Redesigning the Drosophila histone gene cluster: An improved genetic platform for spatiotemporal manipulation of histone function.

Mutating replication-dependent (RD) histone genes is an important tool for understanding chromatin-based epigenetic regulation. Deploying this tool in metazoan models is particularly challenging because RD histones in these organisms are typically encoded by many genes, often located at multiple loci. Such RD histone gene arrangements make the ability to generate homogenous histone mutant genotypes by site-specific gene editing quite difficult. Drosophila melanogaster provides a solution to this problem because the RD histone genes are organized into a single large tandem array that can be deleted and replaced with transgenes containing mutant histone genes. In the last ∼15 years several different RD histone gene replacement platforms have been developed using this simple strategy. However, each platform contains weaknesses that preclude full use of the powerful developmental genetic capabilities available to Drosophila researchers. Here we describe the development of a newly engineered platform that rectifies many of these weaknesses. We used CRISPR to precisely delete the RD histone gene array ( HisC ), replacing it with a multifunctional cassette that permits site-specific insertion of either one or two synthetic gene arrays using selectable markers. We designed this cassette with the ability to selectively delete each of the integrated gene arrays in specific tissues using site-specific recombinases. We also present a method for rapidly synthesizing histone gene arrays of any genotype using Golden Gate cloning technologies. These improvements facilitate generation of histone mutant cells in various tissues at different stages of Drosophila development and provide an opportunity to apply forward genetic strategies to interrogate chromatin structure and gene regulation.

Drosophila melanogaster Set8 and L(3)mbt function in gene expression independently of histone H4 lysine 20 methylation.

Mono-methylation of Lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by H4K20me-binding proteins. We interrogated this model in a developing animal by comparing in parallel the transcriptomes of Set8 null , H4 K20R/A , and l(3)mbt mutant Drosophila melanogaster . We found that the gene expression profiles of H4 K20A and H4 K20R larvae are markedly different than Set8 null larvae despite similar reductions in H4K20me1. Set8 null mutant cells have a severely disrupted transcriptome and fail to proliferate in vivo , but these phenotypes are not recapitulated by mutation of H4 K20 indicating that the developmental defects of Set8 null animals are largely due to H4K20me1-independent effects on gene expression. Further, the H4K20me1 binding protein L(3)mbt is recruited to the transcription start sites of most genes independently of H4K20me even though genes bound by L(3)mbt have high levels of H4K20me1. Moreover, both Set8 and L(3)mbt bind to purified H4K20R nucleosomes in vitro. We conclude that gene expression changes in Set8 null and H4 K20 mutants cannot be explained by loss of H4K20me1 or L(3)mbt binding to chromatin, and therefore that H4K20me1 does not play a large role in gene expression.

Histone locus bodies: a paradigm for how nuclear biomolecular condensates control cell cycle regulated gene expression.

Histone locus bodies (HLBs) are biomolecular condensates that assemble at replication-dependent (RD) histone genes in animal cells. These genes produce unique mRNAs that are not polyadenylated and instead end in a conserved 3' stem loop critical for coordinated production of histone proteins during S phase of the cell cycle. Several evolutionarily conserved factors necessary for synthesis of RD histone mRNAs concentrate only in the HLB. Moreover, because HLBs are present throughout the cell cycle even though RD histone genes are only expressed during S phase, changes in HLB composition during cell cycle progression drive much of the cell cycle regulation of RD histone gene expression. Thus, HLBs provide a powerful opportunity to determine the cause-and-effect relationships between nuclear body formation and cell cycle regulated gene expression. In this review, we focus on progress during the last five years that has advanced our understanding of HLB biology.

Coordinated expression of replication-dependent histone genes from multiple loci promotes histone homeostasis in Drosophila.

Production of large amounts of histone proteins during S phase is critical for proper chromatin formation and genome integrity. This process is achieved in part by the presence of multiple copies of replication dependent (RD) histone genes that occur in one or more clusters in metazoan genomes. In addition, RD histone gene clusters are associated with a specialized nuclear body, the histone locus body (HLB), which facilitates efficient transcription and 3' end-processing of RD histone mRNA. How all five RD histone genes within these clusters are coordinately regulated such that neither too few nor too many histones are produced, a process referred to as histone homeostasis, is not fully understood. Here, we explored the mechanisms of coordinate regulation between multiple RD histone loci in Drosophila melanogaster and Drosophila virilis. We provide evidence for functional competition between endogenous and ectopic transgenic histone arrays located at different chromosomal locations in D. melanogaster that helps maintain proper histone mRNA levels. Consistent with this model, in both species we found that individual histone gene arrays can independently assemble an HLB that results in active histone transcription. Our findings suggest a role for HLB assembly in coordinating RD histone gene expression to maintain histone homeostasis.

Reduced histone gene copy number disrupts Drosophila Polycomb function.

The chromatin of animal cells contains two types of histones: canonical histones that are expressed during S phase of the cell cycle to package the newly replicated genome, and variant histones with specialized functions that are expressed throughout the cell cycle and in non-proliferating cells. Determining whether and how canonical and variant histones cooperate to regulate genome function is integral to understanding how chromatin-based processes affect normal and pathological development. Here, we demonstrate that variant histone H3.3 is essential for Drosophila development only when canonical histone gene copy number is reduced, suggesting that coordination between canonical H3.2 and variant H3.3 expression is necessary to provide sufficient H3 protein for normal genome function. To identify genes that depend upon, or are involved in, this coordinate regulation we screened for heterozygous chromosome 3 deficiencies that impair development of flies bearing reduced H3.2 and H3.3 gene copy number. We identified two regions of chromosome 3 that conferred this phenotype, one of which contains the Polycomb gene, which is necessary for establishing domains of facultative chromatin that repress master regulator genes during development. We further found that reduction in Polycomb dosage decreases viability of animals with no H3.3 gene copies. Moreover, heterozygous Polycomb mutations result in de-repression of the Polycomb target gene Ubx and cause ectopic sex combs when either canonical or variant H3 gene copy number is reduced. We conclude that Polycomb-mediated facultative heterochromatin function is compromised when canonical and variant H3 gene copy number falls below a critical threshold.

Reduced histone gene copy number disrupts Drosophila Polycomb function.

The chromatin of animal cells contains two types of histones: canonical histones that are expressed during S phase of the cell cycle to package the newly replicated genome, and variant histones with specialized functions that are expressed throughout the cell cycle and in non-proliferating cells. Determining whether and how canonical and variant histones cooperate to regulate genome function is integral to understanding how chromatin-based processes affect normal and pathological development. Here, we demonstrate that variant histone H3.3 is essential for Drosophila development only when canonical histone gene copy number is reduced, suggesting that coordination between canonical H3.2 and variant H3.3 expression is necessary to provide sufficient H3 protein for normal genome function. To identify genes that depend upon, or are involved in, this coordinate regulation we screened for heterozygous chromosome 3 deficiencies that impair development of flies bearing reduced H3.2 and H3.3 gene copy number. We identified two regions of chromosome 3 that conferred this phenotype, one of which contains the Polycomb gene, which is necessary for establishing domains of facultative chromatin that repress master regulator genes during development. We further found that reduction in Polycomb dosage decreases viability of animals with no H3.3 gene copies. Moreover, heterozygous Polycomb mutations result in de-repression of the Polycomb target gene Ubx and cause ectopic sex combs when either canonical or variant H3 gene copy number is also reduced. We conclude that Polycomb-mediated facultative heterochromatin function is compromised when canonical and variant H3 gene copy number falls below a critical threshold.

Distinct roles for canonical and variant histone H3 lysine-36 in Polycomb silencing.

Polycomb complexes regulate cell type-specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. We show that mutants of Drosophila replication-dependent (H3.2K36R) or replication-independent (H3.3K36R) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined (H3.3K36RH3.2K36R) mutants display widespread Hox gene misexpression and fail to develop past the first larval stage. Chromatin profiling revealed that the H3.2K36R mutation disrupts H3K27me3 levels broadly throughout silenced domains, whereas these regions are mostly unaffected in H3.3K36R animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive Polycomb response elements located outside of silenced domains but relatively depleted from those inside. We conclude that H3.2 and H3.3 K36 residues collaborate to repress Hox genes using different mechanisms.

Drosophila SUMM4 complex couples insulator function and DNA replication control.

Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underreplicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here, we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier, and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins.

Inside cells, molecules of DNA provide the instructions needed to make proteins. Cells carefully maintain and repair their DNA, and typically make a complete copy of the genome before they divide to ensure that after division, each daughter cell has a full set. Within human, fly and other eukaryotic nuclei, DNA is packaged into structures known as chromosomes. Cells follow precisely controlled programs to replicate distinct regions of chromosomes at different times. To start copying a particular region, the cell machinery that replicates DNA binds to a sequence known as the origin of replication. It is thought that as-yet unknown cues from the cell may lead the replication machinery to bind to different origins of replication at different times. In some circumstances, cells make extra copies of their DNA without dividing. For example, many cells in the larvae of fruit flies contain hundreds of extra DNA copies to sustain their increased sizes. However, the entire genome is not copied during this process, so cells end up with more copies of some regions of the genome than others. A protein called SUUR is required for hindering the replication of the 'underrepresented' regions, but it is not clear how it works. To address this question, Andreyeva, Emelyanov et al. developed a new approach based on liquid chromatography and quantitative proteomics to identify the native form of SUUR in fruit flies. This revealed that SUUR exists as a stable complex with a protein called Mod(Mdg4), which is needed to recruit SUUR to the chromosomes. Further experiments suggested that SUUR and Mod(Mdg4) work together to bind to regions of DNA known as gypsy insulator elements, creating a physical barrier that hinders the replication machinery from accessing some parts of the genome. The findings of Andreyeva, Emelyanov et al. provide an alternative explanation for how individual cells may stagger the process of copying their DNA without relying on the replication machinery binding to various replication origins at different times. Rather, late replication timing may be instructed by an insulator-born delay of the progression of replication over particular genomic regions. This mechanism adds to the list of nuclear processes (chromosome partitioning, transcriptional regulation, etc.) that are known to be directed by insulators and associated architectural proteins.

Coordinating transcription and replication to mitigate their conflicts in early Drosophila embryos.

Collisions between transcribing RNA polymerases and DNA replication forks are disruptive. The threat of collisions is particularly acute during the rapid early embryonic cell cycles of Drosophila when S phase occupies the entirety of interphase. We hypothesize that collision-avoidance mechanisms safeguard this early transcription. Real-time imaging of endogenously tagged RNA polymerase II (RNAPII) and a reporter for nascent transcripts in unperturbed embryos shows clustering of RNAPII at around 2 min after mitotic exit, followed by progressive dispersal as associated nascent transcripts accumulate later in interphase. Abrupt inhibition of various steps in DNA replication, including origin licensing, origin firing, and polymerization, suppresses post-mitotic RNAPII clustering and transcription in nuclear cycles. We propose that replication dependency defers the onset of transcription so that RNAPII transcribes behind advancing replication forks. The resulting orderly progression can explain how early embryos circumvent transcription-replication conflicts to express essential developmental genes.

Distinct developmental phenotypes result from mutation of Set8/KMT5A and histone H4 lysine 20 in Drosophila melanogaster.

Mono-methylation of histone H4 lysine 20 (H4K20me1) is catalyzed by Set8/KMT5A and regulates numerous aspects of genome organization and function. Loss-of-function mutations in Drosophila melanogaster Set8 or mammalian KMT5A prevent H4K20me1 and disrupt development. Set8/KMT5A also has non-histone substrates, making it difficult to determine which developmental functions of Set8/KMT5A are attributable to H4K20me1 and which to other substrates or to non-catalytic roles. Here, we show that human KMT5A can functionally substitute for Set8 during Drosophila development and that the catalytic SET domains of the two enzymes are fully interchangeable. We also uncovered a role in eye development for the N-terminal domain of Set8 that cannot be complemented by human KMT5A. Whereas Set820/20 null mutants are inviable, we found that an R634G mutation in Set8 predicted from in vitro experiments to ablate catalytic activity resulted in viable adults. Additionally, Set8(R634G) mutants retain significant, albeit reduced, H4K20me1, indicating that the R634G mutation does not eliminate catalytic activity in vivo and is functionally hypomorphic rather than null. Flies engineered to express only unmodifiable H4 histones (H4K20A) can also complete development, but are phenotypically distinct from H4K20R, Set820/20 null, and Set8R634G mutants. Taken together, our results demonstrate functional conservation of KMT5A and Set8 enzymes, as well as distinct roles for Set8 and H4K20me1 in Drosophila development.

The awesome power of histone genetics.

Using a genetic platform to generate histone mutants in Drosophila, Regadas et al. (2021) discover a novel mechanism for tissue-specific gene expression requiring a chromatin state defined by acetylation of lysine 14 of H3 but lacking other activating histone post-translational modifications.

Superresolution light microscopy of the Drosophila histone locus body reveals a core-shell organization associated with expression of replication-dependent histone genes.

The histone locus body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of replication-dependent (RD) histone mRNAs, which are the only eukaryotic mRNAs lacking a poly-A tail. Many nuclear bodies contain distinct domains, but how internal organization is related to nuclear body function is not fully understood. Here, we demonstrate using structured illumination microscopy that Drosophila HLBs have a "core-shell" organization in which the internal core contains transcriptionally active RD histone genes. The N-terminus of Mxc, which contains a domain required for Mxc oligomerization, HLB assembly, and RD histone gene expression, is enriched in the HLB core. In contrast, the C-terminus of Mxc is enriched in the HLB outer shell as is FLASH, a component of the active U7 snRNP that cotranscriptionally cleaves RD histone pre-mRNA. Consistent with these results, we show biochemically that FLASH binds directly to the Mxc C-terminal region. In the rapid S-M nuclear cycles of syncytial blastoderm Drosophila embryos, the HLB disassembles at mitosis and reassembles the core-shell arrangement as histone gene transcription is activated immediately after mitosis. Thus, the core-shell organization is coupled to zygotic histone gene transcription, revealing a link between HLB internal organization and RD histone gene expression.

Deciding when to exit.

A new imaging approach can distinguish between cells destined to stop proliferating and those committed to re-entering the cell cycle in live animals.

CDK-Regulated Phase Separation Seeded by Histone Genes Ensures Precise Growth and Function of Histone Locus Bodies.

Many membraneless organelles form through liquid-liquid phase separation, but how their size is controlled and whether size is linked to function remain poorly understood. The histone locus body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of histone mRNAs. Here, we show that Drosophila HLBs form through phase separation. During embryogenesis, the size of HLBs is controlled in a precise and dynamic manner that is dependent on the cell cycle and zygotic histone gene activation. Control of HLB growth is achieved by a mechanism integrating nascent mRNAs at the histone locus, which facilitates phase separation, and the nuclear concentration of the scaffold protein multi-sex combs (Mxc), which is controlled by the activity of cyclin-dependent kinases. Reduced Cdk2 activity results in smaller HLBs and the appearance of nascent, misprocessed histone mRNAs. Thus, our experiments identify a mechanism linking nuclear body growth and size with gene expression.

Drosophila histone locus body assembly and function involves multiple interactions.

The histone locus body (HLB) assembles at replication-dependent (RD) histone loci and concentrates factors required for RD histone mRNA biosynthesis. The Drosophila melanogaster genome has a single locus comprised of ∼100 copies of a tandemly arrayed 5-kB repeat unit containing one copy of each of the 5 RD histone genes. To determine sequence elements required for D. melanogaster HLB formation and histone gene expression, we used transgenic gene arrays containing 12 copies of the histone repeat unit that functionally complement loss of the ∼200 endogenous RD histone genes. A 12x histone gene array in which all H3-H4 promoters were replaced with H2a-H2b promoters (12xPR) does not form an HLB or express high levels of RD histone mRNA in the presence of the endogenous histone genes. In contrast, this same transgenic array is active in HLB assembly and RD histone gene expression in the absence of the endogenous RD histone genes and rescues the lethality caused by homozygous deletion of the RD histone locus. The HLB formed in the absence of endogenous RD histone genes on the mutant 12x array contains all known factors present in the wild-type HLB including CLAMP, which normally binds to GAGA repeats in the H3-H4 promoter. These data suggest that multiple protein-protein and/or protein-DNA interactions contribute to HLB formation, and that the large number of endogenous RD histone gene copies sequester available factor(s) from attenuated transgenic arrays, thereby preventing HLB formation and gene expression on these arrays.

Rif1 Functions in a Tissue-Specific Manner To Control Replication Timing Through Its PP1-Binding Motif.

Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early- and late-replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, we utilized the powerful developmental systems available in Drosophila melanogaster We generated and compared RT profiles from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases are largely independent of transcriptional differences. We also employed a genetic approach in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs.

Phasing in heterochromatin during development.

Constitutive heterochromatin is a prevalent feature of eukaryotic genomes important for promoting cell differentiation and maintaining genome stability. During animal reproduction, constitutive heterochromatin is disassembled in gametes prior to formation of the zygote and then subsequently re-established as development ensues and cells differentiate. Despite progress in understanding the mechanisms that maintain heterochromatin in differentiated cell types, how constitutive heterochromatin is assembled de novo during early development remains poorly understood. In this issue of Genes & Development, Seller and colleagues (pp. 403-417) develop a new technology for inhibiting maternal gene function to identify the H3K9 methyltransferase necessary for initiating constitutive heterochromatin formation during early Drosophila embryogenesis.

H3K9 Promotes Under-Replication of Pericentromeric Heterochromatin in Drosophila Salivary Gland Polytene Chromosomes.

Chromatin structure and its organization contributes to the proper regulation and timing of DNA replication. Yet, the precise mechanism by which chromatin contributes to DNA replication remains incompletely understood. This is particularly true for cell types that rely on polyploidization as a developmental strategy for growth and high biosynthetic capacity. During Drosophila larval development, cells of the salivary gland undergo endoreplication, repetitive rounds of DNA synthesis without intervening cell division, resulting in ploidy values of ~1350C. S phase of these endocycles displays a reproducible pattern of early and late replicating regions of the genome resulting from the activity of the same replication initiation factors that are used in diploid cells. However, unlike diploid cells, the latest replicating regions of polyploid salivary gland genomes, composed primarily of pericentric heterochromatic enriched in H3K9 methylation, are not replicated each endocycle, resulting in under-replicated domains with reduced ploidy. Here, we employ a histone gene replacement strategy in Drosophila to demonstrate that mutation of a histone residue important for heterochromatin organization and function (H3K9) but not mutation of a histone residue important for euchromatin function (H4K16), disrupts proper endoreplication in Drosophila salivary gland polyploid genomes thereby leading to DNA copy gain in pericentric heterochromatin. These findings reveal that H3K9 is necessary for normal levels of under-replication of pericentric heterochromatin and suggest that under-replication at pericentric heterochromatin is mediated through H3K9 methylation.

Lysine 27 of replication-independent histone H3.3 is required for Polycomb target gene silencing but not for gene activation.

Proper determination of cell fates depends on epigenetic information that is used to preserve memory of decisions made earlier in development. Post-translational modification of histone residues is thought to be a central means by which epigenetic information is propagated. In particular, modifications of histone H3 lysine 27 (H3K27) are strongly correlated with both gene activation and gene repression. H3K27 acetylation is found at sites of active transcription, whereas H3K27 methylation is found at loci silenced by Polycomb group proteins. The histones bearing these modifications are encoded by the replication-dependent H3 genes as well as the replication-independent H3.3 genes. Owing to differential rates of nucleosome turnover, H3K27 acetylation is enriched on replication-independent H3.3 histones at active gene loci, and H3K27 methylation is enriched on replication-dependent H3 histones across silenced gene loci. Previously, we found that modification of replication-dependent H3K27 is required for Polycomb target gene silencing, but it is not required for gene activation. However, the contribution of replication-independent H3.3K27 to these functions is unknown. Here, we used CRISPR/Cas9 to mutate the endogenous replication-independent H3.3K27 to a non-modifiable residue. Surprisingly, we find that H3.3K27 is also required for Polycomb target gene silencing despite the association of H3.3 with active transcription. However, the requirement for H3.3K27 comes at a later stage of development than that found for replication-dependent H3K27, suggesting a greater reliance on replication-independent H3.3K27 in post-mitotic cells. Notably, we find no evidence of global transcriptional defects in H3.3K27 mutants, despite the strong correlation between H3.3K27 acetylation and active transcription.