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. 2023 Oct 31;34(12):ar118. doi: 10.1091/mbc.E22-11-0532

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

Ashlesha Chaubal a, Justin M Waldern b, Colin Taylor b, Alain Laederach b,c, William F Marzluff a,b,c,d, Robert J Duronio a,b,c,e,*
Editor: Tom Mistelif
PMCID: PMC10846616  PMID: 37647143

Abstract

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.

INTRODUCTION

Nucleosomes containing an octamer of histone proteins constitute the fundamental building blocks of chromatin and regulate access to, and expression of, the information within eukaryotic genomes. Generating sufficient H2a, H2b, H3, and H4 histone proteins in the correct stoichiometric amounts to assemble nucleosomes during S phase of the cell cycle is imperative for properly packaging the newly replicated DNA and is critical for normal-genome function and stability. Disruptions to this process resulting in either a deficit of histones during S-phase or an accumulation of excess, nonnucleosomal histones can have detrimental effects on cell viability. For example, depletion of H2B or H4 in yeast causes mitotic arrest and disruption of chromosome segregation (Han et al., 1987; Kim et al., 1988), as does reduction of maternal levels of all four core-histone mRNAs in early Drosophila embryos (Sullivan et al., 2001). Similarly, in human cells inhibition of histone protein expression by knockdown of Stem Loop Binding Protein (SLBP), a factor important for histone mRNA 3′ end processing and translation (Wagner et al., 2005; Sullivan et al., 2009), or inhibition of histone incorporation into chromatin by depletion of the histone chaperone CAF-1 (Ye et al., 2003a), results in S phase arrest. Thus, S phase cells need to rapidly produce large amounts of histones and deposit them onto replicating DNA to maintain proper-chromatin structure. Conversely, excess positively charged histones can bind nonspecifically to nucleic acids forming aggregates or sequester histone-binding proteins, thereby resulting in cytotoxicity (Singh et al., 2010). Overexpression of histone genes in budding yeast causes an increased rate of chromosome loss (Meeks-Wagner and Hartwell, 1986; Au et al., 2008) likely by overwhelming the active-degradation system that removes excess histones (Gunjan and Verreault, 2003). Furthermore, excess histones increase sensitivity to DNA damage in budding yeast by interfering with the homologous-recombination machinery (Liang et al., 2012). In early Xenopus and zebrafish embryos, which store large amounts of histone proteins on chaperones, addition of excess histones delays activation of zygotic transcription (Amodeo et al., 2015; Joseph et al., 2017).

All these studies indicate that cells must maintain a balance of not too many or not too few histones, a process referred to as histone homeostasis. Achieving histone homeostasis occurs through several mechanisms, including the cell-cycle regulation of replication-dependent (RD) histone mRNA synthesis via transcription and pre-mRNA processing (Heintz et al., 1983; Harris et al., 1991; Whitfield et al., 2000; Marzluff et al., 2008). The metazoan RD histone mRNAs are the only eukaryotic cellular mRNAs that are not polyadenylated, ending instead in a conserved stem-loop that binds SLBP (Marzluff et al., 2008; Marzluff and Koreski, 2017). Once synthesized histone mRNAs continue to be coordinately regulated in the cytoplasm because SLBP only binds to the 3′ end of RD histone mRNAs (Brooks et al., 2015). SLBP is required for nuclear export (Sullivan et al., 2009) and translation (Sanchez and Marzluff, 2002), and the 3′ end is also the signal for coordinate RD histone mRNA degradation at the end of S phase or when DNA synthesis is inhibited (Pandey and Marzluff, 1987; Meaux et al., 2018). Once synthesized, histone proteins are also efficiently incorporated into chromatin (Mendiratta et al., 2019), and excess histones are degraded (Gunjan and Verreault, 2003). Here, we provide evidence that an additional mechanism for achieving histone homeostasis in Drosophila involves modulation of histone mRNA accumulation in response to differing numbers of histone genes at different genomic loci.

Histones are categorized into two classes, RD or canonical histones and replication-independent histone variants (Talbert and Henikoff, 2017). RD histones comprise the bulk of histones in chromatin and their synthesis is tightly coupled to the cell cycle, only being produced during S phase, whereas variant–histone expression is not coupled to the cell cycle and their location in the genome is reflective of their function (e.g., centromeric histone H3, H3.3, or H2a.Z). Eukaryotic organisms contain multiple copies of RD histone genes, and in metazoans the genes encoding the five RD histone proteins are clustered together at one or more loci (Lifton et al., 1978; Maxson et al., 1983; Marzluff et al., 2002). In D. melanogaster, there is a single RD histone locus on chromosome 2L (HisC) where a 5-kb unit containing one copy of each RD histone gene is tandemly repeated ∼ 100 times (McKay et al., 2015; Bongartz and Schloissnig, 2019). The evolutionarily conserved clustering of RD histone genes almost certainly contributes to the coordinated expression of all five histones and ensures rapid activation of histone gene expression at the beginning of S phase. Clusters of RD histone genes in metazoans are also associated with a phase-separated nuclear body called the Histone Locus Body (HLB; Duronio and Marzluff, 2017), which promotes efficient pre-mRNA processing resulting in the nonpolyadenylated RD histone mRNAs (Tatomer et al., 2016; Hur et al., 2020). The HLB is primarily organized and identified by the orthologous proteins Multi-sex-combs (Mxc) in Drosophila and NPAT in mammals, and contains other evolutionarily conserved factors involved only in RD-histone gene transcription and pre-mRNA processing (Ye et al., 2003b; Dominski and Marzluff, 2007; Yang et al., 2009; Bulchand et al., 2010; White et al., 2011). Mxc is a large (>1800aa) protein composed mostly of intrinsically disordered regions with a structured N-terminal domain that mediates multimerization and is required for HLB formation (Terzo et al., 2015). HLB formation in Drosophila is critical for histone biosynthesis, as depletion of Mxc prevents RD-histone gene expression (White et al., 2011). The HLB is also important for coupling of RD histone gene expression with the cell cycle, as Cyclin E/cdk2-mediated phosphorylation of Mxc/NPAT as cells enter S phase activates histone gene expression (Ma et al., 2000; Wei et al., 2003; Ye et al., 2003b; White et al., 2007; White et al., 2011; Armstrong and Spencer, 2021; Armstrong et al., 2023). Here we explore whether HLBs play a role in coordinating expression from multiple histone genes at nonhomologous loci.

To explore this question, we employed a previously established platform for engineering specific histone genotypes in D. melanogaster (McKay et al., 2015; Meers et al., 2018). Removal of all ∼200 copies of each endogenous RD histone gene by homozygous deletion of HisC is lethal, but this lethality can be rescued by just 12 or 24 copies of each RD histone gene provided by a BAC-based transgenic histone gene array (McKay et al., 2015). This rescue occurs because despite the large difference in gene copy number the overall level of H2A mRNA is similar between the “24x” engineered, transgenic-histone genotype and the “200x” wild-type genotype (McKay et al., 2015), and the total amount of RD histone mRNA that accumulates per gene copy is modulated to achieve histone homeostasis. Moreover, when wild-type HisC and the homozygous 12x transgenic array were present in the same animal (“224x” genotype), the amount of H2A transcript detected from each locus changed such that the total amount of H2A mRNA was comparable to a 200x or a 24x genotype (McKay et al., 2015). This result suggested there is a mechanism for communication among histone genes residing at different loci to achieve a precise level of H2A gene expression. In this study, we demonstrate that such regulation applies to all five RD histone genes. We also provide evidence for functional competition between endogenous histone genes and transgenic-histone arrays that likely results from limited availability of an HLB component(s), suggesting how HLB assembly might contribute to histone homeostasis.

RESULTS

All five RD histone genes modulate expression to maintain histone homeostasis

In D. melanogaster, the deficiency Df(2L)HisCED1429 (hereafter ΔHisC) removes the entire endogenous histone locus on chromosome 2L and causes embryonic lethality when homozygous (Günesdogan et al., 2010). This lethality can be rescued with a BAC-based transgene containing an engineered gene array with 12 tandemly repeated copies of the wild-type histone gene unit (HWT; McKay et al., 2015). The 12xHWT array contains a synonymous polymorphism in the H2A gene resulting in loss of an XhoI site, enabling us to measure the amount of endogenous versus transgenic H2A transcript by restriction enzyme digestion of H2A RT-PCR products (Figure 1A). In this study, we utilized a transgenic-histone gene unit where each gene is similarly marked by the insertion or removal of a restriction site(s) without altering the protein-coding sequence of any RD histone gene (Figure 1A). The transgenic array containing 12 copies of this designer wild-type (DWT) gene unit also rescues lethality caused by homozygous ΔHisC (Koreski et al., 2020).

FIGURE 1.

FIGURE 1.

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All five RD histone genes display homeostatic control of histone mRNA levels. (A) Schematic representation of the endogenous RD histone gene unit of D. melanogaster and the transgenic-histone gene unit DWT. The endogenous-histone gene unit is tandemly arrayed ∼100 times at the HisC locus on chromosome 2L, resulting in ∼200 copies of RD histone genes in a diploid fly. The transgenic array consists of 12 repeating gene units inserted on chromosome 3L, making a total of 24 RD histone gene copies in a homozygous genotype. Each histone gene in the transgenic array is designed to be molecularly distinguishable from the endogenous counterpart through insertion or removal of specific restriction sites as indicated. (B–D) Polyacrylamide gels of restriction digested RT-PCR products from 4–6 h old embryos for (B) the H2A–H2B gene pair, (C) the H3–H4 gene pair and (D) the linker histone, H1. mRNA level for each gene was measured in three genotypes: yw control (“200”, lane one), ΔHisC / ΔHisC; 12XDWT/12XDWT (“24”, lane two), and +/+; 12XDWT/12XDWT (“200, 24″, lane three). Bar plots of band intensity normalized to tubulin and relative to yw from three biological replicates. Values indicate mean and error bars indicate SD.

To test whether all RD histone genes modulate expression to compensate for differences in gene copy number, we measured zygotic RD histone mRNA amounts from 4–6 h old embryos that were either wild-type or carrying a homozygous 12xDWT transgenic array in the presence or absence of the endogenous HisC locus (Supplemental Figure S1, A–D). We compared RNA levels among embryos with the normal number of endogenous histone genes (wild-type, 200x), carrying two copies of 12xDWT transgenic-histone gene array (24x), or that were homozygous both for HisC and the 12xDWT transgenic-histone gene arrays (224x). We amplified cDNA from these three genotypes using primers that recognize each RD histone gene in both the endogenous and transgenic templates, followed by restriction digestion with specific enzymes that differentially digest endogenous versus transgenic PCR products (Figure 1A). Quantitation of band intensities from restriction digested PCR products revealed that the amount of each RD histone mRNA is similar between the 24x genotype and the 200x genotype despite about eight-fold difference in the number of histone genes (Figure 1, B–D).

We next analyzed the relative mRNA contribution from endogenous versus transgenic-histone genes when both were present in the same embryos. Interestingly, the 24xDWT transgenic-histone gene arrays, which when present alone can generate histone mRNA equivalent to or exceeding that made by 200 copies of the endogenous genes, contributes only ∼20–30% of the total RD histone mRNA when present together with the endogenous HisC locus (Figure 1, B–D). We also note that the ratio of the level of transcripts contributed by the transgenic versus endogenous histone loci (1:3–1:4) is still higher than expected from the ratio of number of transgenic-histone genes to endogenous-histone genes (1:8). These data suggest that there must be coordination between the endogenous- and transgenic-histone loci, which are located on different chromosomes, to maintain a particular overall amount of RD histone RNA. Thus, we have established a molecular assay to detect the relative amount of endogenous versus transgenic transcripts for each of the five RD histone genes and observed regulation between different histone gene loci that likely contributes to histone homeostasis.

To quantify the relative level of histone gene expression more accurately we sequenced RT-PCR products using a miSeq platform. We determined the relative amount of transcript from the 12xDWT transgene versus the endogenous HisC locus for each of the core histone genes (H2A, H2B, H3, and H4) using the sequence differences we engineered into the DWT construct (see Materials and Methods and Supplemental Table S1A; note that we were unable to develop this method for H1). The results closely match the quantification of band intensities from restriction digested PCR products, revealing that the ratio of transgenic:endogenous derived transcripts for the H2B, H3, and H4 genes is ∼1:3 and for H2A is ∼2:3 (Table 1). These data are consistent with our previously published results with the H2A gene (McKay et al., 2015; Koreski et al., 2020) and provide evidence for modulation of expression of all five RD histone genes to maintain histone homeostasis.

TABLE 1.

Quantification of relative expression of endogenous (HisC) and transgenic (12XDWT, 12XPR, or 8XHWT) RD histone genes in 3–6 h old embryos determined by either quantification of band intensities of restriction digested RT-PCR products (PCR data) or by direct sequencing (MiSeq data). A) HisC/HisC; 12XDWT. B) ΔHisC/ΔHisC; 12XPR, 8XHWT. All values are average ± SD for either three (PCR data) or two (MiSeq data) biological replicates. ND = not determined.

H2A H2B H3 H4 H1
PCR data 68.9 ± 5.4 80.2 ± 1.8 75.4 ± 3.7 77.9 ± 4.1 80.0 ± 1.1 HisC
31.1 ± 5.4 19.4 ± 1.8 24.6 ± 3.7 22.2 ± 4.1 20.0 ± 1.1 12XDWT
MiSeq data 61.8 ± 2.1 74.1 ± 0.7 76.7 ± 0.2 78.4 ± 0.3 ND HisC
39.2 ± 2.0 25.9 ± 0.7 23.3 ± 0.1 21.6 ± 0.2 ND 12XDWT
PCR data 63.3 ± 3.7 71.5 ± 2.5 71.9 ± 5.3 63.0 ± 1.5 65.6 ± 3.3 12XPR
36.7 ± 3.7 28.5 ± 2.5 28.1 ± 5.3 37.0 ± 1.5 34.4 ± 3.3 8XHWT
MiSeq data 81.6 ± 0.6 77.0 ± 0.1 71.5 ± 1.3 60.2 ± 2.3 ND 12XPR
18.4 ± 0.6 23.0 ± 0.1 29.5 ± 1.2 39.8 ± 2.2 ND 8XHWT
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Different histone loci compete for limiting RD histone-gene expression factors

How do cells coordinate gene expression between distinct histone gene arrays at different loci to achieve a particular overall level of histone mRNA? One potential mechanism could involve competition for shared but limiting histone mRNA biosynthetic factors. In this situation, a histone gene array that is functionally attenuated via mutation may be unable to effectively compete with wild-type HisC, resulting in lower levels of gene expression from that array. We previously showed that a transgene carrying 12 copies of an RD histone gene unit in which the bidirectional H3–H4 promoters are replaced by the wild-type bidirectional H2a–H2b promoters (Figure 2A; Supplemental Figure S1F; 12XPR or promoter replacement [PR]) behaves as an attenuated-histone gene array: it does not form an HLB and is not expressed in the presence of the endogenous RD histone genes at HisC, but in the absence of the endogenous-RD histone genes the 12XPR array forms an HLB, expresses RD histone mRNA, and can rescue the lethality caused by homozygous ΔHisC (Koreski et al., 2020). One difference between H2a–H2b and H3–H4 bidirectional promoters is that the H2a–H2b region lacks the GAGA repeat present in the H3–H4 promoter that binds the zinc-finger protein CLAMP, which promotes RD histone gene expression (Rieder et al., 2017). We also previously showed that the H3–H4 promoter was necessary and sufficient for HLB formation and expression of core RD histone genes in salivary glands when the endogenous histone genes are present (Salzler et al., 2013; Rieder et al., 2017). Thus, the 12XPR is an attenuated RD histone gene array that cannot effectively compete with the endogenous HisC locus but is fully functional when it is the only source of RD histone genes.

FIGURE 2:

FIGURE 2:

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The 12X PR-transgenic array is silenced by ∼100 endogenous histone genes but not by an 8X HWT array. (A) Schematic representation of transgenic RD histone gene arrays inserted on chromosome three. The PR array inserted on chromosome 3L has 12 histone gene units in which the H3–H4 promoter (blue rectangle) is replaced by the H2A-H2B promoter (yellow rectangle). The HWT array inserted on chromosome 3R consists of eight repeating wild-type RD-histone gene units. (B) Polyacrylamide gels of restriction digested RT-PCR products from 3–6 h old embryos. mRNA level for each RD histone gene was measured in three genotypes: ΔHisC / ΔHisC; 12XPR/+ (lane one), ΔHisC /CyO; 12XPR,8XHWT/+ (lane two) and ΔHisC / ΔHisC; 12XPR, 8XHWT /+ (lane three). Asterisk indicates low-molecular weight restriction digested product(∼50bp). (C) Bar plots of band intensity normalized to undigested PCR products from three biological replicates. Values indicate mean and error bars indicate SD.

This result led us to ask whether a wild-type transgenic array with a small number of RD histone genes could also attenuate the expression of 12XPR. We created flies carrying two different transgenic histone arrays by making a recombinant third chromosome carrying the 12XPR transgene on the left arm of chromosome three and a transgene with only eight copies of the HWT gene unit (8XHWT) on the right arm of chromosome three (Figure 2A). Like the DWT transgene, the 12XPR array also carries synonymous polymorphisms in each histone gene enabling us to differentiate between transcripts generated from 12XPR versus 8XHWT (or vs. the endogenous genes at HisC; Figure 2A). We determined the level of expression of all five RD histone genes in 3–6 h old embryos carrying a single copy of the 12XPR array in a homozygous ΔHisC background (Supplemental Figure S1A; Table 1, Supplemental Table S1A). Consistent with our previous results (Koreski et al., 2020), in this genotype the 12XPR transgene produces wild-type amounts of all five RD histone genes (Figure 2, B and C; Genotype one vs. two). Thus, in this genotype replacing the H3–H4 promoter with the H2a–H2b promoter does not have any substantial effect on the expression of the H3–H4 gene pair. Next, we measured the relative expression from the 12XPR array in the presence of both HisC and 8XHWT (Figure 2B; Genotype two) or just 8XHWT (Figure 2B; Genotype three). Including one complement of endogenous histone genes (∼100 copies at the HisC locus located on the CyO balancer chromosome) results in loss of expression of all five RD histone genes from the 12XPR array (Figure 2, B and C; Genotype two). Using the direct-sequencing quantification method described above we calculated that the core histone genes in the 12XPR array are expressed at least 1000 times lower than those at HisC (Supplemental Table S1B). Thus, although the 12XPR array carries intact wild-type H1 and H2a–H2b genes with their normal promoters, the replacement of the H3–H4 promoter with the H2a–H2b promoter results in loss of expression of this entire transgenic locus in the presence of ∼100 copies of the endogenous-histone genes (Salzler et al., 2013; Koreski et al., 2020).

In contrast, the 12XPR transgene is expressed when present with one copy of the 8XHWT transgene rather than with HisC (Figure 2, B and C; Genotype three). In this genotype, the 12XPR and 8XHWT transgenic arrays exhibit an ∼ 70:30 relative contribution, respectively, to the total amount of histone mRNA. These two arrays maintain an overall RD histone gene expression level similar to that of one copy of HisC and therefore can rescue loss of HisC (Figure 2C; Genotype three). We conclude from this experiment that eight copies of the wild-type RD histone gene unit are not able to compete with 12XPR like ∼100 copies do. We hypothesize that this competition is due to limiting amounts of a factor(s) involved in histone mRNA biosynthesis and/or HLB formation that must be distributed between different loci (Koreski et al., 2020). Together, these data provide further evidence for coordination between histone loci that are present on either the same or separate chromosomes.

HLB assembly reflects competition between different RD histone loci

We tested whether the basis for distributing limiting-gene expression factors to multiple RD histone loci is rooted in the mechanism of HLB assembly, which occurs through a combination of ordered and stochastic processes (Duronio and Marzluff, 2017). Recruitment of histone mRNA biosynthetic factors to the HLB is consistent with a “seed and grow” mechanism of assembly, and HLBs display properties of phase separated biomolecular condensates (White et al., 2011; Hur et al., 2020). The H3–H4 promoter and/or nascent histone mRNA provides the “seed” (Rieder et al., 2017; Hur et al., 2020) while multimerization of Mxc (Terzo et al., 2015) provides a scaffold for recruitment of other HLB components (“grow”) resulting in a phase separated nuclear body that facilitates activation of histone gene expression. To explore whether HLB assembly might play a role in coordination or competition between different histone loci (e.g., by assembling these loci into a single body or multiple, distinct bodies), we stained embryos with antibodies against Mxc to visualize HLBs in the different genotypes described above. We first asked whether a single 12XPR locus was competent for HLB assembly in diploid cells by analyzing ΔHisC/ΔHisC embryos containing either one (12XPR/+) or two (12XPR/12XPR) copies of the transgene. We observed a single HLB in all (n = 372) epidermal-cell nuclei of germ band retracted ΔHisC/ΔHisC; 12XPR/+ embryos in which these cells are arrested in G1 phase of the cell cycle, consistent with the presence of a single, unreplicated 12XPR transgene (Figure 3, A and B). In ΔHisC/ΔHisC; 12XPR/12XPR blastoderm embryos essentially all nuclei (∼99%) had either one or two HLBs, which is reflective of paired versus unpaired homologous chromosomes, respectively, at this stage of embryogenesis (Supplemental Figure S2A). We obtained a similar result in control ΔHisC/ΔHisC; 12XDWT/12XDWT embryos (Supplemental Figure S2A). We conclude from these data that HLB formation occurs at the 12XPR transgenic array when the endogenous RD histone genes are absent, consistent with our previous observation that 12XPR can assemble an HLB in highly polyploid-salivary gland nuclei as well as syncytial stage embryos and that 12XPR is active for RD histone gene expression in the homozygous ΔHisC genotype (Koreski et al., 2020).

FIGURE 3:

FIGURE 3:

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HLB assembly at the PR array is impaired by the presence of endogenous histone genes. (A) G1-arrested epidermal cells stained with antibodies against Mxc in germband retracted embryos from three different genotypes: ΔHisC/ΔHisC; 12XPR/12XPR (top panel), ΔHisC/CyO;12XPR/+ (middle panel) and ΔHisC/ΔHisC; 12XPR, 8XHWT/+ (bottom panel). Schematics on the right represent HLB formation at the histone loci (endogenous HisC and transgenic) in each respective genotype. Possible fusion of HLBs at nonhomologous loci located on the same chromosome is also depicted (bottom panel schematic). Red circles represent nuclei with two HLBs. Scale bar, 5 microns. (B) Bar plots represent the number of HLBs detected in each nucleus in embryos from each genotype. “n” indicates the number of nuclei analyzed for each genotype. Values over bars show percentage of nuclei.

We next determined the effect on HLB assembly at 12XPR of introducing one (ΔHisC/CyO) or two (+/+) copies of the wild-type HisC locus. We found in G1-arrested embryonic epidermal cells that most (96%, n = 485) nuclei in ΔHisC/CyO;12XPR/+ embryos contained a single HLB, while a small proportion of nuclei (4%, n = 485) contained two HLBs of differing sizes (Figure 3, A and B). In +/+; 12XPR/12XPR blastoderm embryos essentially all nuclei (n = 5681) contained either two HLBs that appear similarly sized (18%) or one HLB (82%; Supplemental Figure S2B). This result is identical to that obtained in true wild-type Oregon R embryos (Figure 4A). These data suggest that an HLB does not form at 12XPR in the presence of two copies of HisC, but may form with a low frequency when there is a single copy of the histone locus. One possibility is that ∼100–200 copies of each wild-type RD histone gene unit (or of the H3–H4 promoter itself) present at HisC sequester a limiting factor(s) and prevent HLB components from assembling on the 12XPR locus. We conclude that HLB assembly at 12XPR is severely impaired by the presence of wild-type HisC, resulting in very low or no histone mRNA production from 12XPR (Figure 2).

FIGURE 4:

FIGURE 4:

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Transcription occurs at each individual HLB formed at an ectopic-histone gene array. (A) Syncytial-nuclear cycle 14 embryos stained with antibodies against Mxc. Top panel shows Oregon R wild-type embryos, and bottom panel shows embryos carrying a homozygous wild-type transgenic-histone gene array (12XDWT) in the presence of HisC. Colored circles represent nuclei with 1–4 HLBs (1:red, 2:yellow, 3:blue, 4:orange). Bar plots represent the number of HLBs detected in each nucleus, quantified using IMARIS imaging software (See Methods). The “n” indicates number of nuclei analyzed for each genotype. Values over bars show percentage of nuclei. Schematics on the right represent HLB formation at the endogenous (blue second chromosomes) and transgenic (green third chromosomes) histone loci present in each respective genotype. Scale bar, 5 microns. (B) Syncytial-blastoderm embryos from the genotypes indicated above, simultaneously stained for Mxc (left panel) and hybridized with fluorescent probes detecting RD core histone RNAs (i.e., H2A, HB, H3, and H4; middle panel). The panel on the right shows a merge of HLBs and nascent RNA transcripts. The bottom panel shows high-resolution images of HLBs obtained using the Leica SP8 Lightning system where the pinhole was set at 0.6 Airy units for increased resolution. Dashed square represents zoomed-in images of a single nucleus shown in smaller panels on the right. Scale bar, 5 microns.

To test the competition hypothesis, we examined whether only eight copies of HWT genes could attenuate 12XPR HLB assembly. In ΔHisC/ΔHisC embryos simultaneously carrying one copy of the 12XPR transgene and one copy of the 8XHWT transgene on different arms of the third chromosome, we observed that 25% (n = 506) of nuclei had two HLBs in G1-arrested epidermal cells (Figure 3, A and B). This result indicates that an HLB can simultaneously form at both 12XPR and 8XHWT transgenic loci. Furthermore, because 8XHWT does not suppress HLB formation at, or expression from, the 12XPR transgene, our observations suggest that 8XHWT is not as effective as HisC in sequestering factors from 12XPR. At present we cannot explain why ∼75% of nuclei had a single HLB in ΔHisC/ΔHisC; 12XPR, 8XHWT/+ embryos (Figure 3B); one possibility is that these nuclei have HLB formation only at the 12XPR or only at the 8XHWT transgene.

Transcription of RD histone mRNA occurs in individual ectopic HLBs

To determine whether RD-histone gene transcription is always coincident with HLB formation, we performed RNA Flourescence In Situ Hybridization (FISH) to core histone genes while simultaneously staining with anti-Mxc antibodies. As noted above, in nuclei of wild-type blastoderm embryos we observe either one (80%) or two (20%) HLBs (Figure 4A). The fraction of single HLB nuclei is the same as the fraction of paired homologous HisC loci previously determined using DNA in situ hybridization (Hiraoka et al., 1993). Furthermore, we have observed fusion of individual HLBs (two HLBs merging into one) by live imaging embryos carrying GFP-tagged Mxc (Hur et al., 2020). Thus, the distribution of one versus two HLBs in wild-type results from homologous chromosome pairing in early Drosophila embryos. In cellular blastoderm (cycle 14) embryos that are homozygous for both HisC (+/+) and a 12XDWT transgenic histone gene array (12XDWT/12XDWT), we observe a broad distribution of nuclei (n = 4545) with one (26%), two (42%), three (27%), or four (5%) individual HLBs (Figure 4A). Nuclei with four HLBs represent the situation in which neither the homologous HisC loci on chromosome two nor the homologous 12XDWT loci on chromosome three are paired. In these nuclei we observe two larger and two smaller HLBs, consistent with our previous observation that the number of histone genes at a locus determines HLB size (Hur et al., 2020). Nuclei with fewer than four HLBs likely result from homologous chromosome pairing.

To determine whether individual HLBs are active for transcription, we hybridized +/+; 12XDWT/12XDWT embryos with a fluorescent probe set that simultaneously recognizes the four-core histone RNAs (i.e., H2A, HB, H3, and H4). This approach provides a highly sensitive method for detecting nascent-RD histone transcripts. We found that every focus of nascent histone transcripts was associated with an HLB as assessed by Mxc staining (Figure 4B), including in those nuclei with four HLBs. Moreover, and as we have observed previously, these HLBs display a “core-shell” organization with nascent-histone transcripts residing in the core surrounded by Mxc (Figure 4B; High-Resolution; Kemp et al., 2021). Thus, both unpaired HisC and unpaired 12XDWT loci can independently support RD-histone gene transcription in nuclear cycle 14 embryos. In a small number of early interphase nuclei (as assessed by nuclear morphology), we observed an Mxc focus that was not associated with a nascent histone transcript, suggesting that HLB assembly may have occur before detectable RD-histone gene transcription.

D. virilis nonhomologous RD histone loci behave similarly to engineered nonhomologous D. melanogaster loci

Thus far, we have used ectopic-transgenic histone gene arrays to analyze expression and HLB assembly at nonhomologous RD histone loci in D. melanogaster. To examine HLB formation and histone gene transcription in a natural system carrying nonhomologous histone loci, we analyzed early embryos from D. virilis, which contains two RD-histone gene clusters at different loci. In D. virilis, the major RD-histone gene locus (∼30 repeats) is located at the cytogenetic position 25F on chromosome two and the minor locus (∼6 repeats) is located at position 43C on chromosome four (Schienman et al., 1998; Shiotsugu, 2002; Rieder et al., 2017; Figure 5A). The RD-histone gene units in D. virilis exist as either quintets (gene units containing all five RD histone genes) or quartets (gene units containing only core RD-histone genes and lacking the H1 gene; Domier et al., 1986; Schienman et al., 1998).

FIGURE 5:

FIGURE 5:

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HLB formation and transcription can occur independently at both the major- and minor histone loci in D. virilis. (A) Schematic representation of the D. virilis histone loci on chromosome two (major locus) and on chromosome four (minor locus). (B) D. virilis syncytial blastoderm embryos stained with antibodies raised against D. melanogaster Mxc. Colored circles represent nuclei with 1–4 HLBs (1:red, 2:yellow, 3:blue, 4:orange). Bar plots represent the number of HLBs detected in each nucleus, quantified using IMARIS imaging software. The “n” indicates number of nuclei analyzed. Values over bars show percentage of nuclei. (C) D. virilis syncytial blastoderm embryos simultaneously stained for Mxc (left panel) and hybridized with fluorescent probes detecting D. virilis H4 histone mRNA (middle panel). The panel on the right shows a merge of HLBs and nascent H4 mRNA transcripts. Dashed square represents zoomed-in images of a single nucleus shown in smaller panels on the right. Scale bar, 5 microns.

D. virilis syncytial blastoderm embryos stained with antibodies against D. melanogaster Mxc exhibited a distribution of nuclei (n = 1868) with one (23%), two (66%), three (10%) and four (1%) individual HLBs, similar to our engineered system in D. melanogaster (Figure 5B). Nuclei with four HLBs exhibit two larger and two smaller HLBs, implying that HLB formation occurs at both the major and minor locus via a mechanism like D. melanogaster where the number of histone genes determines HLB size at this stage of development (Hur et al., 2020). Furthermore, like D. melanogaster, it is likely that D. virilis nuclei with less than four Mxc foci represent fused HLBs due to pairing of homologous chromosomes.

To test whether nascent transcription can be detected at both these nonhomologous histone loci, we hybridized D. virilis blastoderm embryos that were stained with anti-Mxc antibodies with fluorescent probes that recognize D. virilis histone H4 mRNA. We found that all individual HLBs were active for transcription, including those in nuclei exhibiting three or four HLBs (Figure 5C). Thus, our data demonstrate that both the major- and minor histone loci in D. virilis independently form HLBs and express RD histone genes in the same nucleus.

DISCUSSION

The number of RD histone genes varies widely in different species, ranging from two copies in the yeast Saccharomyces cerevisiae to hundreds of copies in fruit flies and sea urchins (Hentschel and Birnstiel, 1981; Maxson et al., 1983). Furthermore, these genes can either be organized in highly regular tandem repeats at a single locus (e.g., D. melanogaster; Lifton et al., 1978), randomly arrayed in multiple clusters at distinct-chromosomal locations (e.g., mammals; Marzluff et al., 2002; Seal et al., 2022), or distributed as small clusters throughout the genome (e.g., Caenorhabditis elegans Roberts et al., 1987). Because the overall histone level needs to be tightly controlled and coupled with S phase for genomic stability and cell survival, it is likely that coordinate expression from multiple histone loci is actively regulated to maintain histone homeostasis. In this study, we have examined the relationship between expression and HLB assembly at different RD histone loci in Drosophila.

We previously demonstrated that in D. melanogaster embryos, 24 copies of transgenic H2A genes generate an amount of mRNA equivalent to that made by ∼200 endogenous H2A genes (McKay et al., 2015). Here, we extended this analysis to show that the other three core RD histone genes as well as the linker histone H1 gene display this same homeostatic regulation. Because total RNA was measured in our experiments, we cannot distinguish the relative contribution of transcriptional or posttranscriptional (e.g., mRNA half-life) mechanism to the maintenance of overall RD histone mRNA levels. Nevertheless, our data clearly show that individual-histone gene arrays can be simultaneously expressed in both D. melanogaster and D. virilis, indicating that transcriptional regulation contributes to expression homeostasis between distinct RD histone gene loci.

How are all five RD histone genes coordinately regulated? There are no transcription factors known to bind simultaneously to each gene and coordinately regulate them. Rather, there are specific factors associated with specific histone genes (e.g., H2b or H4; Fletcher et al., 1987; LaBella et al., 1988; La Bella et al., 1989; Miele et al., 2005; Isogai et al., 2007; Lee et al., 2010; Guglielmi et al., 2013; Ghule et al., 2016). HLB assembly factors like Mxc are required for expression of each of the five RD histone genes (White et al., 2011), and mammalian NPAT is present at each of the active RD-histone gene promoters (Kaya-Okur et al., 2019). We probed the relationship between HLB assembly and RD histone gene transcription using engineered BAC-based transgenic histone gene arrays, particularly the functionally attenuated 12XPR array in which the bidirectional H3–H4 promoter is replaced by the bidirectional H2a-H2b promoter in each of the 12 histone gene units. This natural H2a-H2b promoter is capable of driving H3–H4 expression and providing RD histone gene function, as 12XPR fully rescues homozygous deletion of the endogenous HisC RD histone gene array (Koreski et al., 2020). Interestingly, in the presence of HisC none of the RD histone genes in 12XPR are expressed, although three of the genes (i.e., H1, H2a, and H2b) are unperturbed and contain their endogenous promoters. Thus, the lack of H2aH2b and H1 expression from 12XPR in the presence of HisC cannot be due to the absence of a key cis element. Rather, we conclude that the lack of HLB assembly, which does not occur at 12XPR in the presence of HisC but does in the absence of HisC, is the reason for the failure of expression. Consistent with this interpretation, 12XPR is expressed in the presence of HisC when integrated in trans with 12XHWT at the same locus on the third chromosome (i.e., the 12XPR/12XHWT genotype), a situation that promotes assembly of a single HLB that includes both transgenes after those homologous chromosomes pair (Koreski et al., 2020).

HLB assembly fails to occur at 12XPR in the presence of HisC because of the absence of the H3–H4 bidirectional promoter. We previously showed that in the presence of the endogenous genes at HisC, HLB components can be recruited to an ectopic RD histone locus by a single H3–H4 gene pair or just the H3–H4 promoter but not by the H2a-H2b and H1 genes (Salzler et al., 2013). In addition, GAGA repeats found only within the H3–H4 promoter and that bind the zinc-finger protein CLAMP are required for ectopic HLB assembly (Rieder et al., 2017). Thus, HLB assembly nucleated by the H3–H4 promoter provides a mechanism for how the H3–H4 promoter can stimulate H2a-H2b and H1 transcription. We hypothesize that the H3–H4 promoters in a histone array provide a strong binding site for the recruitment of HLB components, thereby nucleating HLB assembly and facilitating expression of the entire RD-histone gene array. Note that CLAMP is present in the 12XPR HLBs, but not bound to DNA, suggesting that it interacts with both DNA and an HLB factor(s) in the wild-type array (Koreski et al., 2020), and that CLAMP is present at multiple other loci where Mxc is not recruited (Kaye et al., 2018), indicating CLAMP is not sufficient for HLB assembly.

We also found that unlike the HisC locus, which contains ∼100 histone gene units, a single copy of a transgene containing an array of eight histone gene units did not affect HLB formation or transcription at the 12XPR transgene. One interpretation of this result is that it takes many histone gene units to sequester the limited supply of essential HLB components from the attenuated 12XPR transgene. In this model, the presence of only eight histone gene units is insufficient to bind enough HLB factors to achieve this level of sequestration. In contrast, twelve HWT gene units can effectively compete with HisC, as both loci stably recruited factors to form HLBs simultaneously. However, the amount of expression from 12XDWT is modulated by the presence of HisC, as more RD histone mRNA is produced by 12XDWT in the absence of HisC than in its presence. There is also less expression from the HisC locus in the presence of the 12XDWT. Thus, these distinct loci may compete for limiting factors necessary for RD-histone mRNA biosynthesis.

We suggest that HLB assembly and competition for limiting HLB components between histone gene arrays present at different loci provides a mechanism for coordinating RD-histone gene expression to maintain histone homeostasis. We cannot exclude the possibility that alternative chromatin configurations between the different loci contribute to the effects we observe. A critical function of Drosophila HLBs is to concentrate the factors required for formation of the unique RD histone mRNA 3′ end at the RD histone gene, thereby ensuring efficient processing of all five RD histone mRNAs (Tatomer et al., 2016). An exception to the presence of HLBs in metazoans is C. elegans, which doesn’t have NPAT, FLASH, or U7 snRNA (and thus no HLBs) and forms the RD histone mRNA 3′ end using an RNAi type mechanism after making a polyadenylated pre-mRNA (Avgousti et al., 2012). Thus, nematodes must coordinate expression from multiple histone genes via a mechanism that does not rely on HLB formation.

During early embryonic development when homologous chromosomes begin to pair in Drosophila, individual HLBs associated with each homologous HisC locus come into proximity and fuse into a single HLB, consistent with the liquid droplet properties of a phase separated nuclear body (Hur et al., 2020). Accordingly, we observed nuclei with either one or two HLBs in embryos carrying homologous histone loci in Oregon R, ΔHisC/ΔHisC; 12XPR/12XPR and ΔHisC/ΔHisC; 12XDWT/12XDWT genotypes. Whether the properties of HLB fusion play a role in histone homeostasis by facilitating physical proximity of nonarrayed histone genes in the three-dimensional nuclear space, thereby enabling efficient RD histone gene expression from multiple loci, remains to be investigated. In mammals, a large (Hist1) and a small (Hist2) cluster of RD histone genes resides at two different loci (Marzluff et al., 2002; Seal et al., 2022). These loci independently form HLBs that do not fuse, as homologous chromosomes do not pair, resulting in cells with four HLBs, two larger and two smaller (Ma et al., 2000; Zhao et al., 2000; Armstrong and Spencer, 2021; Armstrong et al., 2023). However, within the large Hist1 cluster, which is not a gene array like Drosophila but a cluster of >50 histone genes with a >1 megabase gap between the two ends of the cluster, there is evidence that the histone genes are in close contact (Quinodoz et al., 2021). This observation is consistent with them being brought close together in the large HLB, while the intergenic DNA is not, to coordinate RD histone mRNA transcription and processing similar to what we have described for Drosophila.

MATERIALS AND METHODS

Request a protocol through Bio-protocol.

Fly strains and genetic crosses

D. virilis (National Drosophila Species Stock center #15010-1051.118) was a gift from Dr. Daniel Matute (University of North Carolina, Department of Biology). The Bloomington Stock Center provided Oregon R (stock #25211) and yw (stock #6599). ΔHisC(Df(2L)HisCED1429),UAS-2xEYFP/CyO and ΔHisC(Df(2L)HisCED1429), twi-GAL4/CyO were a gift from Alf Herzig (Max Planck Institute for Biophysical Chemistry, Molecular Developmental Biology). Other fly stocks are described in (McKay et al., 2015; Koreski et al., 2020). All fly stocks were maintained on standard corn medium. For gene-expression analysis (Figure 2) and HLB formation (Figure 3) embryos were collected as follows: Embryos of the genotype ΔHisC/ΔHisC; 12XPR/+ and ΔHisC/Cyo; 12XPR/+ were obtained by crossing males of the genotype ΔHisC, UAS-2xEYFP/ΔHisC,UAS-2xEYFP; 12XPR/12XPR to females of the genotype ΔHisC,twi-Gal4/CyO; +/+ (Supplemental FigureS1, E and F). Embryos of the genotype ΔHisC/ΔHisC; 12XPR,8XHWT/+ and ΔHisC/CyO; 12XPR,8XHWT/+ were obtained by crossing males of the genotype ΔHisC,UAS- 2xEYFP/ΔHisC,UAS-2xEYFP; 12XPR,8XHWT/12XPR,8XHWT to females of the genotype ΔHisC,twi-Gal4/CyO; +/+ (Supplemental FigureS1, E and F). GFP signal was used to distinguish between the ΔHisC/ΔHisC and ΔHisC/CyO embryonic genotypes.

Histone expression analysis

Total RNA was isolated from embryos using the Trizol reagent (Invitrogen). cDNA was synthesized with random hexamers using SuperscriptII (Invitrogen). PCR was performed using the cDNA template and gene-specific primers for each histone gene. Each reaction was performed at least three times. PCR products were digested using AflII (H1), XhoI (H2a), XbaI (H2a), NruI (H2b), Eco53KI (isoschizomer of SacI; H3) and NgoMIV(isoschizomer of NaeI; H4) (Figure S1F). Digested PCR products were run on an 8% polyacrylamide gel and stained with SYBR gold. Quantitation of band intensities was performed using Image Lab software. Bar plots of band intensities normalized to tubulin and relative to yw (control) were generated in GraphPad Prism (Dotmatics).

Embryo collection and fixation

Embryos were collected on apple juice agar plates and aged at 25°C. Embryos were dechorionated in 50% bleach and then fixed in 4% formaldehyde in phosphate-buffered saline (PBS) with 50% heptane, on a nutator for 15 min at room temperature. The lower formaldehyde layer was removed and replaced with methanol. Embryos were vigorously shaken for 30 s to remove the vitellin membrane. Devitellinized embryos sink to the bottom. The heptane-methanol mixture and the embryos that did not sink were removed and replaced with fresh methanol. These embryos were then stored in methanol at -20°C to be used for immunostaining and FISH experiments.

Immunostaining

Fixed embryos were rehydrated using PBST (PBS + 0.1% TritonX-100) and blocked in Image-iT FX signal enhancer (Invitrogen) for 30m. The signal enhancer was removed and replaced with primary antibodies diluted in PBST at 4°C overnight. The embryos were then washed 3× with PBST, followed by an incubation in secondary antibodies diluted in PBST for 1 h at room temperature. Embryos were then either stained with 4’,6-diamidino-2-phenylindole (DAPI) and mounted in Prolong-Gold antifade (Invitrogen) for imaging or used further for FISH experiments.

Antibodies

Primary antibodies were guinea pig anti-Mxc (1:6000; White et al., 2011) and rabbit anti-GFP (1:1000; Rockland #600-401-215). For FISH experiments rabbit anti-MXC was used (1:500 for D. virilis and 1:1000 for D. melanogaster; White et al., 2011). Alexa Fluor secondary antibodies were goat anti-rabbit-488 and goat anti-guinea pig-647 (at a dilution of 1:1000).

Fluorescent in situ hybridization

Custom Stellaris RNA FISH probes targeting the coding region of core histone mRNA (H2a, H2b, H3, and H4) in D. melanogaster and those targeting the H4 mRNA in D. virilis were designed using the Stellaris RNA FISH Probe Designer (LGC Biosearch Technologies) and labeled with Quasar670. Embryos that were fixed and stained as stated above were incubated in 4% formaldehyde in PBS for 10 min to crosslink bound antibodies, then washed thrice in 2XSSC with 10% formamide. The wash buffer was removed and replaced with hybridization buffer (2XSSC + 10% formamide + 10% dextran sulphate). FISH probes diluted in hybridization buffer (final concentration 50 nm for D. melanogaster and 100 nm for D. virilis) were incubated with the embryos overnight at 37°C. Following hybridization, embryos were washed with wash buffer, stained with DAPI and mounted in Prolong-Gold anti-fade (Invitrogen) for imaging.

Confocal imaging and analysis

All images were acquired with a 63× oil immersion objective using a Zeiss LSM880 confocal microscope with Zen software. High-resolution images of HLBs (Figure 4B, bottom panel) were acquired with a 63× oil immersion objective using the Leica SP8 Lightning system at the highest resolution with LAS X software. Images were analyzed using FIJI and IMARIS (9.7.2) software. Quantitation of the number of HLBs within a nucleus (Figures 4 and 5), was performed in IMARIS as follows: Using the Surface function, nuclei within a blastoderm embryo were converted into individual surfaces by selecting DAPI as the source channel and a seed diameter of 4.5–5 microns. A quality threshold was applied to ensure that every surface generated corresponded to a nucleus. Merged or overlapping surfaces were manually deleted. Using the Spots function, HLBs within nuclei were converted into spots by selecting the Mxc signal as the source channel and a seed diameter of 0.4–0.6 microns. The quality threshold was applied to ensure every spot generated was aligned with an Mxc focus. Spots that did not align with Mxc foci were manually deleted. Finally, HLBs within nuclei were counted using the “Split (spots) into surface objects” extension. The data generated was exported into excel sheets. Bar plots were created in GraphPad Prism. Despite our best effort to manually delete merged or overlapping surfaces and background staining spots that may be considered HLBs by the algorithm, a low level of error was observed (e.g., a nucleus with one HLB that overlaps with another nucleus with two HLBs sometimes can be considered as one surface with three spots. We have included these data in the bar plots. Taking into consideration a high n value, the low level of technical error does not affect the interpretation of our data.

Amplicon RNA-sequencing and bioinformatic quantification of histone cDNA

cDNA was synthesized from total RNA extracted from embryos using random hexamers and Superscript II (Invitrogen). Illumina adaptors and sample-specific bar codes were subsequently added in two consecutive rounds of PCR amplification (24 and 28 cycles, respectively). The libraries were prepared using the small RNA protocol as described previously (Smola et al., 2015). Following library preparation and quality control, the samples were loaded on an Illumina MiSeq subjected to paired-end sequencing using a 600-cycle kit. The reads were demultiplexed using Illumina BaseSpace and the fastq files analyzed.

Because the mutant and HWT genes differ only by several nucleotides, we opted to use an exact-match criterion to quantify relative expression in our sequencing data. We identified unique 25 nucleotide sequences in each of the histone mRNAs and used an exact match regular expression to count reads from each histone gene array. The unique sequences are provided in Supplemental Figure S3, which also include the restriction site changes used for analysis of the PCR products in Figures 1 and 2.

We counted both exact matches and reverse-complement matches in both R1 and R2, and used the raw read counts from the read with the higher quality scores as defined by the MiSeq Illumina sequencer. We then computed relative ratios of read counts and reported these in Supplemental Table S1. Raw fastq files were uploaded to the Sequence Read Archive (PRJNA1005642, SRA, https://www.ncbi.nlm.nih.gov/sra), under project ID PRJNA1005642

PCR Primers used for this study

  • H1_forward 5′-GTCTGATTCTGCAGTTGCAACG-3′

  • H1_reverse 5′-TCCAGTTTTCTTGGCATCC-3′

  • H2a_forward 5′-GGCCATGTCTGGACGTGGAAAAGGT-3′

  • H2a_reverse 5′-GGCCTTAGGCCTTCTTCTCGGTCTT-3′

  • H2b_forward 5′-CTAGTGGAAAGGCAGCCA-3′

  • H2b_reverse 5′-GAGCTGGTGTACTTGGTGA-3′

  • H3_forward 5′-GCTACTAAGGCCGCTCG-3′

  • H3_reverse 5′-GGCATTATGGTGACACGC-3′

  • H4_forward 5′-GCC AAA TCC GTA GAG GGT-3′

  • H4_reverse 5′-GGTCGTGGTAAAGGAGGCA-3′

  • α-tubulin_forward 5′-GGCAGTTCGAACGTATACGC-3′

  • α-tubulin_reverse 5′-GACCACAGTGGGTTCCAGAT-3′

  • attB 5′-AGTGTGTCGCTGTCGAGATG-3′

  • attP 5′-CCTTCACGTTTTCCCAGGT-3′

  • Lamp1_forward 5′-CCTGTGTTATATAAACCCGTGATA-3′

  • Lamp1_reverse 5′-CTAACGAACGTAAGCGACAC-3′

  • Pry4_forward 5′- CAATCATATCGCTGTCTCACTCA-3′

  • PR_verification_forward 5′-CGATGACGCTTGGCGCCAC-3′

  • PR_verification_reverse 5′-CCACCAGTCGATTTGCGAGCAG-3′

Supplementary Material

Click here for additional data file. (3.1MB, pdf)

Acknowledgments

We thank Mark Geisler for developing the D. melanogaster core-histone FISH probes, Sierra Cole for helping develop the PCR assay, and Tony Perdue, the Department of Biology Imaging core, and James Kemp for help with confocal microscopy. This work was supported by GM058921 from the National Institutes of Health to W.F.M and R.J.D. Additional support for this work was provided by NHLBI R01, HL111527, and NIGMS R35 GM140844 to A.L.

Abbreviations used:

BAC

Bacterial artificial chromosome

HLB

Histone Locus Body

RD

Replication dependent.

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E22-11-0532) on August 30, 2023.

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