| Shin-Hae Lee, In-Joo Kim: The authors equally contributed to this work. To whom correspondence should be addressed: Mi-Ae Yoo, Department of Molecular Biology, College of Natural Science, Pusan National University, Busan 609-735, Republic of Korea. Tel: +82-51-510-2278, Fax: +82-51-513-9258 E-mail: mayoo@pusan.ac.kr |
Many epigenetic factors are known to be associated with tumorigenesis, suggesting that cancer is an epigenetic disease (Egger et al., 2004). The structure of chromatin is profoundly altered by neoplasia (Counts and Goodman, 1995), and abnormal expression of epigenetic factors during tumorigenesis has been well documented (Jones, 2005; Pantry and Medveczky, 2009; Tateno et al., 2010). Furthermore, recent studies have revealed that dysregulation of stem cell proliferation is the cause of many cancers (O’Brien et al., 2010), demonstrating that cancer may represent a stem cell-based disease.
Therefore, understanding of the role of epigenetic factors in adult stem cell regulation would provide valuable insights into the mechanisms of tumorigenesis (Rossi et al., 2008). Methyl-CpG-binding protein-2 (MeCP2) is a multifunctional nuclear protein, that plays a critical role in interpreting epigenetic codes, and acts as a bridge factor linked to DNA modification and histone modification (Nan et al., 1998). MeCP2 was initially described as a DNA methylation-dependent transcription repressor (Nan et al., 1998). However, it was recently been reported to function in the chromatin architecture, RNA splicing regulation, and transcriptional activation, as well as DNA methylation-independent gene silencing (Hite et al., 2009).
MeCP2 is ubiquitously expressed in almost all tissues, with varying levels of expression; high in the spleen and lung; lower in the heart, kidney, liver, stomach and small intestine (Shahbazian et al., 2002). The expression level of MeCP2 is elevated during carcinogenesis in several tissues, including the mammary gland, lymphoma and colorectal carcinomas (Billard et al., 2002; Darwanto et al., 2003; Bernard et al., 2006). Combined with the fact that hypermethylation has been observed in virtually every type of human neoplasm (Egger et al., 2004), these evidences suggest the possibility that MeCP2 is involved in tumorigenesis. However, the function of MeCP2 in stem cell regulation in adult tissues remains poorly understood.
Adult Drosophila gut appears to be an excellent model system for the study of stem cell biology (Pearson et al., 2009; Jones and Rando, 2011; Wang and Jones, 2011). Drosophila intestinal stem cells (ISCs) were recently discovered in the adult posterior midgut and hindgut, which have structural and functional similarities to their mammalian counterpart (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006; Takashima et al., 2008). Drosophila ISCs residing in a basal location within the posterior midgut epithelium are the only mitotic cell type and generate two types of differentiated progeny: polyploid enterocytes (ECs) or less frequently diploid enteroendocrine (ee) cells through an intermediate enteroblast (EB) (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). Each of these cell types can be identified using unique markers (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006, 2007; Lee et al., 2009). To control ISC proliferation in adult Drosophila, several signaling pathways including the Wnt (Lin et al., 2008), JAK/STAT (Jiang et al., 2009), EGFR (Jiang et al., 2011) and Hippo (Staley and Irvine, 2010) pathways have been identified. In addition, PVF2, a Drosophila homologue of VEGF, and its receptor PVR stimulate ISC proliferation (Choi et al., 2008a; Park et al., 2009).
Drosophila system is used as a powerful genetic tool to identify the functions of human genes (Xu et al., 2008). Although Drosophila has a different DNA methylation site compared to mammals (Marhold et al., 2004) and the ortholog of MeCP2 has not been reported to exist in Drosophila, the chromatin remodeling factors are conserved in Drosophila and mammal (Ho and Crabtree, 2011). Furthermore, transgenic Drosophila was recently used to identify modifiers involved in the rough eye phenotype induced by overexpression of human MeCP2 (hMeCP2) (Cukier et al., 2008), suggesting that the transgenic Drosophila approach is suitable for investigating the function of hMeCP2 in vivo. Therefore, in this study we investigated the physiological role of epigenetic factor, hMeCP2 in ISC proliferation, using transgenic Drosophila model.
To construct pUAST-hMeCP2 plasmid, the coding region of hMeCP2 was PCR-cloned from human HS683 cDNA and cloned into pcDNA3.1-His (Molecular Probes, OR, USA). The coding sequence was then subcloned into the pUAST vector (Brand and Perrimon, 1993) and confirmed by sequencing.
Transgenic flies were generated using P element-mediated germ line transformation as described previously (Spradling, 1986). Briefly, pUAST-hMeCP2 plasmids were co-injected with pπ25.7wc helper vector into w1118 embryos (Karess and Rubin, 1984), and transformants having plasmid revealed by the red eye color were selected (Spradling, 1986). Lines containing two-copy inserts were established, and three independent lines were isolated.
All Drosophila stocks were consistently maintained at 25°C on standard cornmeal-sugar-yeast medium under a 12 h/12 h light/dark cycle. The food consisted of 79.2% water, 1% agar, 7% cornmeal, 2% yeast, 10% sucrose, 0.3% bokinin and 0.5% propionic acid. To avoid larval overpopulation in culture vials, 25–30 adult flies were cultured in a vial and transferred to new vials containing fresh food every 2–3 days for a period of 50–60 days or longer. In the experiments described here, only female flies were analyzed. The OregonR strain was utilized as wild type.
For ectopic expression of hMeCP2 in Drosophila, we used the GAL4/UAS system. The GAL4/UAS ectopic expression system is valuable machinery for the overexpression of a transgene under specific conditions, such as cell-, tissue-, temperature- and stage-specific conditions, in Drosophila (Duffy, 2002). When the Gal4 transcription factor is expressed under the control of various promoters, Gal4 can drive the expression of a transgene under an UAS sequence (Duffy, 2002). To ectopically express UAS-hMeCP2 in Drosophila, several Gal4 drivers summarized in Table I were used. esg-Gal4,UAS-GFP,tub-Gal80ts was obtained from B. Ohlstein (Ohlstein and Spradling, 2007), and Myo1A-Gal4;UAS-GFP,tub-Gal80ts was obtained from B. A. Edar (Jiang et al., 2009). Heat-shock (hs)-Gal4, daughterless (da)-Gal4 and caudal (cad)-Gal4 were obtained from the Bloomington Drosophila Stock Center (Indiana University). Salivary gland (sg)-Gal4 was obtained from M. Yamaguchi (Kato et al., 2008).
Anesthetized adult flies were sputter-coated with platinum (HITACHI E-1010) and observed under a scanning electron microscopy (HITACHI-S3500N).
For the expression of UAS-hMeCP2 regardless of the developmental stage, the Gal80ts technique was used (McGuire et al., 2004). The flies containing UAS-hMeCP2, tub-Gal80ts and specific-Gal4 driver were developed at 22°C to suppress hMeCP2 expression in developmental stage. After eclosion of crosses, flies were aged 2–5 days to adulthood at 18°C and shifted to 29°C for 1–5 days for the induction of hMeCP2.
Immunostaining of polytene chromosomes was performed as described previously (Kato et al., 2008). Briefly, squashes of salivary glands were stored in phosphate-buffered Saline (PBS)/0.05% Tween 20/1% bovine serum albumin (BSA, Sigma-Aldrich, MO, USA) (PBST-BSA) for 16 h, then followed by incubation with primary antibodies for 16 h at 4°C. After extensive washing with PBST-BSA, samples were incubated with secondary antibodies for 1 h at 25°C. After samples were washed, the samples were mounted with Vectashield (Vector Labs, CA, USA). The images were analyzed using a Zeiss AxioSkop 2Plus microscope (Carl Zeiss Inc., Germany).
Proteins were extracted from whole third instar larvae using PRO-PREP™ protein extraction solution (iNtRON Biotechnology, Korea) and separated by SDS-PAGE. Proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, UK), and incubated with primary antibodies for 16 h at 4°C. After washing with Tris-based-buffered Saline (TBS)/0.1% Tween 20 (TBST), membranes were incubated with secondary antibodies for 45 min at 25°C. Bound antibodies were then detected using the WEST-One™ Western Blot detection system (iNtRON Biotechnology).
Total RNA was isolated from adult whole guts with TRIzol reagent (Molecular Research Center Inc., OH, USA), according to manufacturer’s instructions, and 1 μg total RNA reverse transcribed using M-MLV reverse transcriptase (Promega, WI, USA). For RT-PCR, samples underwent 30 PCR cycles, were separated on 2% agarose gels, and stained with ethidium bromide (Sigma-Aldrich). Real-time PCR was performed using iQTMSYBR® Green Supermix (Bio-Rad, CA, USA). Data were acquired on a DNA Engine Chromo4 instrument (Bio-Rad). Real-time qPCR cycling conditions were as follows: denaturation for 10 min at 95°C; 35 cycles of 30 s at 95°C, 30 s of annealing at 52°C, and 12 s extension at 72°C. Products were then analyzed via melting curve analysis for 10 s at 95°C and 15 s at 65°C, followed by an increase in temperature from 65 to 95°C (0.1°C/s) and continuous fluorescence recording.
The oligonucleotides designed for amplifying the pUAST-hMeCP2 plasmid were as follows: Forward, 5'-TCC CCA GAA TAC ACC TTG CT-3'; Reverse, 5'-CCA ACT ACT CCC ACC CTG AA-3'. The oligonucleotides used for RT-PCR and real-time qPCR were: hMeCP2: Forward, 5'-CGA AAA GGT AGG CGA CAC AT-3'; Reverse, 5'-CGT TTG ATC ACC ATG ACC TG-3'. Oligonucleotide sequences for the analysis of PCNA (Choi et al., 2008a) and rp49 (Biteau et al., 2008) were previously reported.
For immunochemistry, intact guts were fixed in 4% paraformaldehyde (Electron Microcopy Science, CO, USA) for 30 min, postfixed in methanol for 5 min, gradually washed in PBS/0.1% Triton X-100 (PBST), and incubated overnight with primary antibodies in PBST with 1% BSA at 4°C. Samples were then washed in PBST, incubated with secondary antibodies for 1 h at 25°C and then washed and mounted with Vectashield. Images were analyzed using a Zeiss AxioSkop 2Plus microscope.
For cryosectioning, midguts were dissected, fixed in 4% formaldehyde (Sigma-Aldrich) for 2 h at room temperature and infiltrated with 20% sucrose overnight at 4°C. After flash-freezing in Tissue-Tek OCT medium (SAKURA, Japan), 10 μm thick sections were cut on a Leica CM1850 cryostat (Leica Microsystems, Germany). Sections were then blocked in 5% BSA for 30 min and incubated overnight with primary antibody. After treatment with secondary antibody conjugated to fluorescent dye, samples were mounted with Vectashield and analyzed using a Zeiss AxioSkop 2plus microscope.
Flies were cultured on standard media supplemented with 0.2 mg/ml of BrdU (Sigma-Aldrich) for 4–8 days. Intact guts were fixed with Carnoy’s solution, and then incubated in 2N HCl to denature the DNA. Samples were processed for immunostaining as described above.
For immunostaining, antibodies were diluted as follows: rabbit anti-hMeCP2, 1:300 (Affinity Bioreagents, CO, USA); mouse anti-Delta, 1:20 [Developmental Studies Hybridoma Bank (DSHB), IA, USA]; mouse anti-Prospero, 1:100 (DSHB); rabbit anti-PH3, 1:300 (Upstate Biotechnology Inc., MA, USA); mouse anti-BrdU, 1:50 [Santa Cruz Biotechnology Inc. (SCBT), CA, USA]; mouse anti-GFP, 1:300 (Invitrogen, CA, USA); mouse anti-HP1, 1:100 (DSHB); mouse anti-β-gal, 1:50 (DSHB). Secondary antibodies used included Cy3-conjugated goat anti-mouse, FITC-conjugated goat anti-rabbit, FITC-conjugated goat anti-mouse, and Cy3-conjugated goat anti-rabbit (Jackson ImmunoResearch, PA, USA); all were diluted 1:300. For immunoblotting, rabbit anti-MeCP2, 1:2,000; mouse anti-á-tubulin, 1:10,000 (BioGenex, CA, USA) were used in combination with HRP-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies (1:5,000, SCBT).
To detect apoptosis, TUNEL assay was performed using In Situ Apoptosis Detection Kit TMR Red (Roche, CA, USA), according to the manufacturer’s protocol. Briefly, dissected guts were fixed in 4% paraformaldehyde at 25°C, washed in PBST, incubated with the mixture of TdT enzyme and fluorescent dye for 1 h at 37°C, washed and analyzed using Zeiss ZxioSkop 2plus microscope.
To investigate the role of hMeCP2 in Drosophila using the GAL4/UAS system, we generated three independent UAS-hMeCP2 transgenic fly lines. The Gal4 drivers used in this study are summarized in Table I. We verified whether the UAS-hMeCP2 transgenic flies are suitable to induce ectopic expression of hMeCP2 in Drosophila. Extracts from heat-shocked third instar larvae harboring UAS-hMeCP2 and heat shock-inducible hs-Gal4 were analyzed by RT-PCR and Western blotting. Similar levels of hMeCP2 mRNA and protein were detected in all three transgenic lines (Fig. 1A, upper and lower panels, respectively).
 View Details | Fig. 1. Characterization of transgenic flies expressing human MeCP2. (A) Expression of hMeCP2 mRNA (upper panel) and protein (lower panel) in third instar larvae expressing UAS-hMeCP2 under the control of heat-shock (hs)-Gal4. Total RNA or proteins prepared from the third instar larvae of three transgenic fly lines carrying UAS-hMeCP2 and hs-Gal4 (hs>hMeCP2) after heat shock at 37°C for 30 min and incubated at 25°C for 6 h. Then, RT-PCR or Western blot was performed to measure hMeCP2 mRNA or protein level. The lines carrying hs-Gal4 alone (hs>+) was used as a control. Ribosomal protein 49 (rp49) and α-tubulin (α-tub) were used as internal controls, respectively. (B–I) Ectopic expression of hMeCP2 driven by several Gal4 drivers induces developmental abnormalities in Drosophila. (B and C) A rough eye phenotype induced by the hMeCP2 expression in the eyes under the control of GMR-Gal4 driver (developed at 29°C). Insets are magnified images. (D and E) Additional veins formation on the L3, L4 and L5 vasculatures induced by the hMeCP2 expression in wing pouch under the control of MS1096-Gal4 driver (developed at 25°C). Arrows indicate ectopic vein formation. (F and G) Developmental abnormalities by the hMeCP2 expression in whole bodies under the control of da-Gal4 driver at 25°C, including an inhibition of larval growth (data not shown) and death accompanied by melanization in the caudal region at the moment of pupariation (180 h after egg laying). Arrow indicates abnormal melanization. (H and I) Extra bristle formation on the scutellum of thorax by hMeCP2 expression under the control of da-Gal4 driver at 18°C to obtain a less severe phenotype. The hMeCP2-expressing flies have defects in development, such as smaller size (data not shown) with extra bristles present in the scutellum region (I) compared with four large macrochaetes observed in the wild type scutellum region (H). Red arrows indicate scutellum bristles. (J and K) Human MeCP2 bound to euchromatic regions and heterochromatic chromocenter of polytene chromosomes from Drosophila salivary gland. Polytene chromosomes in third instar larvae carrying salivary-specific sg-Gal4 driver alone (J, sg>+) and carrying UAS-hMeCP2 and sg-Gal4 (K, sg>hMeCP2) were immunostained with DAPI for detection of DNA (cyan, J and K) and anti-hMeCP2 (red, J' and K'). Insets show magnified images. Arrow heads indicate chromocenter. Genotype: hs>+ (+/+; hs-Gal4/+), hs>hMeCP2 (+/+; hs-Gal4/UAS-hMeCP2), GMR>+ (GMR-Gal4/+; +/+; +/+), GMR>hMeCP2 (GMR-Gal4/+; +/+; UAS-hMeCP2/+), MS1096>+ (MS1096-Gal4/+; +/+; +/+), MS1096>hMeCP2 (MS1096-Gal4/+; +/+; UAS-hMeCP2/+), da>+ (+/+; da-Gal4/+), da>hMeCP2 (+/+; da-Gal4/UAS-hMeCP2). sg>+ (sg-Gal4/+; +/+; +/+), sg>hMeCP2 (sg-Gal4/+; +/+; UAS-hMeCP2/+). |
Under the several Gal4 drivers, hMeCP2 expression induced developmental abnormalities in Drosophila. When the glass multiple region (GMR)-Gal4 promoter was used to express hMeCP2 in the eyes, a rough eye phenotype developed at 29°C (Fig. 1C). Similarly, hMeCP2 expression in the wing pouch was achieved with expression of hMeCP2 under the control of the MS1096-GAL4 promoter. In this model, formation of additional veins on the L3, -4 and -5 vasculature were observed (Fig. 1E), and these results were consistent with a previous report (Cukier et al., 2008). In addition, when hMeCP2 was expressed in the whole body under da-Gal4 driver, larval growth was inhibited (data not shown) and the individual died accompanied by melanization in the caudal region at the moment of pupariation (180 h after egg laying) at 25°C (Fig. 1G). To obtain a less severe phenotype, hMeCP2 expressing flies under da-Gal4 driver were developed at 18°C. Although there are some escapers, these flies have also defects in development, such as smaller size (data not shown) with extra bristles present in the scutellum region compared with four large macrochaetes observed in the wild-type scutellum region (Fig. 1I). These results indicate that transgenic fly UAS-hMeCP2 is sufficient for expression of hMeCP2 in Drosophila.
Since MeCP2 is a chromatin-associated protein, the distribution of exogenous hMeCP2 on chromatin was investigated in giant polytene chromosomes from the salivary glands of third instar larvae. Expression of hMeCP2 was driven by a salivary gland-specific sg-Gal4 driver (Kato et al., 2008), and DNA was visualized by DAPI staining. Strong DAPI staining of chromocenter heterochromatin was observed (indicated by arrow heads in Fig. 1J), as well as strong staining of the bands of condensed regions of DNA present in the euchromatic chromosome arms (Fig. 1J). Immunostaining of hMeCP2 in polytene chromosomes demonstrated that exogenous hMeCP2 localized to chromocenter heterochromatin as well as hundreds of euchromatic sites (Fig. 1K'). Moreover, at higher magnification, hMeCP2 co-localized with DAPI-stained bands. This indicates that human MeCP2 can bind to both euchromatin and heterochromatin of Drosophila cells.
To overexpress hMeCP2 in the gut of adult Drosophila, da-Gal4 driver was used to ubiquitously express Gal4 in all cell types in the gut. For transgene expression regardless of developmental stage, a temperature-sensitive version of Gal80 under the control of the tubulin promoter (tub-Gal80ts) was used (McGuire et al., 2004). In this system, the UAS-hMeCP2 was expressed under the control of specific Gal4 driver at 29°C, but not 19°C (data not shown). The guts expressing hMeCP2 in whole flies under the control of da-Gal4,tub-Gal80ts (hereafter refer to as da-Gal80ts) were stained with anti-Delta, a marker of Drosophila ISCs (Ohlstein and Spradling, 2007), and anti-Prospero, a marker of ee cells (Ohlstein and Spradling, 2006). ECs were identified based on the presence of large nuclei with polyploidy (Ohlstein and Spradling, 2006).
Under our experimental conditions, hMeCP2 expression under da-Gal80ts was detected in all cell types of the midgut including Delta-positive ISCs, Prospero-positive ee cells and large nuclei ECs at 29°C, although its expression level varied (Fig. 2B). This variation seems to depend on da-Gal4 driver, since the GFP expression under the control of da-Gal4 (UAS-GFP/+;da-Gal4/+) was also varied in all kinds of cells (ECs, ees, EBs, and ISCs) of the gut (data not shown). To investigate the effect of hMeCP2 expression driven by da-Gal80ts on ISC proliferation, staining with anti-phospho-histone H3 (PH3) antibody was performed as a mitosis indicator, and an up to 15-fold increase in the ISC mitotic index was observed (Fig. 2C–E). In addition, hMeCP2 expression driven by the intestine-specific cad-Gal4 driver, which is expressed in all cell types of the midgut (Choi et al., 2008b), increased ISC proliferation by up to 8-fold (Fig. 2E). Consistent with increase in the proliferation indicator, the total cell number of the hMeCP2 expressing guts was 1.6-fold, relative to that of control flies (Fig. 2F). These results demonstrate that exogenous expression of hMeCP2 can induce ISC proliferation in midgut cells of adult Drosophila.
 View Details | Fig. 2. Human MeCP2 induces ISC proliferation of midgut cells in adult Drosophila. (A and B) Ectopic expression of hMeCP2 in all cell types of the adult midgut under the control of da-Gal4 driver combined with tub-Gal80ts for temporally restricted expression (da-Gal80ts). Flies expressing da-Gal80ts driver alone (A, dats>+) and hMeCP2 expression under the control of da-Gal80ts driver (B, dats>hMeCP2) were cultured at 29°C for 5 days and stained with anti-hMeCP2 (green), anti-Delta (red signal in the cytoplasm, ISC marker), anti-Pros (red signal in the nucleus, ee marker), and DAPI (blue, DNA marker). ISC, intestinal stem cell; ee, enteroendocrine cell; EC, enterocyte. (C–E) Increased PH3-positive cells by hMeCP2 expression in whole body or intestine under the control of da-Gal80ts or cad-Gal4, respectively. (C and D) Flies expressing da-Gal80ts driver alone (C, dats>+) and hMeCP2 expression under the control of da-Gal80ts driver (D, dats>hMeCP2) were cultured at 29°C for 5 days and stained with anti-phospho-histone H3 (PH3, green, mitosis marker) and DAPI (blue). Arrows indicate PH3-positive cells. (E) Number of PH3-positive cells in the whole guts were counted in the flies expressing Gal4 drivers alone (gray bars, +) and hMeCP2 with indicated Gal4 drivers (black bars, hMeCP2) at 29°C for 5 days. Results are expressed as the mean±SE from 20–25 midguts analyzed in each case. P-values were calculated using Student’s t-test. **P<0.01, ***P<0.001. (F) Increased total cell number by hMeCP2 expression in the guts under da-Gal80ts driver. Number of total cells in a capture were counted in the flies expressing da-Gal80ts driver alone (gray bar, dats>+) and hMeCP2 with da-Gal80ts driver (black bar, dats>hMeCP2) at 29°C for 5 days. Results are expressed as the mean±SE from 20 midguts analyzed in each case. P-values were calculated using Student’s t-test. **P<0.01. (G–I) Increase in DNA synthesis of the adult midgut by hMeCP2 expression under the control of da-Gal80ts driver. The flies expressing da-Gal80ts driver alone (G, dats>+) and hMeCP2 under da-Gal80ts driver (H, dats>hMeCP2) were fed on 0.2 mg/ml BrdU medium at 29°C for 6 days, and their guts were stained with anti-BrdU (red) and DAPI (blue). (I) Number of BrdU-incorporated cells were counted in the flies expressing da-Gal80ts driver alone (gray bars, dats>+) and hMeCP2 with da-Gal80ts driver (black bars, dats>hMeCP2) at 29°C for 5 days. Total BrdU-incorporated cells including polyploid EC and diploid ISC/EB (total) and BrdU-incorporated diploid ISC/EB (small) were separately counted. The results are expressed as the mean±SE from 10 midguts analyzed in each case. P-values were calculated using Student’s t-test. ***P<0.001. (J) Increase in PCNA expression in the adult midgut expressing hMeCP2 in the whole body under the control of da-Gal80ts driver. Detection of PCNA mRNA levels by real-time qPCR in whole gut extracts from the flies expressing da-Gal80ts driver alone (gray bar, dats>+) and hMeCP2 with da-Gal80ts driver (black bar, dats>hMeCP2) after incubation at 29°C for 5 days. (View PDF for the rest of the caption.) |
In addition, incorporation of BrdU was carried out for the detection of DNA replication. When hMeCP2 was expressed in whole flies under the control of the da-Gal80ts driver, the number of BrdU-positive cells was increased in both large-nuclei ECs and small-nuclei cells including ISCs/EBs (Fig. 2G–I). This result indicates that hMeCP2 can induce DNA synthesis in ECs, in addition to ISC proliferation. Furthermore, mRNA levels of proliferating cell nuclear antigen (PCNA), which is critical for G1/S phase progression, was increased by up to 2.5-fold following expression of hMeCP2 (Fig. 2J). In combination, these results indicate that expression of hMeCP2 induces ISC proliferation in the gut of adult Drosophila.
To further assess the effect of hMeCP2 expression on ISC proliferation, we performed cell-type specific expression of UAS-hMeCP2 using cell-type specific Gal4 drivers. ISC/EB-specific esg-Gal4 and EC-specific Myo1A-Gal4 were used in combination with the tub-Gal80ts. Specific expression of hMeCP2 using esg-Gal4,UAS-GFP,tub-Gal80ts (hereafter referred to as esg-Gal80ts) or Myo1A-Gal4;UAS-GFP,tub-Gal80ts (hereafter referred to as Myo1A-Gal80ts) was detected at 29°C (Fig. 3A–D). The expression of Myo1A-Gal80ts driver visualized with UAS-GFP was suppressed in some ECs by hMeCP2 overexpression, as indicated by arrows (Fig. 3D), while the expression of esg-GFP was correlated with hMeCP2 expression level (Fig. 3B). This indicates a possibility that hMeCP2 modulate the gene expression in ECs.
 View Details | Fig. 3. Expression of hMeCP2 in differentiated ECs affects ISC proliferation. (A–D) Expression of hMeCP2 in ISCs/EBs under esg-Gal4,UAS-GFP driver combined with tub-Gal80ts (esg-Gal80ts) or in differentiated ECs under Myo1A-Gal4,UAS-GFP driver combined with tub-Gal80ts (Myo1A-Gal80ts). Flies expressing Gal4 driver alone (A, esgts>+; C, Myo1Ats>+) and hMeCP2 expression with esg-Gal80ts or Myo1A-Gal80ts drivers (B, esgts>hMeCP2; D, Myo1Ats>hMeCP2) were cultured at 29°C for 5 days and stained with anti-GFP (green), anti-hMeCP2 (red) and DAPI (blue). Arrows indicate GFP-negative/hMeCP2-positive cells. (E–G) Increased PH3-positive cells by hMeCP2 expression in ECs under the control of Myo1A-Gal80ts driver. (E and F) Flies expressing Myo1A-Gal80ts driver alone (E, Myo1Ats>+) and hMeCP2 expression under the control of Myo1A-Gal80ts driver (F, Myo1Ats>hMeCP2) were cultured at 29°C for 5 days and stained with anti-PH3 (green) and DAPI (blue). Arrows indicate PH3-positive cells. (G) Number of PH3-positive cells in the whole guts were counted in the flies expressing Gal4 drivers alone (gray bars, +) and hMeCP2 with indicated Gal4 drivers (black bars, hMeCP2) at 29°C for 5 days. Results are expressed as mean±SE from 35 midguts analyzed in each case. P-values were calculated using Student’s t-test. ***P<0.001. (H and I) Cross-sections of the epithelium of the flies expressing Myo1A-Gal80ts driver alone (H, Myo1Ats>+), and hMeCP2 expression under the control of Myo1A-Gal80ts driver (I, Myo1Ats>hMeCP2) were stained with DAPI. The dashed lines outline the basal epithelial regions. (J-M) Increase in DNA synthesis of the adult midgut by ectopic hMeCP2 expression under the control of Myo1A-Gal80ts driver. The flies expressing Gal4 drivers alone (J, esgts>+; L, Myo1Ats>+) and hMeCP2-expressing flies under the drivers (K, esgts>hMeCP2; M, Myo1Ats>hMeCP2) were fed on 0.2 mg/ml BrdU medium at 29°C for 6 days, and their guts were stained with anti-BrdU (red) and DAPI (blue). (N and O) Increase in ERK activity in ISCs/EBs by hMeCP2 expression in ECs under Myo1A-Gal80ts driver. The guts expressing Myo1A-Gal80ts alone (N, Myo1Ats>+) or hMeCP2 under the control of Myo1A-Gal80ts (O, Myo1Ats>hMeCP2) were cultured at 29°C for 1 day and stained with anti-GFP (green), anti-dpERK (red) and DAPI (blue). Arrows indicate GFP-negative/dpERK-positive diploid cells. Genotypes: esgts>+ (esg-Gal4,UAS-GFP,tub-Gal80ts/+; +/+), esgts>hMeCP2 (esg-Gal4,UAS-GFP,tub-Gal80ts/+; UAS-hMeCP2/+), Myo1Ats>+ (Myo1A-Gal4/+; UAS-GFP,tub-Gal80ts/+), Myo1Ats>hMeCP2 (Myo1A-Gal4/+; UAS-GFP,tub-Gal80ts/UAS-hMeCP2). |
Interestingly, we observed that expression of hMeCP2 in ISCs/EBs was not associated with an increase in the number of PH3-positive cells, whereas hMeCP2 expression in ECs increased ISC proliferation by up to 11-fold (Fig. 3E–G). This result indicates that EC-specific rather than ISC/EB-specific expression of hMeCP2 can induce ISC proliferation.
To assess whether the increase in ISC proliferation by hMeCP2 expression induced tissue deterioration, we cross-sectioned the guts expressing hMeCP2 from the Myo1A-Gal80ts driver. Control guts exhibited a single layer of epithelium (Fig. 3H); however, the guts expressing hMeCP2 in ECs showed intestinal hyperplasia with multilayered epithelium (Fig. 3I).
Furthermore, ISC/EB-specific hMeCP2 expression did not affect the number of BrdU-positive small cells (Fig. 3J and K), whereas hMeCP2 expression in ECs increased the number of BrdU-positive small and large cells (Fig. 3L and M). These results indicate that hMeCP2 expression in differentiated ECs, but not ISCs and EBs, is associated with a greater number of proliferating ISCs.
To more assess the effect of hMeCP2 expression in differentiated EC on ISC, we investigated whether hMeCP2 expression in ECs can activate ISC activity. The activity of extracellular signal-regulated kinase (ERK) in ISCs was reported to be strongly associated with ISC proliferation in Drosophila (Jiang and Edgar, 2009; Biteau and Jasper, 2011; Jiang et al., 2011). In the control guts, dpERK signal, the activated form of ERK, was weakly detected in small nuclei cells at 1 day after temperature shift (Fig. 3N). Guts expressing hMeCP2 in differentiated ECs showed strong activation of dpERK signal in Myo1A-GFP-negative undifferentiated diploid cells, including ISCs (Fig. 3O). This indicate that expression of hMeCP2 in differentiated ECs activate ISCs. Taken together, these results suggest a role of hMeCP2 in EC as a regulator of ISC proliferation.
ECs that received proapoptotic signals or that are stressed by bacterial infection are known to promote compensatory ISC proliferation (Amcheslavsky et al., 2009; Buchon et al., 2009). To examine whether ISC proliferation increased by EC-specific hMeCP2 expression stemmed from EC death, we monitored apoptosis via TUNEL assay. We did not observe any significant change in apoptotic signals in hMeCP2-expressing guts (Fig. 4), indicating that increasing ISC proliferation by EC-specific hMeCP2 expression may be not associated with EC death.
 View Details | Fig. 4. Expression of hMeCP2 in ECs do not induce EC death. Expression of hMeCP2 in differentiated ECs under the control of Myo1A-Gal80ts did not induce apoptosis. Flies expressing Myo1A-Gal80ts driver alone (A, Myo1Ats>+) and hMeCP2 with Myo1A-Gal80ts driver (B, Myo1Ats>hMeCP2) were cultured at 29°C for 5 days and analyzed with TUNEL assay (red). DAPI for detecting nuclei (blue). (C) The flies treated with bleomycin, an apoptosis inducing chemical, were used as positive control (25ug/ml, 2 days). Genotype: Myo1Ats>+ (Myo1A-Gal4/+; UAS-GFP,tub-Gal80ts/+), Myo1Ats>hMeCP2 (Myo1A-Gal4/+; UAS-GFP,tub-Gal80ts/UAS-hMeCP2). |
Next, we hypothesized that ISC proliferation induced by EC-specific hMeCP2 expression may be associated with the epigenetic regulation of EC. To assess the epigenetic modulation mediated by hMeCP2 in ECs, we investigated the distribution of heterochromatin protein-1 (HP1). HP1 is a chromatin-associated protein often used as an indicator of chromatin status (Agarwal et al., 2007), and it has been reported to interact with MeCP2 in mammals (Agarwal et al., 2007). The distribution of HP1 in Drosophila midgut cells has not yet been reported.
In this study, we observed that in normal differentiated ECs, HP1 localized to the nucleus with a single, oval-like condensed distribution (Fig. 5A). Interestingly, when hMeCP2 was exogenously expressed in ECs under the Myo1A-Gal80ts driver, up to 50% of ECs displayed multiple HP1 foci (Fig. 5B and C). Under hMeCP2 expression, HP1 was distributed among several spots, which were co-localized with hMeCP2 (Fig. 5B). The changes of HP1 distribution in hMeCP2-expressing cells may be due to the alteration of nuclear shape. To exclude this possibility we monitored the nuclear shape. To monitor the nuclear shape, we stained the midgut expressing hMeCP2 under Myo1A-GAL80ts driver with anti-laminC (lamC), a component of nuclear structure (Drosophila homolog of laminA) (Gruenbaum et al., 2005). In control guts, lamC was detected in the nuclear boundary of all midgut cells with varied expression level (Fig. 5D). Expression of hMeCP2 in ECs under Myo1A-GAl80ts driver did not show any significant changes in nuclear shapes visualized with lamC (Fig. 5E). These results indicate that hMeCP2 can modulate HP1 distribution in differentiated ECs in Drosophila.
 View Details | Fig. 5. Human MeCP2 modulates chromatin status in differentiated ECs. (A–C) Modulation of the HP1 distribution by the hMeCP2 expression in ECs. The guts expressing Myo1A-Gal80ts alone (A, Myo1Ats>+) and hMeCP2 under the control of Myo1A-Gal80ts driver (B, Myo1Ats>hMeCP2) were cultured at 29°C for 5 days and stained with anti-HP1 (red), anti-hMeCP2 (green) and DAPI (blue). Insets are magnified images. (C) The number of ECs containing multiple foci of HP1. Gray bar shows Myo1A-Gal80ts alone (Myo1Ats>+). Black bar shows hMeCP2 expression in ECs under the control of Myo1A-Gal80ts driver (Myo1Ats>hMeCP2). Results are expressed as mean±SE from 20 midguts analyzed in each case. P-values were calculated using Student’s t-test. ***P<0.001. (D and E) Expression of hMeCP2 did not change nuclear shape in ECs. Flies expressing Myo1A-Gal80ts driver alone (D, Myo1Ats>+) and hMeCP2 under the control of Myo1A-Gal80ts driver (E, Myo1Ats>hMeCP2) were cultured at 29°C for 5 days and stained with anti-lamC (red, nuclear shape maker), hMeCP2 (green) and DAPI (blue). Insets are magnified image. (F and G) Expression of hMeCP2 induced ectopic expression of endogenous Drosophila gene in ECs. Flies expressing Myo1A-Gal80ts driver with pvf2-lacZ reporter gene (F, Myo1Ats >pvf2-lacZ) and hMeCP2 under the control of Myo1A-Gal80ts driver with pvf2-lacZ (G, Myo1Ats>hMeCP2,pvf2-lacZ) were cultured at 29°C for 5 days and stained with anti-β-gal (red), anti-hMeCP2 (green), and DAPI (blue). When hMeCP2 was exogenously expressed in ECs under the Myo1A-Gal80ts driver, the pvf2-lacZ transgene was ectopically detected in the cytoplasm of hMeCP2-positive differentiated ECs. pvf2-lacZ is expressed only in ISCs and EBs but not ECs under normal conditions (white arrows). Arrow heads indicate ectopic pvf2-lacZ expression in polyploid ECs by hMeCP2. Genotypes: Myo1Ats>+ (Myo1A-Gal4/+; UAS-GFP,tub-Gal80ts/+), Myo1Ats>hMeCP2 (Myo1A-Gal4/+; UAS-GFP,tub-Gal80ts/UAS-hMeCP2). Myo1Ats>pvf2-lacZ (Myo1A-Gal4/pvf2-lacZ; UAS-GFP,tub-Gal80ts/+), Myo1Ats>hMeCP2,pvf2-lacZ (Myo1A-Gal4/pvf2-lacZ; UAS-GFP,tub-GAl80ts/UAS-hMeCP2). |
Furthermore, to investigate whether hMeCP2 expression modulates gene expression in enterocytes, we assayed the expression pattern of the pvf2-lacZ reporter gene. We previously reported that pvf2-lacZ is expressed only in ISCs and EBs but not ECs under normal conditions (Choi et al., 2008a). When hMeCP2 was exogenously expressed in ECs under the Myo1A-Gal80ts driver, the pvf2-lacZ transgene was ectopically detected in the cytoplasm of hMeCP2-positive differentiated enterocytes (Fig. 5G), whereas in control guts, pvf2-lacZ was expressed only in small cells, including ISCs and EBs (Fig. 5F). This result suggests that hMeCP2 epigenetically modulate the expression of genes in differentiated ECs.
In this study, we obtained evidences showing that hMeCP2 induces an increase in stem cell proliferation in the adult Drosophila midgut. We also showed that hMeCP2 expression in ECs can activate ISC activity. In mammals, MeCP2 was previously reported to regulate Cyclin D1 expression in developing rat gut (Darwanto et al., 2008), and it induces cell proliferation and growth of embryonic non-neuronal cells (Nagai et al., 2005) as well as prostate cancer cells (Bernard et al., 2006). Furthermore, MeCP2 has been reported to induce secretion of several growth factors such as brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1) and nerve growth factor (NGF) in the central nervous system (Schaevitz et al., 2010). Therefore, our data support a role of MeCP2 in tumorigenesis and stem cell proliferation.
In our study, hMeCP2 expression in ECs was shown to alter the distribution of HP1, supporting the hypothesis that hMeCP2 interacts with Drosophila HP1, similar to the interaction between MeCP2 and HP1 in mammals (Agarwal et al., 2007). In addition, using pvf2-lacZ reporter gene which is expressed only in ISCs and EBs but not ECs under normal conditions (Choi et al., 2008a), we observed that hMeCP2 can induce ectopic gene expression in ECs (Fig. 5G). The chromatin remodeling factors are conserved in Drosophila and mammal (Ho and Crabtree, 2011), although Drosophila has a different methylation site compared to mammals (Marhold et al., 2004), and the ortholog of MeCP2 has not been reported to exist in Drosophila. hMeCP2 protein was reported to genetically interact with several Drosophila chromatin remodeling factors, such as Additional sex combs (Asx), osa, pebble (pbl), and trithorax (trx) (Cukier et al., 2008). Therefore, our data suggest the possibility that hMeCP2 regulates stem cell proliferation via the modulation or/and interaction of chromatin remodeling factors independent with binding to methylated DNA.
It is interesting to note that according to our current data, hMeCP2 expression in undifferentiated ISCs/EBs did not induce an increase in ISC proliferation under unstressed condition, in contrast to hMeCP2 expression in differentiated ECs. A recent study reported that the epigenetic stability of Drosophila follicle stem cells increases during differentiation (Skora and Spradling, 2010). In the same study, the overall epigenetic status of differentiated cells was found to be more consistent than that of stem cells. Therefore, this differential effect of cell-type specific hMeCP2 expression may be due to differences in the epigenetic flexibility of stem cells vs. differentiated cells.
The distinct effect of hMeCP2 expression on ISCs/EBs vs. ECs may be also due to differences in MeCP2 activity. Posttranslational modifications including phosphoylation (Chao and Zoghbi, 2009), ubiquitination (Thambirajah et al., 2009) and SUMOylation regulate the activity of MeCP2 (Thambirajah et al., 2009). In particular, phosphorylation of MeCP2 at Ser80 by homeodomain-interacting protein kinase 2 (HIPK2) (Bracaglia et al., 2009) and Ser421 by CaMKII (Zhou et al., 2006) has been shown to regulate MeCP2 activity. MeCP2 in human has been shown to undergo phosphorylation at Ser423, which corresponds to Ser421 in mice, and this phosphorylation event is maintained in hMeCP2 expressed in Drosophila (Cukier et al., 2008). Therefore, we also hypothesize that the differences in the effects associated with hMeCP2 expression in ISCs/EBs vs. ECs might be based on differential posttranslational modifications between each cell types.
In conclusion, we documented in the present study: expression of hMeCP2 in differentiated ECs induces ISC proliferation, and hMeCP2 can modulate the chromatin status of differentiated ECs in adult Drosophila midgut model system. Our data suggest the hypothesis that hMeCP2 stimulates stem cell proliferation via epigenetic modulation of stem cell niche factors in differentiated cells.
We thank Drs. B. Ohlstein (Columbia University Medical Center), B.A. Edar (Fred Hutchinson Cancer Research Center) for their generous gift of fly strains. We would like to thank the Developmental Studies Hybridoma Bank for antibodies, and the Bloomington Stock Center and the Drosophila Genetic Resource Center for Drosophila stocks. We would also like to thank the Center for Research Facilities of Pusan National University for use of their scanning electron microscope. We thank Prof. B.P. Yu (University of Texas) for helpful comments on the manuscript. We also thank reviewers for valuable comments on the manuscript. This work was supported by a Bio-Scientific Research Grant funded by Pusan National University (PNU-2008-101-217).
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