Fly stocks and crosses

The following stocks are from the Drosophila Stock Center at Bloomington, IN: tara¹/TM3 hypomorphic allele due to P(lacW) insertion [14], tara⁰³⁸⁸¹/TM3 P(Pz) insertion, hypomorphic [17], Pc³/TM1, Ubx^Cbx-1,Ubx¹, hsflp;;FRT82B,ubi-GFP/TM3,Tubulin80-gal4,UAS-GFP/CyO. The Fab-X flies were from G. Cavalli (CNRS, France). Sgs3-Gal4 was from Dirk Bohmann (Rochester, NY). UAS-tara RNAi (Transformant ID: 34362) was from Vienna Drosophila RNAi Center (VDRC; Vienna, Austria). Salivary gland clones were generated by crossing hsflp;;FRT82B,Ubi-GFP/TM3 (females) with FRT82B,tara⁰³⁸⁸¹/TM3 (males).

Immunostaining of the salivary glands

Embryos from the cross between hsflp;;FRT82B,Ubi-GFP/TM3 and FRT82B,tara⁰³⁸⁸¹/TM3 were heat shocked in a 37°C degree water bath for 1hr and left to grow till third instar at 25°C. Salivary gland were dissected in ice-cold PBS and fixed in 4% formaldehyde in PBS for 20 minutes at RT. After washing with 0.3% Triton X 100 in PBS, salivary glands were placed in primary antibody at 4°C overnight. Primary antibodies were diluted in PBS-0.3% Triton x 100, 1% BSA and 1% Normal goat serum. Incubation with secondary antibody was performed at RT for 3hrs and sample mounted with Vectashield DAPI (4’, 6-diamidino-2-phenylindole, Vector Lab, Catalogue# H-1200).

RNA extraction and real-time qPCR

Total RNA from Drosophila embryo was extracted using Trizol and cDNA was synthesized with Superscripts III. Primers

rp49F: TCC TAC CAG CTT CAA GAT GAC

rp49R: CAC GTT GTG CAC CAG GAA CT

taraF: CAAGCGCGCGTAATTCAAAGT

taraR: AAGCGGCATCACAGAACTT

Antibodies used for immunostaining

The following antibodies were used: 4D9 mouse anti engrailed/invected (DSHB, 1:10), 1A2E9 mouse anti-Abd-B (DSHB, 1:10), mouse anti Ubx FP3.38 (DSHB, 1:10), rabbit anti-Pc (Santa Cruz, 1:250), sheep anti-DIG (1:50, Roche cat no. 11333089001). All secondary antibodies are from Molecular Probes (Invitrogen): goat anti-mouse or goat anti-rabbit Alexa fluor 488, Alexa Fluor 546, Alexa Fluor 660. All of these were used at 1:250 dilutions. For the sheep anti-DIG primary antibody, a donkey anti-sheep Cy3 antibody was used at a 1:100 dilution.

Polytene chromosome squash and fluorescent in situ hybridization followed by immunostaining

Salivary glands were dissected from two wandering 3rd instar larvae and fixed for 1 min in a drop of the solution 1 (50ul Triton X-100, 400 μl PBS and 50 μl 37% p-formaldehyde) on a coverslip. Glands were then move to about 40ul of the solution 2 (50ul 37% p-Formaldehyde, 200 μl De-ionized water and 250 μl glacial acetic acid) on another coverslip. After 10mins, the coverslip was taken up on a poly-L-Lysine coated glass slide. The coverslip was tapped with the erasure end of a pencil to break up the salivary glands and spread the chromosomes. The slides were placed in a -80°C freezer for at least 10mins. The coverslip was then removed with a sharp blade and the slides were washed twice in PBS followed by blocking in PBS- 0.3% Triton-X 100 plus 2% BSA (Bovine Serum Albumin Fraction V). For antibody staining, 20 μl of diluted primary antibody was added onto slide area containing the squashed chromosomes (marked with a glass marker) and covered with a 22mmx22mm piece of parafilm followed by overnight incubation at 4°C. The following day, slides were washed twice (15mins each) in PBS-T and incubated at RT for 2 hrs with the secondary antibody. The slides were mounted with Vectashield DAPI.

For FISH, the protocol from Epigenomic NoE (http://epigenesys.eu) was adapted. After polytene squash, the slides were placed in 2xSSC (diluted from 20x stock: 175.3g NaCl, 88.2g Sodium citrate in 1 L deionized water, pH 7.0) for 45mins at 72°C and dehydrated in 70% and 95% ethanol (2x, 5mins each). DNA was denatured with 0.1N NaOH and the slides were washed with 2xSSC for 5mins. The slides were again dehydrated with ethanol and 14 μl probes in hybridization buffer (50% deionized formamide, 0.3M NaCl, 20mM Tris, 5mM EDTA, 1x Denhardt's solution, 10% dextran sulfate, 10mM DTT, 500ug/ml yeast tRNA) was added onto each slide, covered with a glass slide and sealed with rubber-cement. Hybridization was carried out at 37°C for overnight in a moist chamber. After hybridization, slides were washed in 2xSSC and transferred to PBS and immunostaining to detect DIG. FISH was performed with sheep anti-DIG (2hrs RT) and Donkey anti-sheep Cy3 (2hrs RT). Polycomb or anti-flag staining was performed as mentioned before.

The engrailed genomic region specific probe was made using the Roche DIG labeling Kit according to manufacturer recommendation. An 8kb Sph1 fragment from engrailed locus (P(EN1)) plasmid described in [18]) was used to generate DIG labeled probes. This 8kb engrailed genomic DNA also contains the PRE necessary for engrailed regulation. We used an 8kb fragment form the engrailed promoter region [18]. This fragment was generated by SPHI restriction digest of P(EN1) plasmid mentioned in 19.

Chromatin immunoprecipitation from Drosophila larvae

Larvae were grown on apple agar plates at 29°C with excess yeast paste. Approximately 1mg of 2nd instar larvae of the indicated genotypes were crushed in PBS with a pestle. The larval cells were cross-linked at room temperature for 15 minutes with 1% final concentration of formaldehyde, then 0.125mM final concentration of glycine was added and the samples were incubated for another 5 minutes at room temperature. The cells were collected by centrifugation and washed 3 times with PBS-T (1X PBS, pH 7.6 with 0.3% Triton-X). After that, cell lysis buffer was added (50mM HEPES-KOH, 140mM NaCl, 1mM EDTA, pH 8.0, 1% Triton-X, 0.1% Sodium Deoxycholate, 5mM PMSF, 1X PIC) and the cells were sonicated 8 times with 15 second pulses with a Branson S-450 Sonicator set at 40% and output 5. The sample was centrifuged at 13,000xg for 2 minutes at 4°C to remove the cell debris. A portion of chromatin lysate was heated in Elution Buffer (1% SDS, 100mM NaHCO3) at 65°C overnight and input chromatin was prepared using the QIAquick PCR Purification Kit (Qiagen Cat. 28106). The remaining chromatin lysate was incubated overnight in 4°C with 4 μg of anti-Polycomb (Santacruz Biotech, Sc-25762) or an equivalent volume of water for mock treatment. Agarose-G beads were pre-blocked overnight at 4°C in 1.5 μg salmon sperm DNA per 20 μl beads, then 20 μl was added to the chromatin lysate and the samples were kept on a rocking platform overnight at 4°C. The beads were washed three times for 5 minutes each wash in 4°C in Wash Solution (0.1% SDS, 1% Triton-X, 2mM EDTA, 150mM NaCl, 20mM Tris-Cl pH 8.0), then 2 hours at 4°C in Final Wash Solution (0.1% SDS, 1% Triton-X, 2mM EDTA, 500mM NaCl, 20mM Tris-Cl, pH 8.0). The beads were incubated in room temperature for 20 minutes in Elution Buffer and de-cross linked overnight at 65°C. Chromatin was isolated using the QIAquick PCR Purification Kit and 1ul was used for each Sybr Green qPCR reaction. QPCR was performed using standard settings in a Biorad iCycler IQ5 instrument. The following sets of primer sets were used from [19].

engrailed PRE forward: 5'-GTTCACTCCCTCTGCGAGTAG-3'

engrailed PRE Reverse: 5'-GAAAACGCAGATTGAAACGTC-3'

engrailed Gene Forward: 5'-CGCCTTAAGGTGAGATTCAGTT -3'

engrailed Gene Reverse: 5'- GGCGGTGTCAATATTTTGGT-3'

Western blotting

Total lysate was prepared by homogenizing 10 pairs of salivary gland of the indicated genotypes in 20 μl of cell Lysis Buffer and separated on a 10% gel. Pc antibody (Santacruz Biotech, Sc-25762) was used with 1:500 dilutions and anti-rabbit HRP was used as the secondary.

Tara modulates a transgenic PRE-mediated phenotype

Tara has been previously implicated in PcG/TrxG function [14,20]. We have formerly identified tara in a genetic screen as a potential new component of the RNAi pathway [21]. Since transcriptional silencing mediated by PcGs in certain scenarios requires RNAi components like Argonaute and Dicer [22], we wanted to examine whether Tara could potentially connect PcGs to the RNAi machinery. To this end, we used a transgenic assay system that contains a boundary element for the iab-7 Polycomb response element (PRE) from the BX-C locus called Frontoabdominal 7 (Fab-7) [8,23,24] It regulates selector gene Abdominal-B in the wild type context. We used the Fab-X line, a specific transgenic insertion on X of a 3.6kb fragment from Fab-7 locus containing both the boundary and the respective PRE. Fab-x flies harbor two copies in tandem [8,25]. Interestingly, Fab-X silences the nearby scalloped (sd) gene along with the mini-white reporter, resulting in a stumpy wing phenotype (referred to as sd phenotype) [8] (Fig 1B). The silencing of sd occurs only in homozygotes (females in this case) when raised at 29°C, and is dependent on the Polycomb group of proteins and RNAi components like Dicer and Argonaute, suggesting that the interaction between PcG and RNAi is required for the silencing process [8,22].

We tested whether tara can modulate the Fab-X induced sd wing phenotype. We observed that, the heterozygous tara mutant alleles could partially suppress the sd phenotype (Fig 1B). The tara¹ and tara⁰³⁸⁸¹ are two independent alleles both caused by a P-element insertion at the 5’ region of the tara locus. Both alleles are associated with embryonic lethality in homozygotes, and they are phenotypically indistinguishable. Expression of tara from a transgene under an ubiquitin promoter rescues the lethality, suggesting the lethality is due to loss of tara [14,17,20]. In the absence of tara mutations, more than 90% of the Fab-X flies show sd phenotype affecting both wings. In tara loss-of-function heterozygous background, we found that this phenotype is reduced to 25%. On the other hand, tara heterozygosity does not suppress wing phenotypes, which is caused by a hypomorphic scalloped allele sd¹ (Fig 1C) [26], suggesting that the observed suppression was unlikely due to genetic interaction between scalloped and tara. Thus, Tara may play a role in Fab-X induced silencing of the nearby sd gene.

Loss of tara function leads to homeotic transformation

To understand whether Tara plays a role in Pc function, we investigated the biological function of tara during Drosophila development. We examined Drosophila embryos homozygous for the tara mutations. Since both tara loss-of-function alleles show similar phenotype in relation to homeotic transformation in the embryos, we focused on tara⁰³⁸⁸¹ to investigate Polycomb related mechanisms. Previous RNA in situ hybridization indicates that the maternal component for tara is minimal, as the general pattern of its expression is mostly ubiquitous. However, it is not significantly expressed before the developmental stage 5 [14]. Thus, we used zygotic mutant embryos lacking tara for this study.

During development, Drosophila embryos generate unique patterns of cuticular bristles on the ventral side. These "denticle bands” are arranged in three thoracic (T1-T3) and eight abdominal segments (A1-A8) from anterior to posterior region of the embryo [27] (Fig 2A). Each denticle band has a distinct length and shape and it is thought to be representative of the segment’s identity in the embryo (Fig 2B). In wild-type embryos, the A2-A7 bands in the embryonic cuticle appear trapezoidal, whereas the A8 is rectangular. Cuticles of tara⁰³⁸⁸¹ homozygous embryos show homeotic transformation mostly in the posterior segment, such that abdominal segments following the 5th are transformed to resemble the last abdominal segment (A8) (Fig 2B). The A6 band of tara⁰³⁸⁸¹ homozygous embryos is shortened and is rectangular similar to A8, instead of trapezoidal as seen in wildtype embryos, suggesting a homeotic transformation towards a posterior segmental identity. This transformation is reminiscent of the well-characterized Polycomb phenotype [28], albeit less pronounced. For comparison, a null allele of Polycomb (Pc³) is shown [29]. Pc³ embryos show complete homeotic transformation across all segments in the embryo. In these embryos, all other abdominal segments transform into segment A8 (Fig 2A). Double homozygous mutant (Pc³, tara⁰³⁸⁸¹) embryos lacking both tara and Pc do not show further enhancement regarding homeotic transformation, suggesting that the effect of tara might be mediated by Pc. Since tara predominantly causes transformation in the posterior region of the embryo, we quantified the ratio of the length of A4 over A6 bands (Fig 2C). The length here is measured by the distance between two furthest points for each denticle band. Hence, it is the length of the base of trapezoidal or rectangular shape of the specific band measured. In wild-type embryos, the ratio is ~1.16. In Pc³ homozygous embryos, the ratio is ~1. Similar to Pc mutants, the observed ratio in the tara⁰³⁸⁸¹ homozygotes is significantly smaller (~1.03) than that of wildtype embryos. Expectedly, no statistically significant difference was observed between Pc³ and Pc³,tara⁰³⁸⁸¹ double mutant embryos (Fig 2C). The partial homeotic transformation indicates that Tara could be involved in patterning process that is regulated by PcGs.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Taranis loss-of-function causes partial homeotic transformation.

(A) Darkfield images of embryos (10x) are shown anterior up. From left to right are wild type, homozygous mutants for tara⁰³⁸⁸¹, Pc³, and Pc³, tara⁰³⁸⁸¹. N = 15 embryos for each genotype were examined, and representative embryos are shown. Magnified view of the A5-A8 denticle bands are shown below for each genotype. Scale bar 20 μm. (B) 20x Brightfield illumination of the A6 band from each embryo in A is shown in panel B. Scale bar 10μm. (C) The ratio of the denticle band length A4 to A6 for the indicated genotype was show with standard errors (n = 4 for each genotype). p is calculated using Student’s t-Test.

https://doi.org/10.1371/journal.pone.0180026.g002

Tara genetically interacts with of the BX-C gene Ultrabithorax

Since homeotic transformation is attributable to misexpression of Pc-regulated homeotic genes like Ultrabithorax (Ubx), we next asked whether tara could modulate Ubx dependent phenotype. Ubx is a part of the Drosophila BX-C cluster of genes, which act in concert to regulate posterior segmental identity in the embryo [30].

To investigate the role of tara in Ubx regulation, we utilized a gain-of-function allele of Ubx called Ubx^Cbx-1 [31]. The Ubx^Cbx-1 allele arose due to a transposition event causing a 17-kb fragment from Ubx upstream region integrating into a downstream intronic region of the gene, causing upregulation of Ubx in parasegment 5 [32,33]. Interestingly, even with a Ubx loss-of-function in cis (Ubx¹ allele), Ubx^Cbx-1 was able to activate the wildtype counterpart in trans when present in heterozygous condition (Ubx^cbx-1, Ubx¹/+), causing ectopic expression of Ubx in wing disc, a phenomenon known as transvection [34]. The Ubx^Cbx-1, Ubx¹/+ heterozygous flies exhibit partial wing-to-haltere transformation, which is typically enhanced in the PcG mutant background (Fig 3). This phenotype is fully penetrant but varies in expressivity. We categorized the phenotype into four different classes—I to IV—depending on the severity of the transformation (Fig 3). The Class I category wings has a slight curved region on the posterior boundary of the wing, Class II wings show partial loss of the fifth wing-vein and the area between the 4th and 5th vein appear crumpled. The wing morphology is severely affected in Class III, with the presence of bubbles and crumpled posterior margin. The Class IV wings are the most affected as the wing-to-haltere transformation is prominent in the stumpy wing morphology. The severity of wing phenotype is positively correlated with the expression of Ubx in the larval wing disc tissue [32]. At 22°C, about 75% of Ubx^Cbx-1, Ubx¹ heterozygous flies show mild transformation (Class I), and only 9% shows Class II and 15% shows Class III phenotypes, respectively. In the presence of one copy of tara⁰³⁸⁸¹, the majority of the wings fall into the Class II (46%) and Class III (53%) categories, suggesting that loss of tara enhances the wing-to-haltere transformation of Ubx^Cbx-1, Ubx¹ heterozygous flies. The wing phenotype, however, is not as severe as that of Ubx^Cbx-1, Ubx¹/Pc³ flies, in which 86% of the wings are in Class IV category. These results suggest that Tara normally negatively regulates Ubx expression, although Pc appears more important in repressing Ubx.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Tara interacts with Ubx.

tara enhances Ubx dependent wing-to-haltere transformation. The percentage of flies showing the transformation in Ubx^Cbx-1, Ubx¹/+ (A) is increased in the presence of a heterozygous tara loss-of-function allele (B). The wing-to-haltere transformation in the presence of Pc³, a Polycomb allele, is shown in (C).

https://doi.org/10.1371/journal.pone.0180026.g003

Loss of tara function reduces Pc recruitment to PREs

The genetic experiments described above indicate that Tara might be involved in Pc function. The suppression of the Fab-X and Ubx^Cbx-1 phenotypes by tara mutations could have resulted from a failure of Pc recruitment to the PRE in Fab-X or Ubx regulatory regions. Two of the important steps in the canonical PcG functions are trimethylation of histone 3 lysine 27 (H3K27me3) followed by Pc binding to this epigenetic mark via its chromodomain [35]. Tara could conceivably be involved in either of these processes. We next set out to investigate these possibilities. To this end, we first created mitotic clones using the FLP-FRT technique and examined Pc levels in hypomorphic homozygous tara⁰³⁸⁸¹ cells of the 3rd instar larval salivary glands (see Methods). We observed a significant reduction in Pc fluorescence in tara⁰³⁸⁸¹ / tara⁰³⁸⁸¹ cells (Fig 4A, arrows).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. Taranis knockdown results in loss of Pc recruitment.

(A) Mitotic clones in the Salivary gland were induced by heatshock at the embryonic stage and heat-shocked embryos were left to grow until 3^rd Instar. Salivary glands from wandering 3rd instar larvae were dissected and immunostained for Pc. The GFP negative cells are tara⁰³⁸⁸¹ homozygous clones. Note that tara⁰³⁸⁸¹ homozygous cells (white arrow, GFP negative) show less Pc in salivary glands nuclei. GFP positive cells are wild type or heterozygous for tara⁰³⁸⁸¹. Genotype of the larvae is hsflp;;FRT82B,Ubi-GFP/FRT82B,tara⁰³⁸⁸¹. (B) Salivary glands from Sgs3 > taraRNAi or control wandering 3rd instar larvae were squashed and subjected to immunostaining with antibody against Pc to visualize Pc distribution on polytene chromosomes. DNA (DAPI) staining of the chromosome is shown is grey in the merged images. Magnified view of the boxed area on the right indicating reduction in number of Pc bands. Graph depicting average number of Pc bands on polytene chromosome on taraRNAi as well as driver only (Sgs3 >w1118) control.

https://doi.org/10.1371/journal.pone.0180026.g004

Drosophila polytene chromosome shows distinctive pattern of Pc binding as multiple high and low intensity bands. This provides us with the necessary setup to examine Pc binding to chromosomes at higher resolution compared to whole-mount immunostaining in salivary gland. We knocked down tara in the salivary gland by expressing dsRNA against tara using the UAS-Gal4 system followed by immunostaining with anti-Pc and anti-H3K27me3 (see Methods; S1 Fig). The salivary gland-specific Sgs3-gal4 driver was used for this purpose to drive RNAi specifically in the salivary gland. We observed a significant reduction in the number of Pc bands in tara knockdown (Fig 4B bottom panel) compared with control (Fig 4B top panel), corroborating the observation in tara loss-of-function clones. Pc bands were decreased to an average of 44 per nuclei. In comparison, Sgs3 > w1118 (control) shows 63 Pc bands (Fig 4B, graph). Magnified view of the third chromosome is shown in the panel next to the whole chromosome for better comparison. However, tara RNAi appears to decrease the level of Pc in most of the low intensity bands, whereas the number of high intensity bands remains unchanged. Our result indicates that Tara might be important for Pc recruitment at specific target loci but may not be necessary for all the genes regulated by Pc.

We did not observe any significant changes in the levels and distribution of H3K27me3 in polytene chromosomes after knocking down tara (S2 Fig). When compared to the Tubulin80-Gal4 driver only control, there was no significant change in Pc protein levels for embryos with tara knockdown (S3 Fig). Our data indicate that Tara might be involved in Pc recruitment rather than affecting histone methylation of H3K27 or the production of Pc per se.

Misregulation of engrailed in tara loss-of-function embryo

As mentioned previously, Pc function in Drosophila is mediated by cis DNA element called PRE. We have found tara to be important in the transgene Fab-X mediated silencing and possible repression of Ubx, both of which require PRE activity (Figs 1B and 3). We wanted to test whether tara can affect the PREs of other known Pc targets. Drosophila segment polarity gene engrailed (en) is a Pc target with two well-characterized PREs in a 2.4kb region upstream of its transcription start site. The proximal PRE is located 181bp upstream of the transcription start site and is characterized by Pc binding [36]. We first looked at en expression pattern in tara⁰³⁸⁸¹ homozygous embryos. en is expressed in 14 distinct stripes during embryonic development at germ-band extension [37,38]. In each stripe, en expression is limited to 2–3 rows of cells in wildtype embryos (Fig 5A). Pc³ homozygous embryos show a misexpression of en, as the total number of cells expressing en in a stripe increases. We observed a very similar phenotype in the tara embryos homozygous for tara⁰³⁸⁸¹ (Fig 5A). Interestingly, unlike the homeotic transformation phenotype (Fig 2A), the tara⁰³⁸⁸¹ and Pc³ double mutant embryos show a further increase in the number of cells that express en than that of single mutant embryos (Fig 5A). The effect is most discernible in the 13^th parasegment. Similar synergistic relationship is known to exist between PcG proteins in the context of engrailed regulation [39]. To quantify en expression, we counted the number of en-expressing cells in the 13th parasegment of each embryo. In wildtype embryos, the en stripe in the 13th parasegment contains average 17 cells (Fig 5A, white box and right), whereas in tara⁰³⁸⁸¹ homozygous embryo, this number is increased to 22. Pc³ homozygous mutation results in expression of en in average 29 cells, and the tara⁰³⁸⁸¹ Pc³ double mutant shows enhancement as the number of en expressing cells reaches 33 (Fig 5A, white box and right).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. Polycomb target engrailed is derepressed in taranis loss of function.

(A) Embryos showing Engrailed immunostaining. The white-boxed area is shown on the right panel with higher magnification for the corresponding embryo. No. of cells was counted in the boxed regions and plotted for comparison with wildtype. From top to bottom: wild type, tara⁰³⁸⁸¹, Pc³ and Pc³,tara⁰³⁸⁸¹. Scale bar 10 μm left panel 2.5μm right panel, N = 6 (wt, tara) and 8(pc, pc-tara) (B) Salivary glands from Sgs3 > taraRNAi or control wandering 3rd instar larvae were squashed and subjected to FISH for the en locus and followed by immunostaining with antibody against Pc. Top: Sgs3 >w1118; Bottom: Sgs3 > taraRNAi at 29°C. To maximize knockdown, larvae were raised at 29°C. (C) Tubulin80-gal4>UAS-taraRNAi and control 2nd instar larvae were collected for chromatin extraction. Chromatin IP was performed with primers to amplify an en PRE fragment located at 0.2 Kb upstream from the transcription start site and an intergenic fragment located 1.3 Kb downstream. The diagram shows approximate location of the PRE and intergenic region as a negative control (not to scale).

https://doi.org/10.1371/journal.pone.0180026.g005

Tara is required for efficient Pc binding to the engrailed PRE

Next, we asked whether misexpression of en could be attributable to loss of Pc binding to the en locus and PRE. We employed chromatin immunoprecipitation (ChIP) to investigate Pc binding to the en locus. For FISH-I we used the Sgs3-gal4 driver to knockdown tara in the salivary gland and examined polytene chromosomes from salivary glands of wandering third instar larvae. Squashed polytene chromosome from control (Sgs3 >w1118 and Sgs3 > taraRNAi were subjected to FISH-I (see Methods). In the driver-only control, a distinct Pc band (red) is coincident with the FISH signal (green) from the engrailed locus (Fig 5B, top panel). The intensity of this band is lower than a nearby high intensity Pc band. Knocking down tara resulted in a much-reduced Pc signal at the en locus (Fig 5B, bottom panel). The nearby high intensity Pc band, however, does not diminish, consistent with our observation that not all Pc bands are affected by knocking down tara (Fig 4B). This suggests that Tara might be required for Pc binding to a subset of Pc regulated loci including en.

We also examined whether Tara is required for Pc binding to en PRE with Chromatin IP (ChIP). We used the ubiquitous Tubulin80-gal4 driver to globally knockdown tara. Tubulin80 > taraRNAi and control 2nd instar larvae were collected for chromatin extraction. ChIP was performed with primers described in [19]. One primer set was used to amplify the en PRE, located 0.2kb upstream from the transcription start site. The other set of primers were for a fragment located 1.3 kb downstream site in the en gene region (Fig 5C), which does not contain any PRE or Pc binding sequence, hence can be considered as an internal negative control. In Tubulin80 >w1118 larvae, Pc binding to en PRE was observed but not in the control en gene region (Fig 5C). In contrast, Tubulin80 > taraRNAi larvae show reduced Pc binding at en PRE (p = 0.04). As expected, the internal gene control did not show any Pc enrichment. These results suggest that Tara is required for the recruitment of Pc to the endogenous en PRE to modulate its transcription.