Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Jul 7.
Published in final edited form as: Curr Biol. 2014 Jun 12;24(13):1485–1491. doi: 10.1016/j.cub.2014.05.022

Drosophila lipid droplets buffer the H2Av supply to protect early embryonic development

Zhihuan Li 1, Matthew R Johnson 1, Zhonghe Ke 1, Lili Chen 1, Michael A Welte 1
PMCID: PMC4122669  NIHMSID: NIHMS598176  PMID: 24930966

Summary

Assembly of DNA into chromatin requires a delicate balancing act, as both dearth and excess of histones severely disrupts chromatin function [1–3]. In particular, cells need to carefully control histone stoichiometry: if different types of histones are incorporated into chromatin in an imbalanced manner, it can lead to altered gene expression, mitotic errors, and death [4–6]. Both the balance between individual core histones and between core histones and histone variants is critical [5, 7]. Here, we find that in early Drosophila embryos histone balance in the nuclei is regulated by lipid droplets, cytoplasmic fat storage organelles [8]. Lipid droplets were previously known to function in long-term histone storage: newly laid embryos contain large amounts of excess histones generated during oogenesis [9], and the maternal supplies of core histone H2A and the histone variant H2Av are anchored to lipid droplets via the novel protein Jabba [3]. We find that in these embryos synthesis of new H2A and H2Av is imbalanced and that newly produced H2Av can be recruited to lipid droplets. When droplet sequestration is disrupted by mutating Jabba, embryos display an elevated H2Av/H2A ratio in nuclei as well as mitotic defects, reduced viability, and hypersensitivity to H2Av overexpression. We propose that in Drosophila embryos lipid droplets serve as a histone buffer, not only storing maternal histones to support the early cell cycles, but also transiently sequestering H2Av produced in excess and thus ensuring proper histone balance in the nucleus.

Results

Jabba mutant embryos display temperature-dependent developmental defects

Newly laid Drosophila embryos contain large stockpiles of histone proteins and histone mRNAs [9, 10], generated during oogenesis. Certain maternally provided histone proteins (the core histones H2A, H2B, and the histone variant H2Av) are stored on lipid droplets via the droplet protein Jabba [9, 11]. Droplet binding apparently protects the extranuclear histone pool from degradation and thus mediates long-term storage of maternally synthesized histones [11].

Jabba mutant embryos compensate for this loss of histone storage by synthesizing abundant new histones, sufficient to support seemingly normal early embryonic development [11, 12]. We speculated that new histone biosynthesis might be insufficient to deal with unpredictable increases in histone demand and that environmental conditions that speed up development might therefore be particularly challenging to Jabba embryos.

Most of our previous analysis had been done at room temperature (RT, corresponding to ~21°C). At 25°C, embryogenesis is about 38% faster than at 21°C [13]. We therefore examined embryos raised at 25°C for signs of “nuclear falling” [14], an embryo-specific DNA damage response that can be triggered by an insufficient histone supply [11, 15]. For wild-type embryos at 25°C, nuclear falling was rare, just like for wild-type and Jabba mutant embryos at RT [11]; in contrast, about half of the Jabba mutant embryos at 25°C showed nuclear falling (Figures 1A and B). While most of the mutant embryos cellularize and gastrulate, they hatch at lower rates than the wild type (Figure 1D). Three independently derived Jabba alleles gave similar phenotypes (Figure 1C).

Figure 1. At 25°C, Jabba embryos show nuclear falling and reduced hatching.

Figure 1

Open in a new tab

(A) Wild type and Jabbazl01 syncytial blastoderm embryos stained for DNA (blue). At the surface (top), nuclei are slightly sparser in Jabbazl01 embryos. Cross-sectional views (bottom) reveal many nuclei between the cortex and the central yolk in the mutant. Scale bar= 50μm

(B) Blastoderm embryos stained for DNA (blue) and centrosomes (CNN, green): surface view (top) and cross section (bottom). Jabbazl01 embryos have free centrosomes at the surface (arrows) and displaced nuclei between the cortex and the central yolk. Scale bar = 10μm

(C) Quantification of nuclear falling. Embryos prepared as in (A) were classified as displaying massive nuclear falling or as normal (see Experimental Procedures for details). At 25°C, about half of Jabba embryos show such massive nuclear falling. Jabbazl01, JabbaG19631, JabbaDL are independently derived Jabba alleles [11]. Error bars represent standard deviations (SDs). Red bars correspond to the wild type; blue bars to Jabba mutants.

(D) Hatching frequency of embryos from wild-type and Jabbazl01 mothers, crossed to wild-type males, at 21°C or 25°C. Embryos from Jabbazl01 mothers show a reduced rate of hatching at 25°C. Error bars represent SDs. Red bars correspond to the wild type; blue bars to Jabba mutants.

**p<0.01

Increased levels of nuclear H2Av in Jabba embryos

Yeast cells with an insufficient H2B supply package the newly replicated DNA without H2B, but arrest in mitosis due to defective chromosome segregation [2]. In Jabba embryos, the loss of droplet-stored H2A and H2B might similarly cause diminished histone incorporation during S phase. We examined nuclear histone accumulation by immunostaining, focusing specifically on early mitosis, but found no obvious difference in either H2A or H2B between wild-type and Jabba embryos (Figures 2B, S1E-G). The two genotypes also showed essentially no change in the H2B/H3 ratio (Figures S1A and B); histone H3 is not associated with lipid droplets [9] and its levels are unaltered in Jabba mutants [11]. Thus, at the elevated temperature, new synthesis of H2A and H2B is sufficiently upregulated to achieve near normal nuclear accumulation in Jabba embryos.

Figure 2. Jabba embryos contain more H2Av in their nuclei.

Figure 2

Open in a new tab

(A) Comparison of H2Av signal (as determined by anti-H2Av staining) in embryos of various ages in the same field of view. In Jabbazl01 embryos, H2Av is increased in cycle 12 relative to the wild type, but not in cycle 14. Scale bar = 50μm

(B) Wild-type and Jabbazl01 embryos in prometaphase, stained for H2Av (green) and H2B (red). In cycle 10 to 12 embryos (left to right), Jabbazl01 nuclei contain similar amounts of H2B, but more H2Av than in the wild type. Scale bar = 10μm

(C) Quantification of H2Av signal. The nuclear H2B and H2Av signal of at least three embryos in each cycle was quantified using ImageJ. H2Av levels are normalized against H2B. White bars represent the wild type, black bars Jabba mutants.

(D) GFP immunostaining of H2AvGFP/+ and Jabbazl01, H2AvGFP/+ embryos. The Jabba mutants show increased nuclear H2AvGFP in cycles 10 to 13, but when cellularization starts (cycle 14, right), the nuclear H2AvGFP levels are similar to wild type. Scale bar = 10μm

(E) Quantification of GFP signal in (D). Error bars represent SDs of the data collected from 3 different embryos in the same cycle. White bars represent H2AvGFP in the wild-type background, black bars H2AvGFP in a Jabba mutant background.

(F) Jabba mutant embryos and centrifuged wild-type embryos lacking the lipid-droplet layer were compared by Western blotting (see main text for details): Jabba embryos have more nuclear H2Av. Timing of embryo collections and visual sorting was used to enrich for cycle 12 and 13 embryos.

(G) Quantification of Western blotting experiments as in (F). The H2Av/H2B ratio was normalized to the value observed in the wild type. Error bar represents the SD from three repeat experiments. All embryos used were collected and raised at 25°C.

**p<0.01, *p<0.05, NS: not significant

See also Figure S1.

Lipid droplets also store the variant histone H2Av [11]. H2Av replaces a small fraction (5–10% [16]) of H2A molecules in chromatin but has important roles in transcriptional regulation and DNA repair (reviewed in [17]). In Jabba mutant embryos, nuclear H2Av signal was dramatically elevated compared to the wild type (Figures 2B and C). An increased H2Av/H2B ratio was also observed with two additional Jabba alleles (data not shown). Using strains that carry H2AvGFP transgenes, we found that Jabba mutant embryos also display increased nuclear GFP signal - as assessed by GFP fluorescence in living embryos (data not shown) or by anti-GFP immunostaining after fixation (Figure 2D). Increased H2Av accumulation varied with embryonic stage (Figures 2A–D): During cycles 10–13, Jabba mutants had increasingly more H2Av or H2AvGFP in the nuclei than wild-type embryos; by early cycle 14, the two genotypes displayed similar nuclear levels of these histones (Figures 2A, D, E). We conclude that loss of Jabba causes a stage-specific increase in nuclear H2Av levels and alters the balance between H2Av and canonical histones in the nucleus.

We also measured relative H2Av levels biochemically, by comparing histone levels in individually staged blastoderm embryos. We centrifuged wild-type embryos to accumulate the droplets at one end of the embryo in a compact layer [18] that – after fixation – could be removed manually. Western blotting for the lipid-droplet protein LSD-2 demonstrates that our procedure largely depleted lipid droplets (Figure 2F). For Jabba mutants, we used entire embryos as there is no droplet pool of histones. Relatively elevated H2B and H2A levels in the wild type may reflect that our preparations retain some lipid droplets and their associated histones (Figures 2F and S1H). Nevertheless, levels of H2Av were higher in Jabba embryos (Figures 2F and S1C). These results support that in Jabba mutants nuclear H2Av and H2A levels are imbalanced.

Newly synthesized histones can accumulate on lipid droplets

How does the H2Av/H2A ratio in Jabba embryos become abnormal? The role of Jabba in long-term histone storage on droplets provides a paradigm: histones synthesized during oogenesis are stored on lipid droplets until they are needed during embryo development. Both wild type and Jabba mutants also generate new histones during early embryogenesis, as shown by the increase in H2A and H2Av levels over time (Figures 3A, B, C; see also [9]). In Jabba mutants, H2Av levels increase faster than those of H2A, indicating unbalanced synthesis. We propose that in the wild type H2Av generated in excess can be transiently sequestered on lipid droplets; without Jabba, excess H2Av could not be retained in the cytoplasm and thus the H2Av/H2A ratio in the nucleus might directly reflect the rate at which these two histones are produced, leading to a skewed H2Av/H2A ratio in their nuclei.

Figure 3.

Figure 3

Open in a new tab

Histones are newly synthesized in embryos and localize on lipid droplets. (A–C) Western blotting analysis of H2A and H2Av in early Jabba (A) and wild-type (B) embryos. Embryos were sorted according to nuclei density, as visualized by Hoechst staining; tubulin serves as the loading control. (C) Cycle 13 wild-type embryos contain significantly higher H2Av and H2A levels than cycle 1–2 embryos. Protein levels were measured by ImageJ and normalized to tubulin. An unpaired t test was used to statistically analyze the results of three repeated experiments. (D, E) Embryos from mothers expressing Gal4 in their germ line and carrying UAS-H2AvGFP or UAS-H3GFP constructs were centrifuged and stained for GFP. Embryo age was estimated according to the number of nuclei present. (D) Compared with newly laid embryos, stage 5 (~3hrs) embryos show dramatically increased H2AvGFP signal in the lipid-droplet layer (arrow). (E) Although total histone H3GFP signal similarly goes up from newly laid embryos to stage 5, GFP signal is only detected in nuclei, not in the droplet layer. (F) Diagram of the droplet transplantation strategy. Wild-type lipid droplets marked by H2AvRFP are injected into Jabba embryos expressing H2AvGFP, and H2AvGFP from the recipient then accumulates on lipids droplets. (G) Jabbazl01, H2AvGFP blastoderm embryos (“recipient”) injected with lipid droplets from H2AvRFP embryos (“donor”). 15 min post-transplantation, the donor droplets (red, center) are decorated with H2AvGFP (green, left) generated by the recipient. In regions distant from the site of transplantation, H2AvGFP is only detected in the nuclei (arrow) of the recipient and not in the cytoplasm, as the endogenous lipid droplets lack Jabba and fail to bind H2AvGFP (left bottom). Scale bars = 25 μm (top) and 5 μm (mid and bottom). Boxes H and I in panel G are shown in magnified view in subsequent panels.

If this model is correct, then histone loading onto droplets is not restricted to oogenesis but can occur for H2Av newly synthesized in the embryo. We generated females expressing Gal4 in the germline and carrying a UAS-H2AvGFP transgene [19]; embryos obtained from these females were centrifuged, fixed, and stained for GFP. In cleavage stage embryos, we only detected background levels of GFP signal, indicating that only limited amounts of H2AvGFP were produced during oogenesis; by syncytial blastoderm, signal was dramatically increased, suggesting that H2AvGFP was newly produced. This H2AvGFP was present both in nuclei and the lipid-droplet layer (Figure 3D). We conclude that newly synthesized H2Av can indeed localize to lipid droplets in the embryo. Using a UAS-H3GFP construct [20], we also observed an increase in GFP signal from cleavage stages to the syncytial blastoderm; but this newly made H3-GFP was detected just in the nuclei (Figure 3E), consistent with the fact that endogenous H3 fails to accumulate on lipid droplets [9, 11].

As an independent test, we transplanted lipid droplets between embryos from mothers of two different genotypes (Figure 3F). Donor embryos were from females wild-type for Jabba and expressing H2AvRFP; the RFP marks the lipid droplets, and Jabba might provide docking sites for newly made histones. Recipient embryos were from females lacking Jabba and expressing H2AvGFP; these embryos lack maternally provided H2AvGFP proteins stored on lipid droplets [11] but are able to synthesize new H2AvGFP in the embryo. We detected GFP signal on the transplanted H2AvRFP lipid droplets (Figures 3G and H), indicating that they had recruited H2AvGFP. H2Av can thus be loaded onto lipid droplets even in the embryo, and lipid droplets should be able to capture H2Av synthesized in excess.

Jabba mutant embryos are sensitive to extra copies of H2Av

Jabba mutant embryos display an abnormal H2Av/H2A ratio in their nuclei (Figure 2) as well as massive nuclear falling and reduced hatching (Figure 1). These phenotypes may be causally related because females carrying four transgenes expressing H2AvGFP and H2AvRFP fusions lay embryos that display massive nuclear falling (Figure S2) and hatch at very low rates (<1%). As the transgenes are fully functional [21], these defects are likely due to H2Av overproduction.

If the excessive H2Av accumulating in Jabba mutant nuclei is the cause of the embryonic defects, then Jabba mutants should be hypersensitive to H2Av dosage. One copy of H2AvGFP increased the percentage of Jabba embryos displaying massive nuclear falling and decreased the hatching rate; these defects were enhanced by further increase in H2Av dosage (Figures 4A and B). But in a wild-type background, one or two copies of the transgene did not cause nuclear falling. When we reduced expression of endogenous H2Av in Jabba embryos, using the hypomorphic H2Av05146 allele [22], nuclear falling and hatching defects were ameliorated (Figures 4A and B). We conclude that Jabba embryos are hypersensitive to H2Av overexpression and that the embryonic defects observed in Jabba embryos are at least partially due to excessive accumulation of H2Av in nuclei.

Figure 4. Jabba embryos are sensitive to extra copies of H2Av.

Figure 4

Open in a new tab

(A) Females of various genotypes were crossed with wild-type males, and the extent of nuclear falling was quantified as in Figure 1C. Embryos from Jabba mothers with increased H2Av dosage are more likely to show massive nuclear mislocalization. Jabbazl01 and JabbaDL are different Jabba alleles; Df = Df(2R)7158 is a large deletion that encompasses Jabba; H2Av05146 is a hypomorphic allele of H2Av. Error bars represent SDs of three repeated experiments. About 110 embryos were counted for each repeat.

(B) Embryos from Jabba mothers with extra copies of H2AvGFP hatch at reduced rates. Error bars represent SDs; over 100 embryos were counted per experiment (n=5).

(C) Double staining of blastoderm embryos for DNA (blue) and centrosomes (CNN, green). Scale bar = 10μm

**p<0.01, *p<0.05, NS: not significant

See also Figure S2.

Discussion

Balanced levels of core histones and histone variants are crucial for genome stability, correct transcription, and successful mitosis [4–6]. To ensure proper histone balance, the synthesis of different histones thus is well coordinated transcriptionally and post-transcriptionally [23–26]. However, how the histone supply is coordinated post-translationlly remains poorly studied. In Drosophila, lipid droplets employ Jabba to store H2A, H2B, and H2Av synthesized during oogenesis [11]. When synthesis of new histones is impaired, this long-term storage of maternal histones is essential for early embryogenesis [11]. In this study, we show that lipid droplets can also mediate short-term sequestration of H2Av newly synthesized in embryos. Lack of this buffering capacity leads to an abnormal nuclear H2A/H2Av ratio, to hypersensitivity to H2Av overexpression, and to DNA damage and embryonic death. Thus, lipid droplets protect against the detrimental consequences of imbalanced histone production and buffer the H2Av supply.

The existence of this buffering system may not be restricted to Drosophila embryos. Both histones and Jabba may be present on fat body lipid droplets in Drosophila larvae [27], and there are numerous reports of histones on lipid droplets in other species, from yeast and worms to mice and humans (discussed in [11]). Lipid droplets may also buffer the availability of proteins other than histones, as a number of “refugee proteins”, representing proteins from various cellular compartments, can transiently accumulate on lipid droplets [28].

Altered H2Av/H2A ratio in the nuclei of Jabba embryos

Although Drosophila embryos are endowed with an enormous pool of maternally deposited histone proteins, they synthesize further histones during the first few hours of embryogenesis (Figures 3G, I; [9]). As expression of canonical histones and histone variants is regulated by distinct mechanisms (e.g., histone variant mRNAs contain Poly(A) tails, while canonical histones mRNAs lack them [29]), it is presumably challenging to produce these histones in a perfectly coordinated manner. Indeed, we have evidence that synthesis of H2A and of H2Av is imbalanced: in Jabba embryos, the levels of these histones rise at different rates (Figures 3A and B).

After H2A and H2Av have been synthesized, they typically dimerize with H2B, bind to histone chaperones in the cytoplasm, and are imported into the nucleus [23, 30]. If their binding partner H2B is limiting, H2A and H2Av might compete with each other for assembly into heterodimers, leading to skewed incorporation into chromatin. Consistent with this idea, H3 competes with the H3 variant CENP-A for chromatin incorporation at centromeres when the H3/CENP-A ratio is artificially increased [7, 31]. In embryos that lack Jabba, imbalanced production of H2A and H2Av indeed results in an altered H2A/H2Av ratio in the nuclei (Figures 2 and S1). We propose that in the wild type supernumerary H2Av generated due to imbalanced synthesis is sequestered on lipid droplets, allowing the embryo to maintain a proper H2A/H2Av ratio.

In other cells, excess non-chromatin histones are proteolysed with a half-life of 30 minutes [32]. In early Drosophila embryos, the cell cycles are extremely rapid (9–20 min at 25°C) and, in particular, lack G2 phases [33]. Thus, there may simply not be enough time for this surveillance system to function, making buffering on lipid droplets particularly crucial. Intriguingly, we observed an imbalanced nuclear H2A/H2Av ratio in nuclear cycles with relatively short interphases (e.g., less than 6 min in cycle 10); in cycle 14, where the interphase last over an hour, the H2A/H2Av ratio in Jabba nuclei returns to normal.

It is unresolved if the dramatic increase in nuclear H2Av levels in Jabba mutants is associated with a concomitant decrease in nuclear H2A. Since H2A is much more abundant than H2Av, the expected small reduction in H2A accumulation may not be detectable with the sensitivity of our methods (Figure S1). Regardless whether absolute levels of H2A are changed, nuclear H2Av/H2A ratio is clearly altered. This elevated H2Av/H2A ratio might disrupt gene expression since H2Av is involved in both repression and activation of transcription [34–36]. In the wild type, we detected an increase in nuclear H2Av from cycle 10 to cycle 13 (Figures 2A and D), coinciding with the increase in zygotic gene expression through these cycles [37]. Thus, the increased H2Av/H2A ratio in Jabba mutants might result in untimely expression of certain genes; premature activation of zygotic transcription has indeed been linked to mitotic errors and embryonic death [38].

Similarities between Jabba and histone chaperones

We propose that Jabba buffers the histone supply by transiently sequestering excess histones and keeping them available for later use. Such buffering has also been reported for histone chaperones [3, 39]. Similarly, Jabba is responsible for storing maternal histones H2A/H2B/H2Av in Drosophila embryos; a similar function is performed by the chaperone nucleoplasmin in Xenopus embryos [40]. Jabba and histone chaperones share a number of other characteristics. Like chaperones, Jabba exists in transient protein complexes with histones [11], and the bound histones can be released to enter nuclei [9]. Histones binding to lipid droplets – and thus presumably to Jabba – depends on electrostatic interactions [9]; a highly acidic stretch in the C-terminal half of Jabba is an intriguing candidate for mediating this interaction. Although it remains unclear how chaperones interact with histones to assemble nucleosomes, charge complementarity and acidic tracts are considered to be important [41].

In this paper, we provide evidence for the transient sequestration of histone H2Av on lipid droplets via Jabba. Since Jabba recruits H2A and H2B to lipid droplets during oogenesis, we predict that lipid droplets can also buffer overproduction of H2A or H2B; this property does not appear to be critical in early embryos since we did not detect changes in the H2B/H3 ratio in Jabba embryos but may be physiologically important at other developmental stages. Since H3 is absent from embryonic lipid droplets [9, 11], we predict that this buffering function does not extend to the other canonical histones. The mechanistic basis for Jabba’s specificity is an intriguing, unsolved problem.

Jabba embryos are sensitive to high temperature

In Jabba embryos, nuclear falling and hatching defects only become apparent at 25°C, a temperature well within the normal range experienced by Drosophila melanogaster in the wild and not a stressful condition per se [42]. Drosophila embryogenesis is highly streamlined to be as fast as possible [43]. At 25°C, development is even faster than at room temperature, limiting the time available for embryos to remove excess histones. In the absence of lipid-droplet sequestration, this should increase the imbalance in H2A and H2Av accumulation in nuclei even further. At 25°C, nuclear H2Av levels of Jabba embryos are indeed slightly higher than at 21°C (Figures S1C and D).

The reduced hatching rates of Jabba mutants at elevated temperatures suggest that sequestering histones on lipid droplets provides a selective advantage, enabling wild-type flies to thrive across a broader temperature range and to withstand temperature variations. We thus propose that the Jabba-based machinery contributes to thermal robustness of embryogenesis.

Supplementary Material

01

Highlights.

  • In Drosophila embryos, lipid droplets can sequester newly synthesized H2Av.

  • This sequestration protects embryos against H2Av overexpression.

  • Lack of sequestration results in an abnormal H2Av/H2A ratio in the nucleus and DNA damage.

Acknowledgments

We thank Robert Glaser and Timothy Megraw for antibodies, Alexei Tulin, Stephan Heidmann, Kami Ahmed, and the Bloomington Drosophila Stock Center for fly strains. We are grateful to two anonymous reviewers for their feedback that allowed us to improve the manuscript. This work was supported by NIH grant R01GM102155 to MAW.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Kim UJ, Han M, Kayne P, Grunstein M. Effects of histone H4 depletion on the cell cycle and transcription of Saccharomyces cerevisiae. Embo J. 1988;7:2211–2219. doi: 10.1002/j.1460-2075.1988.tb03060.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Han M, Chang M, Kim UJ, Grunstein M. Histone H2B repression causes cell-cycle-specific arrest in yeast: effects on chromosomal segregation, replication, and transcription. Cell. 1987;48:589–597. doi: 10.1016/0092-8674(87)90237-6. [DOI] [PubMed] [Google Scholar]
  • 3.Gunjan A, Verreault A. A Rad53 kinase-dependent surveillance mechanism that regulates histone protein levels in S. cerevisiae. Cell. 2003;115:537–549. doi: 10.1016/s0092-8674(03)00896-1. [DOI] [PubMed] [Google Scholar]
  • 4.Au WC, Crisp MJ, DeLuca SZ, Rando OJ, Basrai MA. Altered dosage and mislocalization of histone H3 and Cse4p lead to chromosome loss in Saccharomyces cerevisiae. Genetics. 2008;179:263–275. doi: 10.1534/genetics.108.088518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Meeks-Wagner D, Hartwell LH. Normal stoichiometry of histone dimer sets is necessary for high fidelity of mitotic chromosome transmission. Cell. 1986;44:43–52. doi: 10.1016/0092-8674(86)90483-6. [DOI] [PubMed] [Google Scholar]
  • 6.Singh RK, Liang D, Gajjalaiahvari UR, Kabbaj MH, Paik J, Gunjan A. Excess histone levels mediate cytotoxicity via multiple mechanisms. Cell Cycle. 2010;9:4236–4244. doi: 10.4161/cc.9.20.13636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Blower MD, Sullivan BA, Karpen GH. Conserved organization of centromeric chromatin in flies and humans. Developmental cell. 2002;2:319–330. doi: 10.1016/s1534-5807(02)00135-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Farese RV, Jr, Walther TC. Lipid droplets finally get a little RE-S-P-E-C-T. Cell. 2009;139:855–860. doi: 10.1016/j.cell.2009.11.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Cermelli S, Guo Y, Gross SP, Welte MA. The lipid-droplet proteome reveals that droplets are a protein-storage depot. Curr Biol. 2006;16:1783–1795. doi: 10.1016/j.cub.2006.07.062. [DOI] [PubMed] [Google Scholar]
  • 10.Lanzotti DJ, Kaygun H, Yang X, Duronio RJ, Marzluff WF. Developmental control of histone mRNA and dSLBP synthesis during Drosophila embryogenesis and the role of dSLBP in histone mRNA 3′ end processing in vivo. Mol Cell Biol. 2002;22:2267–2282. doi: 10.1128/MCB.22.7.2267-2282.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Li Z, Thiel K, Thul PJ, Beller M, Kuhnlein RP, Welte MA. Lipid droplets control the maternal histone supply of Drosophila embryos. Curr Biol. 2012;22:2104–2113. doi: 10.1016/j.cub.2012.09.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Marzluff WF, Tatomer DC. Histones: sequestered by Jabba in fatty storehouse. Curr Biol. 2012;22:R951–953. doi: 10.1016/j.cub.2012.10.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Ashburner M. Drosophila a Laboratory Handbook. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989. p. 181. [Google Scholar]
  • 14.Sullivan W, Minden JS, Alberts BM. daughterless-abo-like, a Drosophila maternal-effect mutation that exhibits abnormal centrosome separation during the late blastoderm divisions. Development. 1990;110:311–323. doi: 10.1242/dev.110.2.311. [DOI] [PubMed] [Google Scholar]
  • 15.Sullivan E, Santiago C, Parker ED, Dominski Z, Yang X, Lanzotti DJ, Ingledue TC, Marzluff WF, Duronio RJ. Drosophila stem loop binding protein coordinates accumulation of mature histone mRNA with cell cycle progression. Genes Dev. 2001;15:173–187. doi: 10.1101/gad.862801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Leach TJ, Mazzeo M, Chotkowski HL, Madigan JP, Wotring MG, Glaser RL. Histone H2A.Z is widely but nonrandomly distributed in chromosomes of Drosophila melanogaster. J Biol Chem. 2000;275:23267–23272. doi: 10.1074/jbc.M910206199. [DOI] [PubMed] [Google Scholar]
  • 17.Baldi S, Becker PB. The variant histone H2A.V of Drosophila--three roles, two guises. Chromosoma. 2013;122:245–258. doi: 10.1007/s00412-013-0409-x. [DOI] [PubMed] [Google Scholar]
  • 18.Tran SL, Welte MA. In-vivo centrifugation of Drosophila embryos. J Vis Exp. 2010;40:2005. doi: 10.3791/2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Kotova E, Lodhi N, Jarnik M, Pinnola AD, Ji Y, Tulin AV. Drosophila histone H2A variant (H2Av) controls poly(ADP-ribose) polymerase 1 (PARP1) activation in chromatin. Proc Natl Acad Sci U S A. 2011;108:6205–6210. doi: 10.1073/pnas.1019644108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Henikoff S, Ahmad K, Platero JS, van Steensel B. Heterochromatic deposition of centromeric histone H3-like proteins. Proc Natl Acad Sci U S A. 2000;97:716–721. doi: 10.1073/pnas.97.2.716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Clarkson M, Saint R. A His2AvDGFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker for Drosophila chromosome behavior. DNA Cell Biol. 1999;18:457–462. doi: 10.1089/104454999315178. [DOI] [PubMed] [Google Scholar]
  • 22.Swaminathan J, Baxter EM, Corces VG. The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin. Genes Dev. 2005;19:65–76. doi: 10.1101/gad.1259105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Marzluff WF, Wagner EJ, Duronio RJ. Metabolism and regulation of canonical histone mRNAs: life without a poly(A) tail. Nature reviews Genetics. 2008;9:843–854. doi: 10.1038/nrg2438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Isogai Y, Keles S, Prestel M, Hochheimer A, Tjian R. Transcription of histone gene cluster by differential core-promoter factors. Genes Dev. 2007;21:2936–2949. doi: 10.1101/gad.1608807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Marzluff WF, Gongidi P, Woods KR, Jin J, Maltais LJ. The human and mouse replication-dependent histone genes. Genomics. 2002;80:487–498. [PubMed] [Google Scholar]
  • 26.Wagner EJ, Burch BD, Godfrey AC, Salzler HR, Duronio RJ, Marzluff WF. A genome-wide RNA interference screen reveals that variant histones are necessary for replication-dependent histone pre-mRNA processing. Mol Cell. 2007;28:692–699. doi: 10.1016/j.molcel.2007.10.009. [DOI] [PubMed] [Google Scholar]
  • 27.Beller M, Riedel D, Jansch L, Dieterich G, Wehland J, Jackle H, Kuhnlein RP. Characterization of the Drosophila lipid droplet subproteome. Mol Cell Proteomics. 2006;5:1082–1094. doi: 10.1074/mcp.M600011-MCP200. [DOI] [PubMed] [Google Scholar]
  • 28.Welte MA. Proteins under new management: lipid droplets deliver. Trends Cell Biol. 2007;17:363–369. doi: 10.1016/j.tcb.2007.06.004. [DOI] [PubMed] [Google Scholar]
  • 29.Marzluff WF. Metazoan replication-dependent histone mRNAs: a distinct set of RNA polymerase II transcripts. Curr Opin Cell Biol. 2005;17:274–280. doi: 10.1016/j.ceb.2005.04.010. [DOI] [PubMed] [Google Scholar]
  • 30.Kusch T, Florens L, Macdonald WH, Swanson SK, Glaser RL, Yates JR, 3rd, Abmayr SM, Washburn MP, Workman JL. Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions. Science. 2004;306:2084–2087. doi: 10.1126/science.1103455. [DOI] [PubMed] [Google Scholar]
  • 31.Castillo AG, Mellone BG, Partridge JF, Richardson W, Hamilton GL, Allshire RC, Pidoux AL. Plasticity of fission yeast CENP-A chromatin driven by relative levels of histone H3 and H4. PLoS Genet. 2007;3:e121. doi: 10.1371/journal.pgen.0030121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Singh RK, Kabbaj MH, Paik J, Gunjan A. Histone levels are regulated by phosphorylation and ubiquitylation-dependent proteolysis. Nat Cell Biol. 2009;11:925–933. doi: 10.1038/ncb1903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Foe VE, Alberts BM. Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. Journal of cell science. 1983;61:31–70. doi: 10.1242/jcs.61.1.31. [DOI] [PubMed] [Google Scholar]
  • 34.Mavrich TN, Jiang C, Ioshikhes IP, Li X, Venters BJ, Zanton SJ, Tomsho LP, Qi J, Glaser RL, Schuster SC, et al. Nucleosome organization in the Drosophila genome. Nature. 2008;453:358–362. doi: 10.1038/nature06929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Weber CM, Henikoff JG, Henikoff S. H2A.Z nucleosomes enriched over active genes are homotypic. Nat Struct Mol Biol. 2010;17:1500–1507. doi: 10.1038/nsmb.1926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Lu J, Ruhf ML, Perrimon N, Leder P. A genome-wide RNA interference screen identifies putative chromatin regulators essential for E2F repression. Proc Natl Acad Sci U S A. 2007;104:9381–9386. doi: 10.1073/pnas.0610279104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Edgar BA, Schubiger G. Parameters controlling transcriptional activation during early Drosophila development. Cell. 1986;44:871–877. doi: 10.1016/0092-8674(86)90009-7. [DOI] [PubMed] [Google Scholar]
  • 38.Perez-Montero S, Carbonell A, Moran T, Vaquero A, Azorin F. The embryonic linker histone H1 variant of Drosophila, dBigH1, regulates zygotic genome activation. Developmental cell. 2013;26:578–590. doi: 10.1016/j.devcel.2013.08.011. [DOI] [PubMed] [Google Scholar]
  • 39.Groth A, Ray-Gallet D, Quivy JP, Lukas J, Bartek J, Almouzni G. Human Asf1 regulates the flow of S phase histones during replicational stress. Mol Cell. 2005;17:301–311. doi: 10.1016/j.molcel.2004.12.018. [DOI] [PubMed] [Google Scholar]
  • 40.Dilworth SM, Black SJ, Laskey RA. Two complexes that contain histones are required for nucleosome assembly in vitro: role of nucleoplasmin and N1 in Xenopus egg extracts. Cell. 1987;51:1009–1018. doi: 10.1016/0092-8674(87)90587-3. [DOI] [PubMed] [Google Scholar]
  • 41.Dutta S, Akey IV, Dingwall C, Hartman KL, Laue T, Nolte RT, Head JF, Akey CW. The crystal structure of nucleoplasmin-core: implications for histone binding and nucleosome assembly. Mol Cell. 2001;8:841–853. doi: 10.1016/s1097-2765(01)00354-9. [DOI] [PubMed] [Google Scholar]
  • 42.Welte MA, Tetrault JM, Dellavalle RP, Lindquist SL. A new method for manipulating transgenes: engineering heat tolerance in a complex, multicellular organism. Curr Biol. 1993;3:842–853. doi: 10.1016/0960-9822(93)90218-d. [DOI] [PubMed] [Google Scholar]
  • 43.Karr TL, Mittenthal JE. Adaptive mechanisms that accelerate embryonic development in Drosophila. In: Mittenthal JE, Baskin AB, editors. Principles of Organization in Organisms; Santa Fe Institutes Studies in the Sciences of Complexity -Proceedings; Urbana: Addison-Wesley publishing company; 1992. pp. 95–108. [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS598176-supplement-01.pdf (1.9MB, pdf)

RESOURCES