ABSTRACT
Controlling the expression of genes using a binary system involving the yeast GAL4 transcription factor has been a mainstay of Drosophila melanogaster developmental genetics for twenty-five years. However, most existing GAL4 expression constructs only function effectively in somatic cells, but not in germ cells during oogenesis, for unknown reasons. A special UAS promoter, UASp was created that does express during oogenesis, but the need to use different constructs for somatic and female germline cells has remained a significant technical limitation. Here we show that the expression problem of UASt and many other Drosophila molecular tools in germline cells is caused by their core Hsp70 promoter sequences, which are targeted in female germ cells by Hsp70-directed piRNAs generated from endogenous Hsp70 gene sequences. In a genetic background lacking genomic Hsp70 genes and associated piRNAs, UASt-based constructs function effectively during oogenesis. By reducing Hsp70 sequences targeted by piRNAs, we created UASz, which functions better than UASp in the germline and like UASt in somatic cells.
INTRODUCTION
Drosophila is an extremely powerful model organism for studies of animal development and disease because of its low maintenance costs, rapid generation time, and expansive collection of tools to genetically modify its cells. One particularly useful tool is the Gal4/UAS two-component activation system, in which the Gal4 transcriptional activator protein recognizes an upstream activator sequence (UAS) to induce the expression of any gene of interest (Fischer et al. 1988; Brand and Perrimon 1993). By controlling the activity of Gal4 with tissue-specific or inducible promoters or the Gal80 inhibitor protein, one can manipulate genes in specific cells or times of development, visualize cell types, probe cell function, or follow cell lineages. One of the most useful applications of these techniques has been to carry out genetic screens by expressing RNAi in targeted tissues or cultured cells (Dietzl et al. 2007; Ni et al. 2008).
The original pUASt vector from Brand and Perrimon (1993), which contains an Hsp70-derived core promoter and SV40 terminator, has undergone several optimizations to improve its expression (Fig 1A). Popular versions, such as the Valium10 or 20 vector used by the Drosophila Transgenic RNAi project (TRiP) (Ni et al. 2009; 2011) and the pMF3 vector used by the Vienna Drosophila Research Center (VDRC) GD collection (Dietzl et al. 2007) added a ftz intron, and the Janelia Gal4 enhancer project used derivatives of pJFRC81, which added a myosin IV intron (IVS), synthetic 5’UTR sequence (syn21) and viral p10 terminator to boost expression levels across all Drosophila cell types (Figure 1A) (Pfeiffer et al. 2012). However, these modifications did not correct UASt‘s major problem-that it drives woefully poor expression in the female germline compared to somatic tissues. Consequently, genetic manipulation in this important tissue has often relied on a special GAL4-activated promoter, UASp, produced by fusing 14 copies of the UAS activator to a germline compatible promoter derived from the P-element, a transposon naturally active in the female germline (Figure 1B) (Rørth 1998). Although UASp expression is qualitatively higher than UASt in the female germline, it is generally known to be lower in somatic tissues.
The lack of a UAS construct that is widely useful in all Drosophila tissues has remained an obstacle to providing optimum genetic tools to the research community. Transgenic RNAi collections were first constructed using UASt and screening of genes for germline functions has relied on increasing the effectiveness of RNAi by co-expressing Dcr2 or expressing short hairpin RNAi from UASp promoters (Ni et al. 2011; Yan et al. 2014; Sanchez et al. 2016). A significant obstacle to obtaining a widely effective GAL4 vector has been the lack of understanding of the reason UASt functions poorly in germ cells, and the paucity of accurate comparisons between the UASp and UASt promoters in the absence of other significant variables.
RESULTS AND DISCUSSION
Difference between UASp and UASt
To study the difference between the UASp and UASt promoters, we first created UASt-GFP and UASp-GFP constructs controlled for other variables between the original UASt and UASp, such as UTR components, introns, terminators, and genomic insertion site. Both constructs were based on pJFRC81 and only varied at the promoter and 5’ UTR of the transcript (Fig 1, red letters). We made these constructs compatible with phiC31-catalyzed recombination-mediated cassette exchange with MiMIC transposons, allowing us to integrate UAS-GFPs into many common sites throughout the genome (Venken et al. 2011). Using a previously established protocol (Nagarkar-Jaiswal et al. 2015), we recombined both UAS-GFPs into several MiMICS, including MI04106, which resides in a region enriched for ubiquitously expressed genes and active chromatin marks (Filion et al. 2010; Kharchenko et al. 2011) referred to as “the gooseneck” by Calvin Bridges for its long stretch of low density in salivary gland polytene chromosome preps (Bridges 1935). Consistent with previous reports, UASt drove significantly stronger expression than UASp in all somatic tissues examined while UASp drove significantly stronger expression in the female germline (Fig 2A,B).
Hsp70 piRNAs repress UASt
We next investigated the reason for the extremely weak UASt expression in the female germline. Several lines of evidence implicated piRNA-directed silencing as a mechanism limiting UASt expression. Drosophila piRNAs are ovary and testis-enriched, 23-29 nucleotide (nt) RNAs that complex with Argonaut family proteins and silence transposons through homologous base-pairing-directed mRNA cleavage and heterochromatin formation (Siomi et al. 2011). Some of the most successful UASt-based genetic screens in the female germline knocked down piRNA biogenesis genes (Ni et al. 2011; Czech et al. 2013; Handler et al. 2013). If piRNAs were silencing UASt, then UASt-RNAi against a piRNA biogenesis gene would boost UASt expression leading to maximal knockdown. Where might these UASt-piRNAs originate from? Previously, Mohn et al (2015) characterized an abundance of germline-specific piRNAs mapping to both Hsp70 gene clusters. Because UASt contains the Hsp70 promoter and 5’UTR, we hypothesized that germline piRNAs against Hsp70 may be targeting UASt. When we searched for UASt sequences in the piRNAs identified by Mohn et. al (2015), we identified abundant piRNAs perfectly homologous to UASt (Fig 2D pink bars, and Fig 2E grey bars). Similar to UASt silencing, these UASt piRNAs are restricted to the female germline because germline-specific knockdown of rhino, a gene required for Hsp70 piRNA production eliminates UASt piRNAs from whole ovaries (Fig 2D) (Mohn et al. 2014).
To directly test whether Hsp70 piRNAs silence UASt, we tested UASt expression in Hsp70Δ flies (Gong and Golic 2004), which completely lack all genetic loci producing piRNAs homologous to UASt (Fig 2D, grey boxes deleted). Despite missing all copies of the inducible Hsp70 gene family and related piRNAs, Hsp70Δ flies have no significant defects in viability or egg production in the absence of heat stress (Gong and Golic 2006). However, Hsp70Δ flies showed greatly enhanced UAStGFP expression. Furthermore, UAStGFP expression was significantly stronger than UASpGFP, which was unaffected by Hsp70Δ (Fig 2C). These results argue strongly that UASt is normally silenced by Hsp70 piRNAs and that UASt is a stronger expression vector than UASp in cells lacking Hsp70 piRNAs.
Construction of UASz
We next attempted to create a new version of the UAS expression vector that works well in both the soma and female germline. We hypothesized that eliminating the part of UASt targeted by piRNAs would boost UASt expression by the same amount as eliminating the piRNAS themselves. Hsp70 piRNAs are homologous to 247 nt of the UASt promoter and 5’UTR. While we could make enough substitutions along this stretch to prevent all possible 23 nt piRNAs from binding, we were afraid this approach might impair important promoter sequences. Instead, we hypothesized that Hsp70 piRNAs might recognize UASt RNA to initiate piRNA silencing. To prevent Hsp70 piRNAs from recognizing UASt RNA, we trimmed down the UASt 5’UTR to be shorter than a single piRNA, from 213 nt to 19 nt (Fig 1A, Fig 2E). We named this UTR-shortened UASt variant “UASz,” because we optimistically hoped it would be the last one anyone would make.
Comparison of UAS vectors
To compare the relative expression levels of our UASz to UASp and UASt, we created all three variants in the same GFP vector backbone (pJFRC81) with a single attB site. We used phiC31 integrase to introduce these UAS-GFP variants into a commonly used genomic site, attP40, and recombined all three inserts with Hsp70Δ to determine the influence of Hsp70 piRNAs on their expression. When combined with Tub-Gal4, a somatic Gal4 driver, UASz was expressed at least 4 times higher than UASp in all somatic tissues tested and was equivalent or greater than UASt in some somatic tissues like the larval epidermis and salivary gland (Fig 3A,C,E). However, UASz was expressed at about 40% of UASt in discs, suggesting some elements of the UASt 5’UTR may boost expression in some tissues (Fig 3C,E). To measure germline expression, we crossed the three UAS-GFPs to vasa-Gal4, which is evenly expressed up to stage 6 of oogenesis. In the germline, UASz was expressed about 4 times higher than UASp at all stages, while UASt was expressed at much lower levels than UASp, except in region 1 of the germarium (Fig 3B,D-F) where piRNA silencing is weaker (Dufourt et al. 2014). We conclude that UASz is a superior expression vector to UASp in all tissues, and is equivalent to UASt in many, but not all, somatic tissues.
Finally, we wanted to test if UASz is still targeted by Hsp70 piRNAs because it contains 63 nt of Hsp70 sequence and about 10% of the putative piRNAs targeting UASt (Fig 2E). We crossed UASzGFP into the Hsp70Δ background and compared UASzGFP levels with or without Hsp70 piRNAs. We observed no enhancement of UASzGFP when Hsp70 piRNAs were removed (Fig 3B,D,F). Therefore, Hsp70 piRNAs likely target the UASt but not UASz 5’UTR, consistent with the model that piRNAs must initially recognize RNA but not DNA.
Is UASz the final, fully optimized iteration of a UAS vector? Probably not. UASt without Hsp70 piRNAs induces about twice the expression of UASz in the ovary (Fig 3B,D,F). This twofold advantage of UASt over UASz in the germline or imaginal discs lacking Hsp70 piRNAs is similar to the twofold advantage of UASt over the UAS fused to the Drosophila Synthetic Core Promoter (Pfeiffer et al. 2010). Perhaps adding back some sequences within the first 203 nt of the Hsp70 5’UTR while avoiding piRNA recognition may improve UASz.
However, the current iteration of UASz remains an unequivocal upgrade over UASp for all applications and UASz should be preferred over UASt if both germline and soma studies are planned from a single vector. Alternatively, one could boost germline expression of an existing UASt construct by crossing it into the Hsp70Δ background.
Current UAS-RNAi collections are heavily biased toward UASt-RNAi-based constructs. To date, the VDRC and DRSC/TRiP RNAi projects used UASt-RNAi to target 12,539 and 8,876 genes, respectively. Germline screens for developmental phenotypes using UASt-RNAi were enriched for phenotypes in germarium region 1 (Yan et al. 2014; Sanchez et al. 2016), where piRNA silencing is weakest (Dufourt et al. 2014) and UASt shows maximum expression (Fig 3D arrow). Perhaps these screens were depleted for developmental defects in later germline stages because of poor UAS-RNAi expression in these stages. Although UASp-RNAi from the Valium22 vector (Fig 1B) increased the efficiency of obtaining phenotypes in a germline screen, only 1,596 genes are currently targeted by this collection (Yan et al. 2014). Additionally, when screening somatic cells, Ni et al. (2011) recommend UASt-RNAi because UASp-RNAi gave incomplete knockdowns. Our results revealed that UASp is equally weak in the germline as somatic tissues when compared to UASz (Fig 3E). Therefore, UASp-RNAi may also generate incomplete knockdowns in the germline. To increase germline RNAi expression, we recommend our UASz-RNAi expression vector (Sup Figure 1), which is compatible with previously generated shRNA oligo cloning (Ni et al. 2011).
MATERIALS AND METHODS
Drosophila strains
Mef2-Gal4 (BL26882) w[*]; Kr[If-1]/CyO, P{w+ GAL4-Mef2.R}2, P{w+ UAS-mCD8.mRFP}2 Tub-Gal4 (BL5138) y[1] w[*]; P{w+ tubP-GAL4}LL7/TM3, Sb[1] Ser[1]
FLP/phiC31int (BL33216) P{hsFLP}12, y[1] w[*] M{vas-int.B}ZH-2A; S[1]/CyO; Pri[1]/TM6B, Tb[1]
Hsp70Δ (BL8841): w[1118]; Df(3R)Hsp70A, Df(3R)Hsp70B
Vasa-Gal4 was obtained from Zhao Zhang‘s lab: y[*] w[*];; P{w+ vas-GAL4.2.6} (Zhao et al.2013)
New stocks created for this study
Bestgene Inc. introduced pMRtGFP and pMRpGFP into yw flies using a P-transposase helper plasmid and we isolated GFP+ insertions by crossing the F0 to a Mef2-Gal4 background and scoring for GFP+ muscles. We introduced UAStGFP or UASpGFP into MI04106 and other MiMIC lines using a cross strategy outlined in (Nagarkar-Jaiswal et al. 2015). Rainbow transgenics introduced pJFRC81 (UAStGFP-attB), pUASpGFP-attB, and pUASzGFP-attB into attP40 using an X-chromosome encoded phiC31 integrase source and we isolated multiple w+, phiC31 minus insert lines by standard fly genetics.
Vectors created for this study
Genescript synthesized pMRtGFP. We created pMRpGFP by replacing the NheI-BglII UASt promoter in pMRtGFP with a SpeI-BglII UASp promoter from Valium22. We created pUASpGFP-attB by replacing the PstI-BglII UASt promoter in pJFRC81 with the PstI-BglII UASp promoter from Valium22. We created UASzGFP-attB by replacing the 259 bp NheI-BglII fragment of pJFRC81 containing the 203 bp Hsp70 promoter with annealed oligos encoding 63 bp from the 5’ end of the same promoter.
Top oligo: 5’ CTAGCGACGTCGAGCGCCGGAGTATAAATAGAGGCGCTTCGTCTACGGAGCGACAA TTCAATTCAAACAAGCAAA 3’
Bottom oligo: 5’ GATCTTTGCTTGTTTGAATTGAATTGTCGCTCCGTAGACGAAGCGCCTCTATTTATAC TCCGGCGCTCGACGTCG 3’
We created UASz by replacing the NotI-syn21-GFP-XbaI fragment in UASzGFP with annealed oligos encoding NotI-sny21-BamHI-XhoI-KpnI-SpeI-XbaI
Top oligo: GGCCGCAACTTAAAAAAAAAAATCAAAGGATCCCTCGAGGGTACCACTAGTT
Bottom oligo: CTAGAACTAGTGGTACCCTCGAGGGATCCTTTGATTTTTTTTTTTAAGTTGC
We created UASz1.1 by replacing the KpnI-EcoRI p10 terminator in UASz with a PCR amplified p10 terminator containing Kpn1-XbaI-EcoRI and ApoI tails.
F primer: 5’ CATGGTACCGCCTCTCTAGAGTGTGAATTCTGGCATGAATCGTTTTTAAAATAACAA ATCAATTGTTTTATAAT
R primer: 5’ GGAAATTTTCGAATCGCTATCCAAGCCAGCT
We created UASz1.2 by destroying the NheI and EcoRI sites in UASz1.1 by cloning annealed oligos into the NheI-EcoRI backbone.
Top oligo: CTAGGAGCGCCGGAGTATAAATAGAGGCGCTTCGTCTACGGAGCGACAATTCAATT CAAACAAGCAAGATCTGGCCTCGAGT
Bottom oligo: AATTACTCGAGGCCAGATCTTGCTTGTTTGAATTGAATTGTCGCTCCGTAGACGAAG CGCCTCTATTTATACTCCGGCGCTC
To create UASzMiR, we cloned a BglII-XhoI fragment containing the MiR1 cassette and ftz intron from Walium22 into the BglII-XhoI backbone of UASz1.2.
Tissue Preparation Imaging and Quantitation
For all experiments, we crossed UAS-GFP or UAS-GFP Hsp70Δ males to control (yw), Tub-Gal4/TM3, homozygous Vasa-Gal4, or homozygous Vasa-Gal4 Hsp70Δ females. For whole larvae imaging, we picked wandering 3rd instar larvae of various genotypes, aligned them on the same glass slide, and placed them the freezer for 30 minutes prior to imaging. For adult ovary or larval tissue imaging, we fixed dissected tissue with 4% paraformaldehyde for 13 minutes (whole ovary) or 20 minutes (larval tissue) and stained with DAPI in PBS + 0.1% Triton X-100. We imaged GFP fluorescence of semi-frozen whole 3rd instar larvae or whole ovaries mounted in 50% glycerol on a Leica Stereoscope equipped with mercury arc light source, GFP filters, and CCD camera. We imaged GFP fluorescence in larval imaginal discs, salivary glands, and epidermis, and manually separated ovarioles mounted in 50% glycerol using a custom-built spinning disc confocal with 20x 0.8 NA lens. For each genotype and tissue type, we acquired a single plane image from at least 4 individuals using Metamorph software and the same laser power, CCD camera gain, and exposure time between equivalent samples. We measured average pixel intensity in 14 bit images of the GFP channel using Image J. We acquired representative images of single planes through single ovarioles for Figure 2 on a Leica Sp8 scanning confocal with 63x 1.4 NA lens and PMT (for DAPI) and HiD (GFP) detectors using identical settings between samples.
UASt piRNA analysis: We clipped and aligned sequenced small RNA libraries from (Mohn et al. 2014) (SRR1187947:control germline knockdown and SRR1187948:rhino germline knockdown) to D. melanogaster Genome Release 6 (Hoskins et al. 2015) or UAStGFP using the Bowtie2 aligner with no filtering for repetitive mappers (Langmead and Salzberg 2012). We visualized piRNA read depth to UAStGFP or both Hsp70 clusters using the Interactive Genome Browser (Robinson et al. 2011).
Supplemental Figure 1
ACKNOWLEDGMENTS
We thank members of the Spradling lab for comments. S.Z.D. was a fellow of the Helen Hay Whitney Foundation.