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Results for “Histone H3.3”

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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↗

PubMed2025

Chromatin assembly by the histone chaperone HIRA facilitates Human Papillomavirus replication.

The circular, double-stranded DNA genomes of Human papillomaviruses (HPV) exist in a nucleosomal state throughout the infectious cycle and rely on host histone epigenetic modifications and chromatin assembly processes to promote various phases of the viral life cycle. Here, we show that the histone H3.3 chaperone HIRA and its associated complex members are recruited to HPV replication factories during the late phase of the HPV life cycle. HIRA is also recruited to HPV replication factories generated by amplification of a replicon with a minimal origin and expression of the viral replication proteins E1 and E2, demonstrating that the E1 and E2 proteins are sufficient for HIRA recruitment. Downregulation of HIRA expression reduces HPV31 DNA amplification and viral transcription in differentiated keratinocytes. Histone H3.3 that is highly phosphorylated on serine residue 31 is also enriched at sites of HPV replication and this modification links the DNA damage response to chromatin that supports rapid gene activation. We propose that deposition of histone H3.3 generates viral minichromosomes that are highly primed to support the late stages of the HPV life cycle.

H3.3 phosphorylation↗

PubMed1992

Netropsin specifically enhances RNA polymerase II termination at terminator sites in vitro.

We describe an in vitro system that emulates the specific and efficient transcriptional termination associated with the human gastrin gene terminator in vivo. The system involves a dC-tailed DNA template containing the gastrin gene terminator sequence, purified RNA polymerase II, and purified elongation factor TFIIS. In this system, the basal level of termination by RNA polymerase II at the gastrin gene terminator is specifically enhanced by netropsin, an (A + T)-rich minor groove-binding peptide. This enhanced termination is maintained even with TFIIS, which normally suppresses termination at this site. In vitro termination is terminator sequence-specific. Mutant sequences that reduce or abolish termination in vivo show corresponding reductions in activity in the in vitro system. This in vitro emulation of in vivo activities of wild-type and mutant terminators strongly suggests that netropsin and a putative termination factor may share some aspects of their biochemical mechanisms. The general applicability of this system to the study of RNA polymerase II elongation and termination is suggested by the enhancement of termination seen at both the gastrin and human histone H3.3 gene terminators.

Base Sequence↗

PubMed1992

cDNA cloning of Entamoeba histolytica histone H3.

E. histolytica has an unusual nuclear structure. Although nucleosome-like particles were observed, no information about histones was available so far. In this paper we describe a cDNA clone with significant homology to H3 histones that was isolated from a lambda Zap library of pathogenic E. histolytica. The complete cDNA encodes a 15 kDa polypeptide, which is shorter by one residue than the human homologue. The amino acid sequence has only 69% identity with human H3.3 histone. Our results indicate that this divergence may contribute to the unusual chromatin structure of E. histolytica.

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↗

PubMed2026

Cohesin prevents local mixing of condensed euchromatic domains in living human cells.

The human genome is folded into chromatin loops by the cohesin complex, forming functional chromatin domains that underlie transcription and DNA replication/repair. However, how cohesin organizes these domains in living cells, especially in active euchromatin, remains elusive. Here, to address this question, we combined single-nucleosome imaging/tracking and super-resolution three-dimensional structured illumination microscopy with euchromatin-specific labeling of histone variant H3.3. Using this nanoscopic approach, we revealed that euchromatin forms condensed domains that are constrained by cohesin-mediated loops. This organization refines the classical view of euchromatin as largely open, in line with emerging evidence. Transcription machinery appears to be located near the condensed domain surfaces/borders. Cohesin loss increased nucleosome-level fluidity within these domains without altering their overall compaction, leading to local mixing of domains and compromising transcriptional insulation. These findings suggest a physical role of cohesin in maintaining the integrity of condensed euchromatic domains and ensuring proper higher-order regulation of gene expression.

Humans↗

PubMed2025

Identification of Plant Chromatin Interaction Networks Using IP-MS and co-IP.

Proteins often act in concert to perform their function. Thus, the identification of protein complexes is crucial if we want to understand how they work. In this chapter, we present a highly sensitive protocol for the immunoprecipitation of nuclear chromatin-linked proteins in Arabidopsis thaliana that does not rely on time-consuming nuclei extraction. Interaction partners are identified using mass spectrometry and confirmed by co-immunoprecipitation. To help solubilize chromatin-bound proteins and eliminate nonspecific interactions of proteins binding the same DNA stretch, we include an enzymatic digestion step to remove DNA before immunoprecipitation. Our protocol offers a simplified process using optimized buffers, which facilitates quick and effective immunoprecipitation. The outcome is high-quality eluates that are ideal for identifying proteins through MS.

Chromatin↗

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↗