Cycling off: a novel mechanism for differential cell cycle-dependent histone gene expression

In this Outlook, Hodkinson and Larschan discuss a study by Geisler et al. in this issue of Genes & Development that defines the role of Drosophila Mute in the precise control of replication-dependent histone gene expression during the cell cycle. The authors highlight the functions of the various factors within the histone locus body that regulate histone gene expression and drive pleiotropic phenotypes.

Abstract

The precise regulation of the replication-dependent histone genes during S phase is essential for packaging newly synthesized DNA and maintaining genome integrity. Identifying the roles of specialized factors that regulate histone biosynthesis is crucial for a comprehensive understanding of this tightly regulated process. In this issue of Genes & Development, Geisler and colleagues (doi:10.1101/gad.353837.126) investigate a previously identified repressor, Mute, and determine that it prevents the accumulation of histone mRNAs outside of S phase by antagonizing Mxc phosphorylation. They also demonstrate a novel mechanism where Mute differentially regulates histone transcript levels. Beyond the repressive mechanism of Mute, Geisler and colleagues highlight how factors can integrate multiple, distinct functions to meet the critical regulatory needs of the histone genes.

During every round of cell division, DNA replication and histone gene expression must be coupled to ensure proper organization of the newly duplicated genome. The canonical replication-dependent (RD) histone proteins form octamers called nucleosomes that package DNA synthesized during S phase. Therefore, precise, cell cycle-dependent regulation of histone biosynthesis is essential to maintain overall genomic integrity.

Factors that regulate histone gene expression are concentrated in a phase-separated biomolecular condensate known as the histone locus body (HLB). Since the discovery of the HLB, substantial research efforts have focused on identifying HLB components and defining their specific roles in the mechanism of RD histone biogenesis. In Drosophila, the histone locus is organized into a single series of ∼100 repetitive arrays, each containing the five RD histone genes (Bongartz and Schloissnig 2019), thereby facilitating the recruitment and localization of HLB factors to a single genomic region. RD histone gene transcription spikes at the start of S phase, followed by a rapid reduction in mRNA levels when DNA replication is inhibited (Harris et al. 1991), preventing accumulation outside S phase when histone proteins are no longer in high demand. Additionally, RD histone mRNAs lack introns and are nonpolyadenylated and therefore require specialized processing prior to translation. To meet these complex regulatory needs, the HLB concentrates a variety of distinct, specialized factors that play roles in HLB formation and stability, histone gene transcription, and mRNA processing.

Previous work has identified several factors that localize to the HLB and play defined roles in regulating RD histone biosynthesis. For example, the essential scaffolding factor Mxc (NPAT) localizes specifically to HLB foci and is phosphorylated by Cyclin E/CDK2 to activate histone gene expression at the beginning of S phase (White et al. 2011). Another well-defined HLB factor, FLASH, also localizes only to HLBs, associates directly with Mxc, and is necessary for RD histone mRNA 3′ end processing (Tatomer et al. 2016). Many screens for HLB components have identified regulators of histone gene transcription (White et al. 2011) and factors essential for the specialized mRNA processing (Wagner et al. 2007). Less is known about the mechanisms that directly repress RD histone gene expression; however, several candidate negative regulators have been reported.

In this issue of Genes & Development, Geisler et al. (2026) build on their group's well-established studies on HLB factors and further probe a previously identified repressor of RD histone gene expression, called Mute. Mute is a highly disordered protein that was first identified in muscle, where it colocalized with Lsm10 and Lsm11 and showed a genetic interaction with SLBP (stem–loop binding protein), all of which are essential for RD histone mRNA processing (Bulchand et al. 2010). Additional data suggested that Mute represses transcription of RD histone genes, as evidenced by higher levels of properly processed mRNAs in mute mutants (Bulchand et al. 2010). Geisler et al. (2026) expanded these findings by demonstrating that Mute colocalizes with Mxc, confirming that it is sequestered to the HLB and the RD histone genes alone, thereby further validating that Mute has a role as a histone gene regulator. While loss of Mute does not disrupt HLB formation, confirmed by uninterrupted Mxc and FLASH localization, it does increase nascent RD histone transcript levels, suggesting improper regulation of histone gene transcription. This is an important distinction in understanding the function of factors in the HLB, as mutations in Mxc, for example, cause misregulation of histone gene expression by destabilizing HLB formation as a whole rather than directly affecting transcript synthesis (White et al. 2011).

Restricting RD histone gene expression to S phase is a critical regulatory responsibility of the HLB. Geisler et al. (2026) determined that Mute plays an important role in restricting histone gene transcription to S phase. They demonstrated that, without Mute, RD histone mRNA persists into G2 and mitosis rather than exhibiting the normal sharp decrease in levels toward the end of S phase. The timing of histone gene transcription is controlled by Cyclin E/CDK2 phosphorylation of Mxc, and therefore changes to this mechanism can affect histone gene regulation (Ma et al. 2000). Geisler et al. (2026) go on to show that loss of Mute causes accumulation of phosphorylated Mxc in HLBs after S phase, which would initiate expression and residual RD histone mRNA outside of DNA synthesis. They hypothesize that Mute may be recruiting an inhibitor of Cyclin E/CDK2 that specifically limits the phosphorylation of Mxc to S phase, thereby restricting histone mRNA synthesis to S phase. This poses an important future direction for understanding HLB factor function. HLB factors may not always act alone in their role of regulating RD histone genes but instead recruit partners to achieve overall positive or negative effects on histone gene regulation.

Because the core RD histone proteins form a strict stoichiometric octamer complex, it is currently hypothesized that the five canonical histone genes are coregulated to maintain accurate protein concentrations. Importantly, Geisler et al. (2026) demonstrated that while constraining RD histone gene transcription to S phase, Mute also differentially impacts the expression of certain histone genes. They show that when Mute is mutated, the maximum transcriptional output of H4 and H3 differs from that of H1, H2A, and H2B. This is the first evidence of an HLB factor that regulates the RD histone genes independently rather than collectively and is a monumental discovery in understanding the mechanistic underpinnings of HLB. The data from Geisler et al. (2026) also present a unique view of how HLB factors can play multiple roles within the body. They show definitively that Mute aids in the repression and restriction of all RD histone gene transcription to S phase; however, they also determined that mutations in Mute impact certain RD histone mRNA outputs distinctly. It is likely that additional HLB members that have been defined to regulate histone biosynthesis can serve multiple functions within the HLB, and defining these roles is the only way to gain a nuanced, comprehensive understanding of histone gene regulation.

Many HLB factors were first described to have visible phenotypes that do not necessarily directly relate to histones but may instead be a subsequent consequence of improper histone biosynthesis. Mutations in Mxc were first described as polycomb, or multisex combs, phenotypes, but it was later determined that Mxc localizes only to the HLB, thereby establishing that this phenotype was a pleiotropic effect of misregulated RD histone gene expression (White et al. 2011; Terzo et al. 2015). Similarly, mutations in Mute were described to cause changes in muscle; however, Geisler et al. (2026) further investigated these Mute phenotypes, showing that they are also a downstream result of improperly regulated histone transcription rather than a direct effect on genes that impact muscle. This highlights an important distinction between defining the role of HLB factors in regulating histone gene expression and characterizing their indirect regulatory effects.

Overall, Geisler et al. (2026) established a more comprehensive understanding of the regulatory mechanisms by which Mute restricts histone gene expression to S phase and regulates the quantity of each histone gene's transcriptional output. This study provides critical insights into one of the repressive components of the HLB and the concept of differential regulation of the five RD histone genes. On a broader scale, this study suggests that accounting for factors within a biomolecular condensate that may play distinct, independent roles is an important consideration when defining their mechanism of action.