shown that HP1 binding and H3K9 methylation are interdepen- dent [40]. However, detailed analyses of HP1 distribution using cytoimmunochemical approaches and high resolution mapping based on the DamID technique have shown that although HP1 and the H3K9me2 mark generally overlap in the chromocenter and pericentric regions, there are also numerous sites on the chromosome arms where HP1 binding is not associated with di- or trimethylation of H3K9. In some cases this binding is dependent on RNA [39,50,51]. Our cytological results indicate that VIG and HP1 overlap mostly outside of the chromocenter, including sites of active transcription (Figure 4D). Destabilization of HP1 binding in those locations seems unlikely to result in significant overall changes in H3K9me levels. The HP1/VIG immunosignals could mark sites of endogenous small RNA precursors and could be involved in regulatory processes affecting heterochromatin indi- rectly. In the Su(var)205 mutant, the resulting HP1 deficit does not result in loss of H3K9 methylation; Su(var)3-9 HKMTase and H3K9me2 are still present at the chromocenter, but are also found in euchromatin, resulting in an almost ubiquitous distribution of H3K9me2 [41]. This suggests that HP1 is probably not the only protein involved in tethering HKMTases to chromatin, although a role for HP1 in sequestering H3K9 methylation to heterochro- matin is clear [52]. Therefore our results showing that vig mutation causes HP1 destabilization without an effect on H3K9 methylation level is not without precedent. The developmental profile generated for vig and vig2 expression shows that the two genes have activity peaks at different stages of development. Vig2 is very abundant in ovaries, where several different HKMTases (dSETDB1, Su(var)3-9, and G9a) work together to methylate H3K9 [53–55]. Depletion of Vig2 results in a decreased level of H3K9me2 in adult flies; however, there was no effect on the amount of HP1 observed in the nuclei. Of the three HKMTases, only Su(var)3-9 is known to recruit HP1 molecules directly, while the others utilize alternative mechanisms, yet to be identified. The point of intersection with Vig2 remains to be identified. Despite the differences in their action, both VIG and Vig2 appear to be new components of an extensive network embracing heterochromatin, small RNAs and transcription regulation. Studying the specific functions of VIG and Vig2 in detail will be important in understanding the dynamics of heterochromatin establishment and maintenance. Materials and Methods Fly strains and genetic experiments Fly stocks were maintained at 22uC, 70% humidity on a cornmeal sucrose-based medium [56]. The w*;P{EP}vigEP812/CyO stock was obtained from the Szeged Drosophila Stock Centre; * homozygous mutant flies are viable, females are sterile. w ; P {FRT(whs)}2A P{neoFRT}82B PBac{GAL4D,EYFP}CG11844PL00470 (vig2PL470) was obtained from the Bloomington Drosophila Stock Center (BL #19518); homozygous mutant flies are viable and fertile. vasAS is described in [35]. X chromosomes of the above mentioned lines were replaced by the y w chromosomes. In the vigEP812/CyO stock the 2nd chromosome balancer was changed to CyO, P{w+mC GAL4}, P{w+mC UAS-GFP} for polytene chromosome immunofluorescence experiments. w; Su(var)3-906 has been described elsewhere [57]. The PEV reporters B79, J545, J448 have been characterized previously [32,33]. To assess modification of PEV, genetic crosses were conducted as follows: virgin females carrying the mutation of interest (y1w67c23; vigEP812/CyO, or y1w67c23; vig2PL470, or y1w67c23; vasAS/ VIG and Vig2 Impact Chromatin CyO) or ‘wild type’ y1w67c23 flies were crossed to males carrying the PEV reporters y1/Y; +; P(y+)B79, y1/Y; +; P(y+)J545, y1/Y, P(y+)J448; +; +. Male progeny from WT and mutant mothers were compared. Adult abdominal cuticle preparation Flies were immobilized on the adhesive side of a piece of tape and covered with PBST. The abdomens were sliced off, dissected along the dorsal midline, and cleaned. These abdomen cuticle halves were transferred to a drop of mounting media [Shandon Immu-Mount (Thermo Fisher Scientific) supplemented with chloral hydrate (53% v/v) and lactic acid (9% v/v)], spread on a microscope slide, covered by a cover slip, left to dry overnight on the slide warmer at 42uC, and subsequently photographed. Real time RT-PCR RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s recommendations. For quantita- tive real time one-step RT-PCR we used the QuantiTect RT-PCR Kit (Qiagen), following the instructions provided by the manufac- turer for the SmartCycler PCR machine (Cepheid). Standard curves were generated using 0.5–500 ng of total RNA for each primer pair. The expression level of the RpL32 gene was used for normalization. Primers used: Su(var)205 (HP1): ACCATTTCTGCTTGGTC- CAC and CAAGCGAAAGTCCGAAGAAC; vig: TTCGCT- GTCGTTCTCCTTCTCCTTCT and AAAGAGCTGACCT- TGGACGA; vig2: GCGTCAATTCAACAATCGTG and CCC- GGTCGTCTTTAAGTCCT; RpL32: ATGGTGCTGCTATC- CCAATC and GTCGCCTGCGTTCTCAAG. Antibodies Rabbit polyclonal antisera directed against the N-terminal 10– 14 AA of VIG and Vig2, CSH1801, CHS1803, and CSH2542 were raised as previously described [11]. Anti- HP1 [38] anti-HP1 (WA191 [58]), anti-Vasa (Lasko and Ashburner, 1990) and anti- HP2 [59] have been previously described. Anti-H2B (# 07–371); anti-H3K9me2 (# 07–441); anti-H4 (# 07–108) were purchased from Upstate; anti-Actin (JLA20) was obtained from DSHB; anti- Dynein was purchased from Sigma (D5167). Secondary HRP-conjugated antibodies were purchased from Thermo Scientific. Polytene chromosomes squashes and immunostaining Polytene chromosomes were prepared as described [60]. VIG deficient 3rd instar larvae were selected based on the absence of fluorescence from vigEP812/ CyO, P{w+mC GAL4}, P{w+mC UAS- GFP} stock. HP1 distribution patterns were identified using the C1A9 monoclonal antibody diluted 1:10. VIG and Vig2 were detected using the CSH1803 antibody at a 1:5 dilution. Secondary antibodies (Molecular Probes) were labeled with Alexa Fluor 594 (red) and Alexa Fluor 488 (green). The images of HP1 and VIG or Vig2 distribution were captured simultaneously (Figure 4, colored panels); then red and green color channels were separated using Corel PHOTO-PAINT software to create the black and white images of HP1, VIG, and Vig2 individual distributions. Western analysis Total protein extracts were obtained as follows: 3–4 day old flies were collected, ground in 10 mM Tris pH 8 with ‘Complete’ Protease Inhibitor Cocktail (Roche), and left on ice for 309. 26 Laemmli loading buffer (LLB) (BioRad) was added and the mixture boiled for 5 min. Nuclei were prepared as described PLoS ONE | www.plosone.org 9 July 2009 | Volume 4 | Issue 7 | e6182