Baculovirus-encoded protein expression for epigenomic profiling in Drosophila cells

Abstract

The expression and genome-wide mapping of epitope-tagged DNA- and chromatin-binding proteins in cultured cells has become a powerful strategy for epigenome characterization, especially in Drosophila, where cell lines derived from numerous tissues are now available. However this strategy relies on establishing transfected cell lines, which is time-consuming and introduces variability. Here we show that baculovirus-encoded proteins can be efficiently produced following infection of Drosophila cell lines of different types. Using chromatin affinity purification, we show that epitope-tagged proteins produced in baculovirus-infected cells provide genome-wide profiles of the histone variant H2Av that are comparable to those produced by plasmid-transfected cells. The ability to express multiple epitope-tagged proteins for epigenome analysis from a single culture, and to do this in a variety of Drosophila cell lines, significantly extends the range of epigenome analysis.

Introduction

Recent technological advances have made genome-wide profiling of epigenomic features a routine and cost-effective procedure¹. Using tiling arrays or short-read sequencing, high-resolution maps of transcription factor binding and chromatin features can be readily obtained for entire genomes of the size of Drosophila². However, in contrast to the rapid improvements in readout technologies, only incremental progress has been made in obtaining suitable quantities of high-quality chromatin needed for genome-wide studies. For applications involving chromatin immunoprecipitation (ChIP), antibodies with high affinity and specificity are necessary to obtain reliable results³. Even when excellent antibodies are available, comparisons between profiles based on different antibodies may be uncertain because of affinity or other differences between them. An alternative strategy is to use epitope-tagged proteins encoded on transgenes. In such cases, a common affinity reagent, such as a high-affinity antibody against the tag, is used to pull down chromatin without the complications of using different antibodies for different epitopes. Transgenic approaches are also required for alternative chromatin profiling methods, such as DamID⁴ and biotin-tagging⁵.

Cell lines are especially desirable for epigenomic profiling, because unlike tissues with multiple cell types, they provide large amounts of uniform sources of chromatin that can be conveniently purified using rapid and simple methods. Drosophila cell lines have been especially popular for this purpose, and the embryonic S2 and Kc167 lines have been used in hundreds of studies for decades⁶. More recently, several other cell lines derived from embryos, imaginal disks and adult tissues have been introduced, extending the benefits of cell lines to study many developmental processes⁷. To perform epitope tagging in cell lines, methods must be developed for the uptake and expression of transgenes. Transgenic methods thus far have been limited to S2 and Kc cells, where plasmid-based vectors are taken up by cells using a variety of transfection methods^{8, 9}. Even for these cells, transfection has numerous limitations, including unpredictable efficiency, the need for selection and the long time required to obtain permanently transformed lines. Once established, these lines can differ from one another in phenotype, or expression can become extinguished with time, which complicates interpretation of results. Ideally, what is desired is a system for the rapid introduction of transgenes that express at predictable levels in a variety of cell lines, such that profiling and other applications can be reliably and generally performed.

To address this need, we have adapted a previously described system for delivery of exogenous proteins expressed from baculovirus genomes in Drosophila cells¹⁰. Unlike baculovirus infection of native host (moth) cells, which results in lysis, fly cells express baculovirus-encoded proteins but remain intact¹¹. We have found conditions for efficient baculovirus infection, such that a single batch of cells can be infected in parallel with different constructs, followed shortly thereafter by epigenomic profiling without selection. We show that epitope-tagged histone variants express at levels that depend upon the multiplicity of infection. Importantly, we find that this system is adaptable to cell lines for which no transgenic methods have been described. Expression levels are similar to what we have observed with good transfected cell lines, and the results of chromatin profiling are comparable. We have recently adapted this system for epigenomic profiling of histone variants as part of our NIH/NHGRI-supported modENCODE project².

Results

Expression and biotinylation of baculovirus-encoded proteins in Drosophila cells.

To test use of baculovirus-encoded proteins for epigenomic analysis, we chose a histone variant, H2A.Z, which has been the subject of numerous studies in a variety of eukaryotes, including Drosophila^{12, 13}. H2Av, which is both the Drosophila H2A.Z ortholog and its H2A.X, replaces its canonical counterpart, H2A, and is known to be enriched around promoters¹⁴. We used a biotin-tagging strategy, in which expression of the Escherichia coli BirA gene encoding biotin ligase specifically biotinylates the lysine of the 23-aa Biotin Ligase Recognition Peptide (BLRP) fused to the N-termini of both H2Av and H2A. BLRP-H2Av and BLRP-H2A flanked by the D. melanogaster metallothionein promoter on the 5’ side, and the alcohol dehydrogenase (ADH) 3’ UTR on the 3’ side, were cloned into a Gateway plasmid vector. By using the Cu⁺⁺-inducible metallothionein promoter, we could control transgene expression upon addition of CuSO₄ to the medium¹⁵. We separately cloned the E. coli BirA gene, flanked by the D. melanogaster Actin-5C promoter and the ADH 3’ UTR into the same Gateway vector (Figure 1A). Cloning was followed by site-directed recombination into the linear baculovirus vector and transfection into SF9 cells. To produce the first, low-titer “P1” recombinant virus stock, the transfected SF9 cells were incubated for several days with ganciclovir, a nucleoside analog that incorporates and inhibits DNA replication upon phosphorylation by a thymidine kinase if recombination does not occur. (Figure 1B). To amplify virus, fresh SF9 cells were infected with the P1 stock and incubated again with ganciclovir for several days to generate a more concentrated “P2” virus stock that was plated for individual plaques, each of which was assayed for expression. The best expressing isolate for each protein was amplified and used to prepare stocks for infection of S2 cells. Stocks were titered (Figure 1C), and the optimal multiplicity of infection (MOI) was determined using a western blot assay (Figure 1D).

Figure 1. Flow chart describing the method.

Schematized Procedure for generating, amplifying and analyzing baculovirus. (A) A gene of Interest (blue) is cloned into the entry vector enabling recombination of the Gene of Interest into the baculovirus DNA, which can then be transfected into SF9 cells. Non-recombinant baculovirus is selected against by the addition of ganciclovir. (B) This first, low-titer P1 stock is collected by spinning out the cells and a portion is used to re-infect a fresh plate of SF9 cells, again adding ganciclovir to generate a more concentrated P2 stock. At this point, individual virus particles are isolated by performing a plaque assay with serial dilutions of the virus and individual virus plaques are picked out, vortexed in a small volume of medium and used to infect a fresh batch of cells. Baculovirus isolates are assayed for expression by western blot assay to determine which will be amplified for further use. (C) To determine the concentration of virus, SF9 cells are infected with a range of diluted virus and after 36 hours the average diameter of the cells in each infection is used to estimate the number of plaque forming units (pfu) per microliter (Reference ¹⁷, supplemental figure 2). (D) Titered virus stock is used to infect D. melanogaster S2 cells with increasing amounts of virus per cell and the resulting induced cultures are analyzed by western assay to determine the multiplicity of infection (MOI) at which cells are saturated.

When we infected S2 cells growing in their standard serum-free medium, and expressed the BLRP-H2Av protein by inducing the metallothionein promoter with Cu⁺⁺, we detected very little protein above background by western analysis (Figure 2A, lane 4). However, addition of serum to the medium led to a much stronger signal, and so we subsequently added serum in all of our experiments. We note that addition of serum to baculovirus stocks is recommended for storage to reduce proteolytic degradation¹⁶, so it is likely that the weak signal observed when cells were grown in serum-free medium resulted from proteolysis in the absence of competitor protein in the medium. When we compared the signal for baculovirus-produced biotinylated BLRP-H2Av to that from Cu⁺⁺-induced stably transformed cells that had been induced for the same length of time (48 hours), we found that the baculovirus-infected cells expressed at a somewhat reduced level compared to the stable lines (Figure 2A, lanes1 and 3). This reduced level is not attributable to effects of infection on cell viability or proliferation, because infected cells showed similar viability levels (95%) and similar proliferation rates (~20 hr per cell cycle, Figure S1) to uninfected cells. It is notable that we consistently obtained this level of expression after only one hour of infection, in contrast to the ~6-week period required to obtain each stable cell line.

Figure 2. Efficient expression and biotinylation of BLRP-tagged H2Av.

Western analysis of cell extracts. A) Lane 1, S2 cells stably transformed with BLRP-H2Av; Lane 2, uninfected S2 cells; Lane 3, virally infected S2 cells with serum; Lane 4, virally infected S2 cells lacking serum. B) Lane 1, virally infected D16 cells; Lane 2, stably transformed S2 cells. Viral infection was at MOI = 100 for 1 hr followed by Cu⁺⁺ induction of transformed and infected cells and a 48-hr incubation. Probing was performed with IRD700-streptavidin (green) to detect BLRP-H2Av, and with anti-histone H3 followed by IRD800 (red), to detect the H3 loading control. White patches in outer lanes are from saturated pixels.

Embryonic cell lines, such as S2 and Kc167, are easily transformed with plasmids, allowing sufficiently high levels of expression from stable lines, however, it is not clear that cell lines derived from later stages of development are equally amenable to transformation and high-level expression. The ability of baculovirus to infect mammalian cells^{17, 18} implies a broad host range and suggests that efficient infection might be achieved with a wide variety of Drosophila cell lines. To test this, we infected ML-DmD16-c3 (D16), a cell line derived from the dorsal mesothoracic disc of a third instar larva, and assayed for protein expression by western analysis. Indeed, high-level expression of H2Av expression was seen (Figure 2B). We also tested a line derived from the central nervous system of a third instar larva (ML-DmBG3-c2), and found a similar high level of expression (Figure S2). Although we tested one line that showed no expression (CME W1 cl.8+), this line requires the addition of cell extract for growth, and it is possible that the extract either degraded the virus or inhibited infection. We conclude that the baculovirus infection system is generally applicable to Drosophila cell lines.

Optimizing the multiplicity of infection

To determine the relationship between amount of virus and level of expression, we infected both S2 and D16 cells with increasing multiplicity of infection (MOI) and assayed by western analysis. With S2 cells, we observed that expression was first detectable at MOI = 2 plaque-forming units (pfu) per cell, and increased with increasing virus concentration, up to MOI = 100, whereupon expression plateaued for both H2A and H2Av (Figure 3A–B)¹⁹. With D16 cells, maximal expression was reached at MOI = 20 for BLRP-H2A and 6 for BLRP-H2Av (Figure 3C–D). The similar dependence of expression on viral titer for both BLRP-H2A and BLRP-H2Av suggests that the cell line, and not the gene sequence, determines efficiency. We also observed saturation in ML-DmBG3-c2 cells at an MOI similar to that of an S2-derived isolate (S2-DGRC) (Figure S2).

Figure 3. Expression and biotinylation of BLRP-tagged histones at different MOI.

S2 cells were infected with viruses containing either a BLRP-H2A (A) or a BLRP-H2Av (B) construct, with an increase in viral titer represented in each lane from left to right, where the far left lane in each series is from uninfected cells. Arrowheads indicate MOI at which saturation was reached in each series. C) and D) Same as A) and B) except using D16 cells. The asterisk (*) in each image marks an abundant biotinylated endogenous protein band that serves as a loading control.

We next asked whether the dependence of expression on MOI resulted from an increase in the fraction of infected cells, in the number of active viruses per cell, or in the level of biotinylation of BLRP. To test whether incorporation per cell was efficient, we constructed a virus expressing FLAG-H2Av under the metallothionein promoter and infected S2 cells for 1 hr, 6 hr or 24 hr at high MOI, induced with Cu⁺⁺, and stained cells using an anti-FLAG antibody (Figure S3). We then counted the number of DAPI-stained cells with and without FLAG signal, and observed that ~50–75% of the cells were infected with no consistent change in FLAG signal with increasing time of infection at two different MOI levels (66 and 100) (Table 1). Therefore, half or more of the S2 cells are competent to take up virus particles regardless of total exposure to virus. This is consistent with earlier studies showing that S2 cells take up abundant virus at high MOI^{10, 11}. To determine whether the increase in signal with increasing MOI is due to an increasing number of active viruses per cell, we compared cells infected with either a combination of BLRP-H2Av and BirA viruses or FLAG-H2Av alone at high MOI. Since both BLRP-H2Av and BirA are needed to detect the BLRP tag, whereas only a single virus is needed to detect FLAG, we can distinguish these two situations by comparing the total fraction of nuclei expressing the tag after infection at the same MOI for the same length of time. We found that a far greater fraction of FLAG-H2Av infected cells showed signal than BLRP-H2Av infected cells in all comparisons. Since the same number of H2Av-containing viruses are estimated to be present in both cases, we attribute the low number of cells with signal when both viruses are present to a low rate of biotinylation. In confirmation of this interpretation, we note that there were consistent increases in the fraction of labeled cells with increasing time of exposure to virus (Table 1). We conclude that competent cells efficiently take up and express virus, but that the relatively low efficiency of biotinylation limits the signal detected when both viruses are present.

Table 1.

Cytological assay of infected S2 cells at different viral exposure times

Virus, MOI, Exposure Time	Nuclei with signal (%)	Images analyzed
S2 cells, no virus	0.8	4
BLRP-H2Av/BirA Stable Cell line no Cu++	0.7	3
BLRP-H2Av/BirA Stable Cell line plus Cu++	24.5	10
BLRP-H2Av/BirA, 100 MOI, 1h	10.0	10
BLRP-H2Av/BirA, 100 MOI, 6h	20.3	10
BLRP-H2Av/BirA, 100 MOI, 24h	29.1	10
BLRP-H2Av/BirA, 200 MOI, 1h	28.2	4
FLAG_H2Av, 66 MOI, 1h	47.3	10
FLAG_H2Av, 66 MOI, 6h	62.6	10
FLAG_H2Av, 66 MOI, 24h	49.1	10
FLAG_H2Av, 100 MOI, 1h	59.0	10
FLAG_H2Av, 100 MOI, 6h	74.2	10
FLAG_H2Av, 100 MOI, 24h	66.6	10

Images from both DAPI and GFP signals were analyzed using NIH ImageJ (http://rsb.info.nih.gov/ij) by counting all nuclei in the DAPI channel and nuclei with recombinant protein signal in the GFP/FITC channel and compared to determine the percentage of nuclei infected.

Baculovirus-encoded and plasmid-encoded H2Av produce similar chromatin profiles

To provide a challenging test of our protocol, we chose to profile epitope-tagged H2Av. Ectopically produced H2Av becomes diluted with endogenous H2Av, which is very highly expressed, as expected for a protein that is present in 20% of all nucleosomes²⁰; therefore, a relatively high level of epitope-tagged H2Av expression is needed to obtain yields sufficient for genome-wide chromatin profiling. We sought to determine whether baculovirus-encoded H2Av provides epigenomic profiles that are comparable to those produced by plasmid-encoded H2Av. To make this comparison, we performed genome-wide profiling of H2Av from cells that were induced to produce BLRP-H2Av, encoded by a stably integrated transgene, and FLAG-H2Av, encoded by a transgene introduced by baculovirus infection. Sufficient quantities of pulled-down chromatin were obtained in both cases to allow for genome-wide profiling on microarrays by hybridization of DNA labeled by random priming without PCR amplification. We found the resulting profiles to be similar overall (Figure 4), where the Pearson correlation coefficient between log-ratio means for 25-bp intervals from −3 kb to + kb is r = 0.86. This is above the acceptable level for replicate arrays (r = 0.8) adopted for our modENCODE project², and so the observed difference between these two samples is likely to be due to sample-to-sample variation within an experiment. H2Av is highly enriched at the +1 nucleosome of actively transcribed genes relative to the transcription start site and shows depletion over gene bodies and depletion at the transcriptional termination site (Figure 4A and C). At inactive genes, the profiles for baculovirus-encoded and plasmid-encoded H2Av are also very similar and remain relatively featureless, with no enrichment at the +1 nucleosome (Figure 4B and D). These profiles resemble those reported for H2Av in previous studies^{12, 13}, confirming that baculovirus-encoded epitope-tagged chromosomal proteins are satisfactory for epigenome profiling.

Figure 4. Comparison between baculovirus-encoded FLAG-tagged and transfected BLRP-tagged H2Av genome-wide profiles.

The baculovirus-expressed FLAG-H2Av profile closely resembles the stably transfected BLRP-H2Av cell line profile. A) Ends analysis obtained by pulldown of FLAG-H2Av and BLRP-H2Av containing nucleosomes profiled as the log₂-ratio of pulldown/input, +/− 1.5 kb from the 5’ and 3’ end of active genes. Both profiles show strong enrichment at the +1 nucleosome downstream of the transcription start site and depletion at the 3’ transcription termination site. B) Same as A except for inactive genes. C) Heat maps of FLAG-H2Av log₂-ratio of pulldown/input sorted by decreasing gene expression, +/− 1 kb from 5’ and 3’ end of 9247 genes. D) Same as C except with BLRP-H2Av pulldown/input.

Discussion

Taking full advantage of technological improvements in epigenomic methods is a major challenge for Drosophila developmental biologists, who must cope with the difficulty of obtaining pure cell types. Fortunately, cell lines provide essentially unlimited amounts of pure material, and in theory virtually eliminate cell-to-cell variation. Furthermore, the availability of an increasing selection of Drosophila cell-type-specific lines makes them attractive for a wide variety of developmental studies. Viruses have been widely used as vectors for introducing transgenes into mammalian cell lines for epigenomic and other in vivo studies^{21, 22}, but thus far this strategy has not been applied to Drosophila.

We have extended the use of baculovirus-encoded protein expression to provide a general tool for epigenomic profiling. Our strategy has several advantages over the production of stable cell lines. 1) Starting from a single culture, multiple infections provide multiple biological replicates with minimal variation between them, an important factor for obtaining reproducible epigenomic data. 2) The use of a single viral stock for infection of diverse cell lines broadens the scope of epitope-tagging strategies, which thus far have been limited to embryonic cell lines. 3) The high efficiency of infection leads to expression in most cells at high MOI. 4) Our system does not involve selection, so that there is no opportunity for differential selection between individual cell lines that could lead to divergence and be mistaken for differences between the encoded proteins. 5) Liquid nitrogen storage, resuscitation and maintenance of the numerous independent stably transformed cell lines is eliminated with the baculovirus system, which requires only refrigeration to maintain viral stocks. 6) Our system allows for a high level of flexibility in controlling expression level via Cu⁺⁺ induction and viral MOI, a feature that should be especially useful for epigenomic profiling methods that are sensitive to levels of transgene expression, such as DamID, which requires very low levels of constitutive expression in order to avoid saturation. Thus, the baculovirus system of gene expression, which is widely used to produce large quantities of proteins for in vitro studies, finds a new application to complement traditional in vivo studies.

Materials and Methods

Baculovirus constructs

Three plasmid constructs that we had previously used for transfection and inducible expression in Drosophila cells^{5, 13} were cloned into a Gateway vector. To create BLRP-H2A and BLRP-H2Av expression plasmids, we digested pRMHa3-BLRP-H2A and pRMHa3-BLRP-H2Av with PvuII (NEB) to release a composite insert consisting of the D. melanogaster metallothionein promoter, in-frame fusions of a 23-aa Biotin Ligase Recognition Peptide (BLRP), a TEV cleavage site, and the coding sequence of a D. melanogaster histone H2A or H2Av gene, and the D. melanogaster ADH 3-’UTR derived from the pRMHA3 plasmid. To create a BirA expression plasmid, we digested plasmid pRMHa3-BirA with NdeI and AclI to release a composite insert consisting of D. melanogaster Actin-5C promoter and the ADH 3’ UTR flanking the BirA coding sequence. Excised fragments were gel-purified and made blunt-ended and A-tailed by incubation with Ex-Taq Polymerase (Takara) and deoxynucleoside triphosphates for 30 minutes at 68°C, and cloned into the pCR^®8/GW/TOPO^® TA Cloning vector (Invitrogen). To create a FLAG-H2Av expression plasmid, we first digested plasmid pRMHa3-BLRP-H2Av with NcoI and NotI to generate an insert containing the coding region of H2Av and the 3’ UTR but lacking the BLRP and TEV cleavage sequences. We then directionally cloned in annealed oligonucleotides containing the 66-bp 3x FLAG sequence flanked by NcoI and NotI restriction sites (Sigma). We PCR amplified the resulting metallothionein promoter-FLAG-H2Av-ADH 3’ UTR composite sequence (Invitrogen, Platinum Taq), which we ligated into the pENTR/D-TOPO vector (Invitrogen).

A recombination reaction between each Gateway (GW) entry clone and the Baculovirus N-terminal Linear DNA Gateway^® Cassette (Invitrogen) was performed according to the manufacturer’s instructions, using the recommended procedures for increased efficiency. Each recombination event was confirmed via PCR using a primer designed to the polyhedrin promoter 5’-ATTCATACCGTCCCACCATC-3’ and a primer specific to each gene of interest.

Transfection and virus amplification

Transfection of SF9 cells was performed as described in the BaculoDirect^™ Baculovirus Expression Systems protocol. After 3 rounds of amplification to increase virus concentration, the activity of the system was confirmed by adding both inducible metallothionein-BLRP-Histone and constitutively expressed pAC5-BirA virus to a sample of SF9 cells with the addition of copper to 0.5mM. Cells were harvested after a 48 hour induction time, pelleted and lysed in 2xSDS loading dye before analysis via SDS-PAGE. Biotinylated BLRP-tagged histones were detected by western analysis with streptavidin-HRP. Virus particles were subjected to serial dilution and plaque assays in order to isolate individual virus, and amplification was performed before final testing of individual clones for confirmation of expression.

Virus titering

The concentration of virus in each baculovirus stock was determined using a method in which the average diameter of a sample of cells infected with a known dilution of virus stock provides a measure of the virus concentration in each dilution²³ We followed the protocol as described, except that we scaled it down to a final volume of 1mL per dilution, and increased the infection period from 24 hours to 36 hours to compensate for the lower incubation temperature of 22°C used in our lab.

Maintenance of cell lines

SF9 cells (Invitrogen 11496015) were maintained in SF900-II SFM medium (Invitrogen 10902–088) supplemented with L-Glutamine (Sigma G7513) to a final concentration of 18mM and Antibiotic-Antimycotic 100X liquid (Invitrogen 15240–062) to a final concentration of 1x. Cells were passaged to a density no less than 1.5×10⁶ cells/mL and grown to late log phase.

S2 cells (Invitrogen 10831–014), and stably transformed cell lines, S2-BLRP-H2A and S2-BLRP-H2Av, were maintained in HYQ-SFX Insect Medium (Fisher SH30278.02), and supplemented with L-Glutamine to 18mM (Sigma G7513) and heat-inactivated FBS to 5% (Invitrogen 16140–063). Cells were seeded at a density of 8×10⁵ to 1×10⁶ cells/mL and were passaged at mid-to-late log phase.

ML-DmD16-c3²⁴ and ML-DmBG3-c2²⁵ cells were obtained from the Drosophila Genomics Resource Center (DGRC, https://dgrc.cgb.indiana.edu/). Cells were maintained in Shields and Sang M3 Medium supplemented with 5mL of BPYE (10%Yeast Extract & 25% Bacto-Peptone) +10% heat-inactivated Fetal Bovine Serum (FBS) + 10 μg/ml insulin (Sigma I9278). Cells were not diluted to more than 2.0×10⁶ cells/mL and were passaged strictly in mid-log phase.

DGRC cells were maintained in Schneider’s Medium (Sigma S0146) +10% heat-inactivated FBS. Cells were seeded at 1.0×10⁶ cells/mL and grown to mid-to-late log phase.

Comparing infected cells to stable cell line via immunocytology

Immunofluorescence analysis was used to determine the percentage of cells expressing recombinant protein. The Flag-H2Av construct was included to compare efficiency between it and the two-part BLRP/BirA system. S2 cells were infected with baculovirus at a 2:1 BLRP-H2Av to BirA ratio and at a final MOI of 100 for 1, 6 or 24 hours, after which time the virus was removed, the medium was replaced and expression was induced with Cu⁺⁺. In parallel, S2 cells were infected with the single baculovirus construct FLAG-H2Av at 66 MOI and 100 MOI and treated the same as described above. After a 60-hour incubation at 22°C, cells were collected and nuclei were extracted²⁶ and fixed to a microscope slide. BLRP-H2Av containing nuclei were probed with streptavidin, Alexa Fluor^® 488 conjugate (Jackson ImmunoResearch Labs, Inc.) at 1:2,500 before staining with DAPI. The nuclei containing FLAG-H2Av were probed with anti-FLAG primary antibody (Sigma F1804) at 1:2,000 and goat anti-mouse FITC-IgG (Jackson ImmunoResearch Labs, Inc.) at a 1:100 dilution and stained with DAPI prior to viewing on Nikon E800 fluorescent microscope. For control samples uninfected S2 cells were used as well as the stable BH2Av cell line, at passage number 22 in its maintenance cycle, induced and uninduced. At collection the viability of all samples was at least 94%.

Testing expression in Drosophila cells

To assess the expression of recombinant baculovirus constructs, mid-log cultures were seeded at minimal density required for growth. After allowing cells to adhere, the medium was removed and a mixture of the necessary viruses was added in a volume of Grace’s Medium (Sigma G8142) sufficient to cover the cells. Following a one-hour infection period the medium was replaced with each cell lines’ own maintenance medium, including 0.5mM Cu⁺⁺ to induce. Induction was allowed to proceed for 48 hours, after which cells were assayed for viability and number on a Beckman Coulter Vi-Cell-AS Automated Cell Viability Analyzer (Beckman 6605769) prior to harvesting. The medium was removed via centrifugation and cells were resuspended in 2x SDS loading dye to a final concentration of 2×10⁴ cells/μL, heated to 100°C for 10 minutes and allowed to return to room temperature. Electrophoresis was performed on an 18% SDS-PAGE gel so that the relative concentration of biotinylated recombinant protein could be compared between samples. Detection was by western analysis using IRDye −800 CW Streptavidin (LI-COR^® 926–32230) and viewed on a LI-COR^® Odyssey Infrared Imaging System. Anti-H3 (Abcam ab1791) primary antibody followed by IRDye^™-680 secondary (LI-COR^® 326–32221) was used to confirm equal loading of samples.

Chromatin affinity purification

We plated 6 × 10⁷ D. melanogaster S2 cells and stable BLRP H2Av S2 cells in 150cm² flasks with 30 mL growth medium. Once adherent, the S2 cells were transduced with FLAG H2Av baculovirus at 200 MOI for 2 hours with occasional agitation. We changed the media and induced both the FLAG H2Av transduced and stable BLRP H2Av cells with 0.5mM CuS0_4. After 72 hours at room temperature we counted and harvested the cells for chromatin isolation. We prepared nuclei as previously described²⁶, and chromatin was extracted using a modification of our previous protocols^{5, 13}. Briefly ~3 × 10⁸ cells were resuspended in HM2 [10mM Hepes at pH 7.0, 2mM MgCl₂, 0.5mM phenylmethanesulphonylfluoride (PMSF)] to a concentration of 0.75 × 10⁸ cells/mL. NP-40 was added to a final concentration of 0.6% while gently vortexing. After cells were visibly lysed the nuclei were pelleted by centrifugation at 800 RPM for 10 minutes at 4˚C. The supernatant was aspirated and the nuclei were washed and resuspended in 900ul HM2. CaCl₂ was then added to ~1mM, the solution was warmed to 37˚C in a water bath for 2 minutes and the chromatin was digested with 1U of micrococcal nuclease (MNase) for 10 minutes with intermittent pipetting. The reaction was stopped with 5mM EDTA on ice for 30 minutes, the nuclei were pelleted at 2,000 RPM for 10 minutes and the supernatant was saved (S1). Pelleted nuclei were resuspended in 600ul HE (10mM Hepes .25mM EDTA 0.5mM phenylmethanesulphonylfluoride [PMSF]) passed 4x through 20 and 26 ga needles, spun at 2,000 RPM for 10 minutes, and the supernatant was collected (S2)²². We combined S1 and S2 fractions, clarified the sample by centrifugation for two minutes at 13,200 RPM, and took out 150ul (input). We performed affinity purification with 50ul of packed Anti-FLAG affinity agarose gel or Streptavidin-sepharose overnight at 4˚C on a rotator (Sigma, GE). We washed the beads 3× 10 minutes with HE plus 0.1% Triton X-100 and 2× 10 minutes with HE alone. DNA was purified as described^{5, 13}.

Microarray Analysis

DNA samples were labeled with Cy3 and Cy5 as described¹³. Briefly, 1.5μg of DNA from input and pull-down was labeled in 50μl strand displacement reactions for 16 hrs at 37˚C. The reaction was terminated with the addition of 5μl of 0.5M EDTA and the labeled DNA was mixed with 5.7μl 5M NaCl and 60ul isopropanol, 10 minutes at room temperature. DNA was collected by centrifugation at 13,200 RPM for 10 minutes, the supernatant was aspirated, and the pellet washed with 500μl cold 80% ethanol, dried and dissolved in 50μL water. Cy3 input and Cy5 pull-down samples (30μg each) were mixed and reduced to 12.5μL volumes for hybridization and scanning by the Fred Hutchinson Center Genomics Shared Resource. Samples were hybridized on single custom designed 2.1 million feature isothermal microarrays (GEO GPL6888) purchased from NimbleGen, Inc following the manufacturer’s protocols. Data were analyzed by quintile and heat map analysis as described¹³. Microarray data are available from GEO (GSE21416).

Abbreviations:

ChIP

chromatin immunoprecipitation

BLRP

Biotin Ligase Recognition Peptide

BirA

biotin ligase

MOI

Multiplicity of infection

pfu

plaque-forming units