Drosophila linker histone H1 coordinates STAT-dependent organization of heterochromatin and suppresses tumorigenesis caused by hyperactive JAK-STAT signaling
© Xu et al.; licensee BioMed Central Ltd. 2014
Received: 24 March 2014
Accepted: 8 July 2014
Published: 28 July 2014
Within the nucleus of eukaryotic cells, chromatin is organized into compact, silent regions called heterochromatin and more loosely packaged regions of euchromatin where transcription is more active. Although the existence of heterochromatin has been known for many years, the cellular factors responsible for its formation have only recently been identified. Two key factors involved in heterochromatin formation in Drosophila are the H3 lysine 9 methyltransferase Su(var)3-9 and heterochromatin protein 1 (HP1). The linker histone H1 also plays a major role in heterochromatin formation in Drosophila by interacting with Su(var)3-9 and helping to recruit it to heterochromatin. Drosophila STAT (Signal transducer and activator of transcription) (STAT92E) has also been shown to be involved in the maintenance of heterochromatin, but its relationship to the H1-Su(var)3-9 heterochromatin pathway is unknown. STAT92E is also involved in tumor formation in flies. Hyperactive Janus kinase (JAK)-STAT signaling due to a mutation in Drosophila JAK (Hopscotch) causes hematopoietic tumors
We show here that STAT92E is a second partner of H1 in the regulation of heterochromatin structure. H1 physically interacts with STAT92E and regulates its ectopic localization in the chromatin. Mis-localization of STAT92E due to its hyperphosphorylation or H1 depletion disrupts heterochromatin integrity. The contribution of the H1-STAT pathway to heterochromatin formation is mechanistically distinct from that of H1 and Su(var)3-9. The recruitment of STAT92E to chromatin by H1 also plays an important regulatory role in JAK-STAT induced tumors in flies. Depleting the linker histone H1 in flies carrying the oncogenic hopscotch Tum-l allele enhances tumorigenesis, and H1 overexpression suppresses tumorigenesis.
Our results suggest the existence of two independent pathways for heterochromatin formation in Drosophila, one involving Su(var)3-9 and HP1 and the other involving STAT92E and HP1. The H1 linker histone directs both pathways through physical interactions with Su(var)3-9 and STAT92E, as well with HP1. The physical interaction of H1 and STAT92E confers a regulatory role on H1 in JAK-STAT signaling. H1 serves as a molecular reservoir for STAT92E in chromatin, enabling H1 to act as a tumor suppressor and oppose an oncogenic mutation in the JAK-STAT signaling pathway.
KeywordsHeterochromatin Linker histone H1 JAK-STAT signaling Tumor suppressor Melanotic tumors Drosophila melanogaster
The genomes of eukaryotes are packaged into a nucleoprotein complex called chromatin[1, 2]. Compaction of the DNA is achieved primarily through its association with a small family of proteins called histones. There are five major classes of histones: the core histones H2A, H2B, H3, and H4 and the linker histone usually referred to as H1. The nucleosome core particle is the basic repeating unit of chromatin in which approximately 145 bp of DNA is wrapped around an octamer of the four core histones. The linker histone H1 binds to the nucleosome core particle near the site at which DNA enters and exits the core particle, organizing an additional of approximately 20 bp of DNA to form the chromatosome. The binding of H1 stabilizes the core particle and facilitates folding of nucleosome arrays into higher order structures[4, 5].
The structure of chromatin is dynamic and undergoes changes in compaction during the cell cycle and during development. Importantly, among the five classes of histones, the H1 linker histone exhibits the greatest mobility, shuttling between the chromatin and the nucleoplasm with a residence time in chromatin of approximately 3 min[6, 7]. Within the nucleus, chromatin exhibits variable levels of packaging. Chromatin is organized into densely packaged, generally silent regions called heterochromatin and more loosely packaged, transcriptionally active euchromatin. Heterochromatin identity is established through modifications of epigenetic landscape of the genome and recruitment of specialized protein factors[9, 10]. An important breakthrough in our understanding of the molecular basis for heterochromatin formation came with the discovery that the H3 histones in heterochromatin are modified by methylation on lysine 9 in the H3 N-terminal tail. This modification is catalyzed by the histone methyltransferase Su(var)3-9 and it is recognized and bound by heterochromatin protein 1 (HP1). Recently, we reported that the linker histone H1 in Drosophila is also required for heterochromatin formation and that it recruits Su(var)3-9 to heterochromatin by directly interacting with it. H1 also has been shown to interact with HP1[14–17].
Another factor that has been linked to heterochromatin stability in Drosophila is the DNA binding protein STAT92E[18–20]. Flies have a single STAT (STAT92E) and a single Janus kinase (JAK) that together constitute the JAK-STAT signaling pathway in Drosophila[21–23]. Perturbations of this pathway, including depletion of STA92E or expression of a mutant hyperactive JAK (hop Tum-l ), lead to heterochromatin instability. The hop Tum-l mutation also leads to the formation of blood cell tumors[24, 25]. We show here that the H1 linker histone directly interacts with STAT92E and regulates its roles in both heterochromatin formation and tumorigenesis. Our results identify a second pathway of heterochromatin formation that is distinct from that of H1 and Su(var)3-9. Our observations also establish linker histone H1 as a tumor suppressor in flies.
Results and discussion
Hyperactive JAK/STAT signaling disrupts pericentric heterochromatin
Previous reports implicated the JAK-STAT signaling pathway in heterochromatin stability and heterochromatin protein1 (HP1) localization in Drosophila. Tumorous-lethal (Tum-l), an oncogenic allele of hopscotch (hop) encoding a constitutively hyperactive mutant of Drosophila JAK, was observed to disrupt heterochromatic silencing and HP1 localization in heterochromatin. Loss of Drosophila STAT (STAT92E) was found to have very similar effects. Based on the observation that HP1 and STAT92E interact, it was proposed that the two proteins colocalize within heterochromatin and that unphosphorylated STAT92E regulates HP1 localization and heterochromatin stability.
Interestingly, although hyperactive JAK clearly disrupts HP1 localization and formation of a single chromocenter in polytene chromosomes, it does not lead to a reduced amount of the H3K9 dimethyl mark in pericentric heterochromatin. In contrast, depletion of H1 causes a marked reduction in pericentric H3K9Me2 signal (Figure 1). Thus, H1 and STAT92E may share some but not all roles in regulation of heterochromatin structure and activity.
H1 regulates localization of STAT92E in chromatin
To further investigate the basis for H1 and STAT92E co-localization in chromatin, we analyzed their distribution in salivary gland nuclei in which H1 was depleted by RNAi. As expected, we observed reduced H1 abundance in polytene chromosomes of H1-depleted larvae (Additional file1: Figure S2A). Strikingly, we also found that specific STAT92E staining of polytene chromosomes, including the chromocenter and euchromatic arms, is almost completely lost upon H1 depletion (Figure 2B). In control experiments, STAT92E or H1 localization is not substantially affected in animals with a homozygous null mutation of Su(var)3-9 or with HP1 depleted by RNAi (Figure 2B and Additional file1: Figure S2A). The observed mis-localization of STAT92E in H1-depleted larvae is corroborated by chromatin IP (ChIP) experiments: the occupancy of both H1 and STAT92E at multiple genomic loci is strongly decreased upon H1 knockdown (Figure 2C).
We also analyzed the distribution of H1 and STAT92E in polytene chromosomes of hop Tum-l mutant larvae and in animals with STAT92E depleted by RNAi. RNAi-mediated depletion almost completely eliminates STAT92E presence in polytene chromosomes (Additional file1: Figure S2C). Interestingly, hop Tum-l mutation also results in reduction and re-distribution of the STAT92E-specific signal in polytene chromosomes (Additional file1: Figure S2C). In contrast to the effects of H1 depletion on STAT92E distribution, neither STAT92E depletion nor its hyperphosphorylation had a discernable effect on H1 localization (Figure 2D). These results indicate that linker histone H1 strongly contributes to STAT92E tethering to chromatin. H1 is required for the apparent ubiquitous localization of STAT92E and thus may act upstream of STAT92E in regulation of normal chromosome architecture and heterochromatin structure proposed previously[18, 19].
H1 physically interacts with STAT92E
To further support this model, we examined interactions of H1 and STAT92E in the context of chromatin. To this end, we reconstituted defined oligonuclosomal substrates that did or did not contain H1 and used in vitro ChIP to analyze association of purified recombinant STAT92E with H1-containing and H1-free chromatin. The plasmid template used for oligonucleosome reconstitution is not known to contain specific STAT92E recognition sequences. Whereas the presence of nucleosomes inhibited non-specific STAT occupancy at the DNA substrate, addition of H1 to chromatin stimulated STAT92E binding (Figure 3B). Thus, STAT92E physically interacts with H1, both as a free protein and as a component of reconstituted chromatin. These observations are similar to our recent discovery of an interaction between H1 and Su(var)3-9, as well as recruitment of Su(var)3-9 to H1-containing chromatin (Figure 3B).
H1 linker histones consist of a short unstructured N-terminal domain (NTD), a central winged helix-like globular domain (GD) and a long unstructured C-terminal domain (CTD)[1, 2]. Particular residues within the GD and regions within the CTD contribute to H1 binding to nucleosomes in vitro. To determine which region(s) contribute to the H1-STAT92E interaction, we performed in vitro binding experiments with GST fusions of the individual H1 domains and His-tagged STAT92E (Figure 3C). We observed that the H1 CTD interacts with STAT92E, whereas the H1 NTD and GD do not interact with STAT92E. Interaction of full-length H1 with STAT92E appears to be stronger than that of the isolated H1 CTD, suggesting that the structure of the CTD required for interaction with STAT92E may be influenced by one or both of the other H1 domains. Interestingly, we recently found that the CTD of the murine H1d subtype is required for its interactions with DNMT1 and DNMT3B. Thus, the C-terminus of H1 may encompass interaction module(s) for multiple binding partners of linker histone H1.
H1 suppresses tumorigenesis caused by hyperactive JAK/STAT signaling
H1 regulates hematopoietic tumor formation caused by hyperactive JAK
hop Tum-l /+
hop Tum-l /+; pINT1-Nau/Act-GAL4
pINT1-H1 6F /+; Tub-GAL4/+
hop Tum-l /+; pINT1-H1 4M /Act-GAL4
hop Tum-l /+; UAS-H1/Act-GAL4
hop Tum-l /+; UAS-STAT/Act-GAL4
hop Tum-l /+; UAS-STAT(Y704F)/Act-GAL4
Importantly, depletion of H1 in the wild type background does not lead to tumorigenesis (Table 1), which suggests that H1 does not play a direct role in the JAK-STAT transcriptional response. To confirm this observation, we made use of a transgenic allele in which a GFP reporter is placed under control of a STAT-responsive promoter containing 10 STAT92E binding sites. We examined GFP expression by GFP autofluorescence of whole larvae or western blot of larval lysates (Figure 4B,C). Upon hyperphosphorylation in the hop Tum-l mutant, STAT92E becomes transcriptionally active and strongly activates GFP expression. Quantitation of western data indicates about fourfold activation of the transgene upon STAT92E hyperphosphorylation (Figure 4D), which is further increased by H1 depletion in the hop Tum-l background. On the other hand, H1 depletion alone does not activate transgene expression (Figure 4B,C,D). Thus, H1 does not appear to play a substantial role in direct regulation of normal transcriptional targets of phosphorylated STAT92E.
Rather, our results are consistent with a model in which H1 is required to maintain sequence-independent, ectopic localization of STAT in chromatin. Upon H1 depletion, excess STAT92E is released from the ectopic sites. The released STAT is in an unphosphorylated, transcriptionally inactive form and is unable to activate STAT-responsive genes (Figure 4). On the other hand, in the presence of the mutant hop Tum-l allele, the STAT released by H1 depletion becomes available as a substrate for phosphorylation by hyperactive JAK, which results in additional activation of endogenous STAT targets and increased tumorigenesis. If this model is correct, then overexpression of STAT itself in the hop Tum-l mutant should also result in a similar, enhanced tumor formation by virtue of an increased JAK substrate availability. Indeed, as expected, UAS-controlled STAT92E overexpression driven by Actin-GAL4 leads to a fourfold increase of the tumor index in the hop Tum-l background (Table 1). However, when a non-phosphorylatable mutant of STAT, STAT92E(Y704F), is overexpressed under similar conditions, the tumorigenic effect of hop Tum-l is not affected.
The tumor suppressor function of HP1 and Su(var)3-9 is dependent on H1
hop Tum-l /+
hop Tum-l /+; ht-HP1/+
hop Tum-l /+; ht-HP1/pINT1-H1 4M , Act-GAL4
hop Tum-l /+; ht-Su(var)3-9/+
hop Tum-l /+; ht-Su(var)3-9/pINT1-H1 4M , Act-GAL4
H1 and STAT92E cooperate in the establishment of heterochromatin structure
H1 depletion results in profound changes of polytene chromosome architecture and heterochromatin structure, activity, and biochemical composition. For instance, H1-depleted larvae largely lose the pericentric H3K9 dimethyl mark (Figure 1) and. However, we previously observed that overexpression of the H3K9-specific HMT, Su(var)3-9, partially ameliorates this defect.
The results of this and other studies[18, 19] suggest that ectopic localization of unphosphorylated STAT92E in chromatin may play an important role in proper polytene chromosome morphology in larvae, specifically the formation of heterochromatic chromocenter. The ectopic localization of STAT requires linker histone H1, depletion of which brings about STAT92E release from ectopic sites and simultaneous disruption of the chromocenter. Thus, in turn, it is possible that H1-mediated effects on heterochromatin structure may depend, at least in part, on STAT92E.
A new paradigm for tumor suppression: linker histone H1 as a molecular reservoir for an oncogenic transcription factor
Li and colleagues have proposed that the oncogenic effect of the hop Tum-l allele and STAT hyperphosphorylation is a direct consequence of the resulting disruption of heterochromatin, which then causes global defects in gene regulation. A recent study in mammalian cells also proposed a role for unphosphorylated STAT5A in stabilization of heterochromatin and tumor suppression via repression of multiple oncogenes. Our results do not support this model. The strongest evidence against the disruption of heterochromatin as a principal cause of tumorigenesis is our finding that H1 depletion produces disruptions in heterochromatin that are comparable to or stronger than those caused by the hop Tum-l mutation, yet H1 depletion alone does not result in tumorigenesis (Table 1). Also importantly, although overexpression of non-phosphorylatable STAT92E(Y704F) largely restores pericentric heterochromatin in H1-depleted salivary glands (Figure 5A), it does not act as tumor suppressor in hop Tum-l background (Table 1). Therefore, tumor formation and the heterochromatin structural abnormalities observed in the hop Tum-l mutant are likely independent phenomena.
Instead, our results are consistent with a model in which linker histone H1 serves as a molecular reservoir for STAT92E. We propose that, normally, unphosphorylated STAT92E resides along with H1 in numerous loci throughout chromosomes, including pericentric heterochromatin, where the two proteins stabilize HP1 binding (Figure 6A, top left). The association of STAT92E with these ectopic loci is dependent on H1, but independent of STAT92E canonical DNA recognition elements. Hyperphosphorylation of STAT92E prevents its efficient association with ectopic sites, directs it to specific DNA elements and causes abnormal transcriptional activation and tumorigenesis (Figure 6A, top right). H1 depletion alone leads to release of unphosphorylated STAT92E from the chromatin reservoir and disruption of normal pericentric structures. However, in the absence of hyperactive JAK, it does not result in tumorigenesis, because the normal level of JAK kinase activity is limiting, and generation of higher levels of activated STAT92E is not achieved (Figure 6A, bottom left). Depleting H1 in the presence of hyperactive hop Tum-l kinase, though, leads to excessive production of phosphorylated STAT92E and enhanced tumorigenesis (Figure 6A, bottom right).
Linker histone H1 directs two alternative pathways of heterochromatin formation
We reported previously that Drosophila H1 interacts with and recruits Su(var)3-9 to promote heterochromatin formation. The results reported here provide evidence for another, alternative pathway of H1-dependent heterochromatin formation, which involves H1 interaction with STAT92E and its recruitment to ectopic sites in chromatin. Eviction of STAT92E from its chromatin reservoir can be achieved by H1 depletion or STAT hyperphosphorylation. However, although hyperphosphorylation of STAT92E disrupts the structure of pericentric heterochromatin, it does not substantially affect H3K9 dimethylation present in HP1-positive foci (Figure 1). Thus, STAT92E appears to be dispensable for Su(var)3-9 localization or activity. Conversely, a null mutation of Su(var)3-9 does not affect STAT92E localization in polytene chromosomes (Figure 2B). We conclude that STAT92E function in the establishment or maintenance of heterochromatin structure is independent of H3K9 methylation by Su(var)3-9. On the other hand, H1 directs formation of heterochromatin structures via both pathways, one that involves H3K9 dimethylation and the other that utilizes STAT-dependent stabilization of HP1 (Figure 6B).
Our analyses reveal that unphosphorylated STAT92E is an abundant and nearly ubiquitous chromatin component (Figure 2A and Additional file1: Figure S3). Its level of expression approaches 10%–20% of that of heterochromatin protein HP1, or close to 1 molecule per 100 nucleosomes in the genome, much higher than expected for a sequence-specific transcription factor. The storage and sequestering of excess inactive STAT in the nucleus is achieved through association with H1-containing chromatin and allows for rapid activation of the JAK-STAT regulatory cascade by external stimuli. At the same time, STAT92E appears to stabilize particular chromatin conformations, such as pericentric heterochromatin in the chromocenter of larval salivary gland chromosomes in Drosophila, and physically interacts with H1 and HP1, heterochromatin components. In the future, it will be interesting to analyze molecular interactions in a putative tripartite STAT-H1-HP1 complex in the context of chromatin and to examine how STAT92E modulates the structure of the chromatin fiber in vitro.
By using polytene chromosome analyses in Drosophila salivary gland cells, we performed studies of chromatin defects associated with hyperactivation of STAT. Although a connection between heterochromatin integrity and tumorigenesis by JAK-STAT effectors has been proposed recently[18, 19], we discovered a new major connection between STAT and linker histone H1 that alters the existing model of STAT-dependent maintenance of heterochromatin and provides mechanistic insight into its regulation. We provide evidence that STAT92E specifically helps to maintain a particular feature of pericentric heterochromatin, namely the chromocenter region of polytene chromosomes in Drosophila larvae. Furthermore, we report direct physical interactions of STAT92E with H1 and HP1, key structural components of heterochromatin, and discern molecular mechanisms of STAT-dependent regulation of heterochromatin formation. These observations lead us to propose a coordinate role for STAT, linker histone H1 and HP1 in the maintenance of heterochromatin integrity. Our studies have also revealed that, as a result of its involvement in STAT-dependent organization of chromatin and sequestering STAT in the nucleus, the linker histone H1 acts to suppress tumorigenesis caused by hyperactive JAK-STAT signaling.
Fly strains and genetics
Flies were grown on standard corn meal, sugar, and yeast medium with Tegosept. Stocks were maintained at 18°C. Crosses were performed in an environmental chamber at 29°C. For polytene chromosome staining, all animals were incubated at 29°C throughout their life cycles. Canton-S flies were used as wild type controls. The following fly stocks were obtained from the Bloomington Stock Center and are described in FlyBase: hop Tum-l , UAST-STAT92E-GFP, UAST-STAT92E-RNAi, 10xSTAT92E-GFP, Actin-GAL4/CyO, HP1-shRNA, Su(var)3-9 1 /TM3,Sb, and Su(var)3-9 2 /TM3,Sb. ht-HP1 and ht-Su(var)3-9 flies were generous gifts from Dr. G. Reuter (Martin Luther University, Halle-Wittenberg, Germany). Nau-RNAi transgenic flies were generously provided by Dr. B. Paterson (NIH). UAS-STAT(Y704F) allele was a generous gift of Dr. W. Li (University of California at San Diego). H1 knockdown was achieved by pINT1-H1[4 M] transgene expression driven by Actin-GAL4.
To analyze the tumor index (TI), hop Tum-l flies were crossed to Canton-S flies, ht-HP1 or ht-Su(var)3-9 flies. Alternatively, hop Tum-l and Actin-GAL4/CyO flies were crossed to pINT-1-H1 4M or pUAST-H1 flies. hop Tum-l , ht-HP1 and hop Tum-l , and ht-Su(var)3-9 flies were crossed to pINT-1-H1 4M , Actin-GAL4/CyO flies. TI was calculated based on observations from F1 adult flies reared at 29°C as described. p values were calculated by the Mann-Whitney U test using GraphPad Prism software.
Indirect immunofluorescence (IF) analyses of polytene chromosomes were carried out as described. DNA was stained by adding 1.5 μg/ml DAPI (Vectashield, CA, USA) to the mounting medium. The following antisera were used at the indicated dilutions: monoclonal mouse anti-Drosophila HP1, C1A9 (1:50, Developmental Studies Hybridoma Bank); goat anti-Drosophila STAT, dF-20 (1:50, Santa Cruz Biotechnology); affinity-purified rabbit Drosophila H1 antiserum (1:5,000) and affinity-purified rabbit anti-H3K9me2 (1:100, Abcam). Appropriate Cy2- and Cy3-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA) were used at 1:200. Specificity of IF staining was verified by appropriate controls, such as staining with secondary antibodies only and staining of polytene chromosomes from H1 and STAT92E knockdown animals (see for example Figure 5B, and Additional file1: Figure S2C).
For GFP autofluorescence analyses, wild type; hop Tum-l ; pINT1-H1 4M and hop Tum-l ; and pINT1-H1 4M flies were crossed with flies carrying 10xSTAT92E-GFP transgene. The F1 L3 larvae were placed on a glass slide and immobilized on ice for 10 min.
Fluorescent images were acquired on a Zeiss Axioplan microscope (Carl Zeiss, Oberkochen, Germany) equipped with Zeiss Digital Microscopy Camera AxioCam ICC1and AxioVision Digital Image Processing Software (Carl Zeiss). Stereoscopic images were acquired on a Zeiss SteREO Discovery V8 microscope (Carl Zeiss).
Recombinant proteins and GST pull-down
Recombinant Drosophila His6- and FLAG-tagged HP1 protein was purified as described. Full-length Drosophila STAT92E cDNA was amplified by PCR from an EST clone (RE13194) (Drosophila Genomic Research Center) and cloned into pFastBac1 vector (Invitrogen) in-frame with a C-terminal His6-tag. Details of cloning are available on request. STAT92E-His6 baculoviruses were prepared using BacToBac System (Invitrogen). The recombinant STAT92E-His6 was synthesized in Sf9 cells and purified by TALON His-Tag Purification Resin (Clontech). GST fusions of H1, H2A, and HP1 were expressed in E. coli (BL21(DE3)pLys strain) and purified by glutathione-Sepharose chromatography as described. To prepare GST fusions of H1 globular (amino acid residues 41–119), N- (1–40) and C-terminal (120–256) domains, corresponding PCR products were cloned into in pGEX 4 T-1 (GE Life Sciences). The purified proteins were analyzed by SDS-PAGE, and concentrations were determined by Coomassie staining along with BSA protein mass standards (Pierce).
In GST pull-down assays, purified recombinant STAT92E-His6 was incubated with GST or GST fusion proteins and purified on glutathione-Sepharose as described. STAT92E-His6 binding to GST fusion proteins was detected by anti-His6 western of the pull-down samples. Additionally, the pull-down samples were examined for the presence of GST fusion proteins by SDS-PAGE and Coomassie staining.
Reconstitution of chromatin and ChIP
Reconstitution of H1-containing and H1-free chromatin was carried out as described. For in vitro ChIP analyses, approximately 0.5 pmol purified STAT92E-His6 or Su(var)3-9-His6 protein was incubated with 0.2 pmol supercoiled plasmid DNA (3.2 kb), H1-containing or H1-free chromatin in 20 μl of reaction buffer (50 mM Tris–HCl, pH 7.9, 5 mM MgCl2, 4 mM DTT and 2 μg/ml BSA) for 15 min at 27°C. The material was cross-linked for 10 min at room temperature, and the cross-linking was terminated by addition of 9.8 μl of 2.5 M glycine. The material was incubated with 2 μl rabbit polyclonal anti-His6 antibody, ChIP grade (Abcam) in 400 μl reaction buffer overnight at 4°C. After immunoprecipitation and cross-link reversal, the DNA was isolated by QIAquick PCR purification kit (Qiagen, Valencia, Santa Clarity, CA, USA). Samples were analyzed quantitatively by real-time PCR (ViiA™ 7 system, Applied Biosystems, Grand Island, NY, USA) as described[13, 14]. For H1 and STAT92E qChIP in vivo, chromatin was prepared from H1-depleted and control whole larvae, immunoprecipitated as described above and analyzed by real-time PCR as described previously[13, 14]. Primer sequences are available upon request. Each sample was analyzed in three independent real-time PCR reactions.
Semi-quantitative western analyses of H1, tubulin, and GFP in Drosophila salivary gland or whole larval lysates were carried out as described. For quantitation of STAT92E in vivo, Drosophila embryonic SK (Soeller-Kornberg) extracts were prepared as described. SK extract was boiled in Laemmli loading buffer for 5 min and centrifuged. An aliquot of SK extract was loaded on a 10% SDS-PAGE gel, along with 0.2–20 pmol purified His6-tagged STAT92E or 2–200 pmol purified His6- and FLAG-tagged Drosophila HP1 protein. The following primary antibodies were used at the indicated dilutions: rabbit anti-Drosophila H1 (1:5,000); mouse monoclonal anti-tubulin, E7 (1:500; Developmental Studies Hybridoma Bank); mouse anti-GFP (1:1,000, Santa Cruz Biotechnology); mouse anti-Drosophila HP1, C1A9 (1:3,000), and goat anti-Drosophila STAT dF-20 (1:50). The infrared dye-labeled secondary antibodies were used at 1:10,000 (LI-COR Bioscience, Lincoln, NE, USA). Images were obtained and quantitated using the LI-COR Odyssey Infrared Imaging System.
green fluorescent protein
heterochromatin protein 1
polymerase chain reaction
quantitative chromatin immunoprecipitation
signal transducer and activator of transcription
upstream activation sequence.
We are grateful to M. Keogh, W. Li, B. Paterson, and G. Reuter for the antibodies and fly stocks, N. Baker for the IF reagents, and A. Jenny for the access to Zeiss SteREO Discovery V8 microscope. We thank X. Lu for expression constructs, E. Vershilova for expert technical assistance, and B. Bartholdy for help with statistical analyses. This work was supported by grants from the National Institutes of Health to DVF (GM074233) and AIS (GM093190 and CA079057). NX was supported in part by the NIH IRACDA/K12 training grant (1K12GM102779-01).
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