Novel Eukaryotic Double Histone Fold Motifs Sharing High Sequence Identity with Histones H3 and H4 in Single-Domain Proteins and in Proteins Featuring Remarkable Histone Fold Multiplets

G-quadruplex structures act as a novel recognition motif for the meiosis-specific histone methyltransferase PRDM9

The histone methyltransferase PR domain containing protein 9 (PRDM9) is a key determinant of meiotic recombination in humans. It deposits activating histone marks thereby promoting recruitment of the meiotic recombination machinery. It recognizes DNA through a repetitive zinc-finger array that binds specific sequence motifs whose complementary G-rich strands can form DNA secondary structures, particularly G-quadruplexes (G4s). These may present an additional binding platform for PRDM9 and contribute to the formation of a chromatin environment permissive for meiotic recombination. We investigated the relationship between PRDM9 binding sites and G4 motifs using computational analyses of predicted and experimentally validated G4s and found that G4 motifs are among the most prevalent features at PRDM9 binding sites, with the strongest enrichment observed for highly stable G4s, largely independent of loop length. Using electrophoretic mobility shift assays, we further examined whether PRDM9 can bind short, single-stranded G4-forming oligonucleotides in addition to its canonical double-stranded DNA targets. PRDM9 directly bound folded G4 structures, and binding increased with G4 stability. This relationship was observed across different G4 motifs and following stabilization of the same G4 by increasing the potassium concentration or adding a G4-stabilizing ligand. PRDM9 also bound an artificial G4-forming sequence absent from the human genome, which was abolished when mutating the G4 motif to avoid structure formation. Together, these results support a model in which stable G4 structures facilitate PRDM9 recruitment by creating and discrete increased local chromatin accessibility, thereby contributing to the initiation of meiotic recombination. Graphical abstract

Reading the maps: Organization and function of chromatin types in Drosophila

Single-Nucleosome Mapping of Histone Modifications in S. cerevisiae

Identifying critical lysines in mammalian histone H3 with high-throughput CRISPR prime editing.

Histone post-translational modifications are fundamental to genome regulation, yet dissecting the functions of individual histone marks in mammals remains challenging due to the presence of multiple histone gene copies. Here we develop a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) prime editing platform enabling precise, reversible and combinatorial mutagenesis of canonical and noncanonical histone H3 genes within their native genomic context. Using systematic lysine-to-arginine substitutions benchmarked against synonymous controls, we identify key residues, including H3K4, H3K9, H3K14, H3K18 and H3K79, whose mutation compromises fitness in mouse embryonic stem cells. We further show that H3K56, linked to genome stability in yeast and Drosophila, has a conserved role in mammalian cells. Through analysis of selected double mutants, we uncover functional crosstalk across residues, with combinations such as H3K27R + H3K36R impairing stem cell self-renewal and altering transcription. Altogether, this study establishes a functional map of histone H3 lysines in mammals and provides a broadly applicable platform for systematic dissection of chromatin regulation.

Are components of the histone gene expression machinery functionally repurposed in terminally differentiated cells?

The expression of metazoan replication-dependent histone genes is controlled by NPAT and U7 snRNP. NPAT activates transcription of histone genes during S-phase, whereas U7 snRNP is a multi-subunit endonuclease that cleaves the resultant transcripts at the 3' end, yielding mature histone mRNAs. In cycling cells, NPAT and U7 snRNP with its four unique components, U7 snRNA, Lsm10, Lsm11 and FLASH, are highly enriched in Histone Locus Bodies (HLBs), nuclear condensates formed near histone gene loci. Here, we show that in muscle and neural cells that have ceased to replicate their chromatin and permanently exited the cell cycle, HLBs are dismantled and NPAT, FLASH and Lsm11 are detected in the cytoplasm. This observation suggests that in postmitotic cells, NPAT and U7 snRNP become repurposed for functions unrelated to generating histone mRNAs. We identified a highly conserved region in Lsm11 that engages in various protein-protein interactions and likely acts as a universal platform that controls the assembly, localization and function of Lsm11 complexes, including U7 snRNP, during cell growth and differentiation. Since the assembly of U7 snRNP requires SMN, the protein mutated in spinal muscular atrophy (SMA), our results may provide a new perspective on pathophysiology of this neuromuscular disorder.

Adventures through wonderland: the wild world of histone gene organization, evolution, and regulation in over 250 species.

Histone proteins are critical for the compaction and organization of DNA. In eukaryotes, these proteins are typically encoded by a multigene family. Replication-dependent histones are highly conserved, both in their amino acid sequence and in their S-phase linked expression. Despite striking protein and regulatory similarities, histone genes often reside in wildly different organizations within genomes, even those of closely related species. At the same time, distantly-related species sometimes share organizational themes. How each species similarly regulates histone genes despite organizational differences remains unclear, and a multi-species approach is necessary to address this gap in knowledge. Here, we distill, categorize, and describe the histone gene organizations of over 250 species spanning 11 phyla. Our comprehensive curation of histone gene organizations provides new insights into the divergence and evolutionary changes of this ancient, conserved gene family. Additionally, we suggest that histone gene organization may contribute to subsequent regulation, as well as misregulation in disease.