation of neuroprogenitor cell growth and maturation in the ventricular and subventricular zones. These findings indicated that genetic and epigenetic programs coordinate the "hand-in-glove" development of the CSF-ventricular system with that of the cerebral cortex and establish a genetic connection between CCV and neurodevelopmental disorders, potentially explaining why some patients with hydrocephalus continue to exhibit CCV and neurodevelopmental disorders despite CSF shunting. We suggest that combined brain imaging and whole-exome sequencing could enable early detection of, and intervention for, autism and other neurodevelopmental disorders.

Humans↗
PubMed1978

The helical model of the nucleosome core.

A model of the nucleosome core is proposed based on a topologically linear array of histones attached sequentially to DNA. The linear complex folds helically forming a spring-like particle. Different variants of the particle are discussed (cylindrical springs with and without histone-histone contacts between turns of the helix, solenoidal spring). The model is consistent with known data about the nucleosome structure. Histones H3 and H4 have a special role in the model which is related also to the superstructure of chromatin.

Chromatin↗
PubMed1978

The DNA sequence of sea urchin (S. purpuratus) H2A, H2B and H3 histone coding and spacer regions.

The DNA sequence of two cloned segments of the histone gene repeat unit of the sea urchin S. purpuratus has been determined. One sequence contains the contiguous H2B and H3 genes and their interdigitated spacer regions; the other comprises the H2A gene and flanking spacer sequences. Analysis of the coding regions reveals a methionine residue within the H2A protein. H2A, which generally lacks this amino acid, contains methionine only in a protein variant which is synthesized in early sea urchin embryogenesis. We thus conclude that the cloned DNA represents a set of genes which is active early in development. Codon selection is markedly skewed and similar for each of the three genes. The DNA sequences are co-linear with known histone protein sequences and-unlike several other eucaryotic genes-do not show any insertions in the coding regions. The spacer regions are relatively AT-rich although GC cluster are scattered throughout. Several short stretches of homology are found in regions both upstream and downstream from the protein coding segments. The conservation of these sequences and their location at analogous sites suggest that they are involved in gene transcription or in mRNA translation. No tandem or dispersed repeats were found, with the exception of the remarkable sequence having the structure located in the spacer between the H2A and H1 genes.

Animals↗
PubMed1977

Studies of human histone messenger RNA. II. The resolution of fractions containing individual human histone messenger RNA species.

Polyribosomal 4 to 18 S RNA from S phase HeLa S-3 cells has been fractionated by chromatography on oligo(dT)-cellulose and resolved into multiple discrete components by continuous elution preparation electrophoresis. The human histone messenger RNA (mRNA) species associated with various polyadenylated [poly(A(+))] and nonpolyadenylated [poly(A(-))] components of 4 to 18 S RNA were determined by translation of these RNA fractions in vitro using a Krebs II ascites cell-free system followed by resolution of histones synthesized in vitro on polyacrylamide gels containing Triton X-100. The results of these studies indicate that poly(A(-)) 4 to 18 S RNA from S phase HeLa polyribosomes contains: (a) large quantities of discrete 7.4 and 8 S RNA species which are not functional histone mRNA; (b) a discrete 8.6 S RNA fraction which contains the templates of human histone H4; (c) 9.2 to 10.7 S RNA which contains mixtures of incompletely resolved histone H2B, H2A, and H3 mRNA (These mRNA species do not closely correspond to discrete RNA subfractions resolvable by our techniques.); (d) discrete 12 and 13 S RNA fractions which contain templates of human histone H1 polypeptides. The present studies also indicate that the mRNA templates of histone variants H3.2 and H3.3 have a slightly lower electrophoretic mobility than H3.1 mRNA and that H2A.2 mRNA has a slightly lower electrophoretic mobility than H2A.1 mRNA. In addition, appreciable quantities of H3.2, H3.3, and H2A.2 mRNA are bound to oligo(dT)-cellulose in 0.5 M KCl. These results indicate that mRNA species of the same histone class differ slightly in primary structure and are consistent with the hypothesis that some histone mRNA species contain short tracts of poly(A).

Chromatography, Affinity↗
PubMed2026

Establishment of a Common Marmoset Lineage Carrying a Frameshift Mutation in SETD1A, a Schizophrenia Risk Gene.

Appropriate histone modifications are essential for maintaining functional chromatin structure and gene expression, and dysfunction of their regulators has been linked to a variety of diseases. Among these modifications, trimethylation of lysine 4 on histone H3 (H3K4me3) is a well-characterized epigenetic mark enriched at transcription start sites of actively transcribed genes. H3K4me3 regulates gene transcription by recruiting transcription factors, facilitating chromatin accessibility, and preventing DNA methylation. In mammals, methylation of H3K4 is catalyzed by a family of histone methyltransferases including SET domain containing 1A (SETD1A), which is primarily responsible for genome-wide deposition of H3K4me2/3. Loss-of-function variants in SETD1A, highlighting its critical role in brain development and cognitive function, are strongly associated with schizophrenia (SCZ) and other neurodevelopmental disorders, but the underlying mechanisms remain largely unclear. To better understand the epigenetic and neurobiological consequences of SETD1A dysfunction, non-human primate models can serve as a useful tool because of their close evolutionary relationship to humans and highly developed cognitive abilities. In this study, we established a genetically engineered common marmoset (Callithrix jacchus) lineage carrying a frameshift mutation in SETD1A, which is, to the best of our knowledge, the first non-human primate lineage carrying a mutation in an epigenetic regulatory gene associated with SCZ, and confirmed germline transmission of the mutant allele. In a comparison between fibroblasts derived from one SETD1A mutant and one wild-type marmoset, the mutant showed a lower SETD1A protein level, modest differences in H3K4me3 deposition, and broader differences in gene expression profiles. Although these molecular observations require validation using additional biological replicates, the establishment of this SETD1A mutant marmoset lineage provides a valuable platform for bridging molecular mechanisms with primate neurobiology and for investigating the role of epigenetic regulation in the pathophysiology of neuropsychiatric and neurodevelopmental disorders.

Animals↗
PubMed2023

H3.3 contributes to chromatin accessibility and transcription factor binding at promoter-proximal regulatory elements in embryonic stem cells.

BACKGROUND: The histone variant H3.3 is enriched at active regulatory elements such as promoters and enhancers in mammalian genomes. These regions are highly accessible, creating an environment that is permissive to transcription factor binding and the recruitment of transcriptional coactivators that establish a unique chromatin post-translational landscape. How H3.3 contributes to the establishment and function of chromatin states at these regions is poorly understood. RESULTS: We perform genomic analyses of features associated with active promoter chromatin in mouse embryonic stem cells (ESCs) and find evidence of subtle yet widespread promoter dysregulation in the absence of H3.3. Loss of H3.3 results in reduced chromatin accessibility and transcription factor (TF) binding at promoters of expressed genes in ESCs. Likewise, enrichment of the transcriptional coactivator p300 and downstream histone H3 acetylation at lysine 27 (H3K27ac) is reduced at promoters in the absence of H3.3, along with reduced enrichment of the acetyl lysine reader BRD4. Despite the observed chromatin dysregulation, H3.3 KO ESCs maintain transcription from ESC-specific genes. However, upon undirected differentiation, H3.3 KO cells retain footprinting of ESC-specific TF motifs and fail to generate footprints of lineage-specific TF motifs, in line with their diminished capacity to differentiate. CONCLUSIONS: H3.3 facilitates DNA accessibility, transcription factor binding, and histone post-translational modification at active promoters. While H3.3 is not required for maintaining transcription in ESCs, it does promote de novo transcription factor binding which may contribute to the dysregulation of cellular differentiation in the absence of H3.3.

Animals↗
PubMed2025

Revisiting the question: When is a centromere not a kinetochore?

Centromeres have been the focus of extensive research for almost a century, so it may come as a surprise that a consistent definition and nomenclature for these structures remains elusive. In recent times, centromeric chromatin is most frequently defined by the presence of nucleosomes containing the H3 variant CENP-A and is typically synonymous with the site of the inner-kinetochore. However, crucial mammalian centromere proteins including CENP-B and INCENP have well defined distributions that show very little overlap with CENP-A. Additional protein localisations spanning the primary constriction or forming a band below CENP-A chromatin have been reported. Together, these observations suggest a complex and multi-layered chromatin organisation that is not well served by the canonical dichotomy of 'centromeric' and 'pericentromeric' chromatin. Strikingly, this is not a new observation but was made soon after the discovery of CENP proteins, including in a 1991 publication titled 'When is the centromere not a kinetochore?'. Here we revisit this question, which has become more pertinent following technical innovations in long-read sequencing and super-resolution microscopy. We present a model of centromere organisation for monocentromeres that incorporates additional complexity. We then use this model to reconceptualise diverse centromere forms in other eukaryotes including regional centromeres, holocentromeres and centromeres that lack key proteins including CENP-A. In this way, we hope to move towards a unified understanding of centromeric chromatin.

Centromere↗