Histone demethylase dUTX antagonizes JAK-STAT signaling to maintain proper gene expression and architecture of the Drosophila testis niche

Abstract

Adult stem cells reside in microenvironments called niches, where they are regulated by both extrinsic cues, such as signaling from neighboring cells, and intrinsic factors, such as chromatin structure. Here we report that in the Drosophila testis niche an H3K27me3-specific histone demethylase encoded by Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (dUTX) maintains active transcription of the Suppressor of cytokine signaling at 36E (Socs36E) gene by removing the repressive H3K27me3 modification near its transcription start site. Socs36E encodes an inhibitor of the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway. Whereas much is known about niche-to-stem cell signaling, such as the JAK-STAT signaling that is crucial for stem cell identity and activity, comparatively little is known about signaling from stem cells to the niche. Our results reveal that stem cells send feedback to niche cells to maintain the proper gene expression and architecture of the niche. We found that dUTX acts in cyst stem cells to maintain gene expression in hub cells through activating Socs36E transcription and preventing hyperactivation of JAK-STAT signaling. dUTX also acts in germline stem cells to maintain hub structure through regulating DE-Cadherin levels. Therefore, our findings provide new insights into how an epigenetic factor regulates crosstalk among different cell types within an endogenous stem cell niche, and shed light on the biological functions of a histone demethylase in vivo.

Fig. 1.

dUTX prevents Zfh1-expressing cells from overpopulating the niche and represses Zfh1 expression in the hub cells. (A) Schematic of the Drosophila testis niche. CySCs, cyst stem cells; GSC, germline stem cell. (B-C′) Immunostaining using antibodies against Arm (blue), Vasa (green) and Zfh1 (red) in (B,B′) wt and (C,C′) dUTX testes. Arrows point to overpopulating Zfh1-expressing cells with nuclei that directly contact the hub (C′). Hub area is outlined (white dotted line). Scale bars: 10 μm.

Fig. 2.

dUTX acts as a histone demethylase in CySCs and/or early cyst cells to repress overpopulation of Zfh1-expressing cells around the hub and ectopic Zfh1 expression in hub cells. (A-C′) Immunostaining using antibodies against Arm (blue), Vasa (green) and Zfh1 (red). Hub area is outlined (white dotted line). (A) c587-Gal4. (A′) c587-Gal4; UAS-dUTX shmiRNA; arrows point to Zfh1-expressing cells with nuclei that directly contact the hub. (B,B′) c587-Gal4; UAS-dUTX and (C,C′) c587-Gal4; UAS-dUTX^JmjC, both in a dUTX background. (D) Percentage of testes with overpopulating Zfh1-expressing cells around the hub. (E) Percentage of testes with ectopic Zfh1 expression in hub cells. P-value calculated using Fisher’s test. Scale bars: 10 μm.

Fig. 3.

dUTX removes the repressive H3K27me3 histone modification at the Socs36E genomic locus and allows active transcription of Socs36E. (A) Socs36E mRNA measured by qRT-PCR in three independent biological replicates, normalized by fringe. (B) Anti-H3K27me3 ChIPed DNA analyzed by qPCR, normalized to input (percentage input) and then compared between dUTX testes and wt controls, based on three independent biological replicates. P-value calculated using Student’s t-test. Error bars represent s.d. (C,D) Immunostaining using antibodies against Arm (blue), Vasa (green) and Zfh1 (red). Hub area is outlined (white dotted line). (C) upd-Gal4; UAS-Socs36E-cDNA transgene in a dUTX background; arrows point to overpopulating Zfh1-expressing cells with nuclei that directly contact the hub. (D) c587-Gal4; UAS-Socs36E-cDNA transgene in a dUTX background. Scale bars: 10 μm.

Fig. 4.

dUTX is required in CySCs and early cyst cells to prevent hyperactivation of the JAK-STAT signaling pathway. (A-B′) Immunostaining using antibodies against Arm (green) and Stat92E (red). Hub area is outlined by white dotted line and stem cell zone by yellow dotted line. (A,A′) wt testis. Arrow points to a gonialblast that is positive for anti-Stat92E staining. (B,B′) dUTX testis. (C) Stat92E mRNA measured by qRT-PCR in five independent biological replicates, normalized by fringe. P-value calculated using Student’s t-test. Error bars represent s.d. (D-E′) Immunostaining using anti-Arm (green) and anti-Stat92E (red) in (D,D′) c587-Gal4 control and (E,E′) c587-Gal4; UAS-dUTX shmiRNA testes. (F,F′) Immunostaining using anti-Arm (green) and anti-Zfh1 (red) in dUTX; Stat92E/+ testes. Scale bars: 10 μm.

Fig. 5.

dUTX acts in germ cells to maintain proper hub size. (A-B′) Immunostaining using antibodies against Arm (blue) and Vasa (green). Hub area is outlined (white dotted line). (A,A′) wt testis. (B,B′) dUTX testis displays enlarged hub. Arrows indicate GSCs with disrupted GSC-hub interface. (C) Quantification of average hub area: 94±18.65 μm² in wt testes versus 181±55.5 μm² in dUTX testes. (D) Quantification of average area of individual hub cells: 8.5±1.1 μm² in wt testes versus 12.7±2.2 μm² in dUTX testes. (E) Quantification of average hub area in testes from males of the following genotypes: nos-Gal4 control (96±20.35 μm²); nos-Gal4; UAS-dUTX shmiRNA (170±41.7 μm², P<0.01); upd-Gal4 control (100±21.4 μm²); upd-Gal4; UAS-dUTX shmiRNA (99±20.3 μm², P>0.05); c587-Gal4 control (109±7.7 μm²); c587-Gal4; UAS-dUTX shmiRNA (121±31.4 μm², P>0.05). P-value calculated using Student’s t-test. Error bars represent s.d. Scale bars: 10 μm.

Fig. 6.

dUTX controls hub size through regulating DE-Cadherin levels in GSCs and model of dUTX function in the testis niche. (A-B′) Immunostaining for Arm (blue) and Vasa (green). Hub area is outlined (white dotted line). (A,A′) dUTX; nos>DE-Cad^dCR4h testis. (B,B′) dUTX; nos>DE-Cad^DEFL. Arrows indicate GSCs with disrupted GSC-hub interface. (C) Quantification of the average GSC-hub interface in testes from males of the following genotypes: wt (4.3±0.4 μm); dUTX (5.9±0.9 μm); dUTX; nos>DE-Cad^dCR4h (4.9±0.5 μm); dUTX; nos>DE-Cad^DEFL (6.5±0.8 μm); dUTX/+; nos>DE-Cad^dCR4h control (4.6±0.3 μm); dUTX/+; nos>DE-Cad^DEFL control (4.5±0.4 μm). (D) DE-Cadherin mRNA measured by qRT-PCR in three independent biological replicates, normalized by RpL32. (E) Quantification of percentage of testes with average hub area of 60-200 μm², 200-300 μm² or exceeding 300 μm², from the following males (left to right): wt; dUTX mutant; dUTX; nos>DE-Cad^dCR4h; dUTX; nos>DE-Cad^DEFL; dUTX/+; nos>DE-Cad^dCR4h control; dUTX/+; nos>DE-Cad^DEFL control. (F) Outline of dUTX functions in the Drosophila testis niche. dUTX negatively regulates the JAK-STAT signaling pathway in CySCs and hub cells. dUTX also regulates DE-Cadherin levels in GSCs to maintain hub architecture (see Discussion for details). P-value by Student’s t-test. Error bars represent s.d. Scale bars: 10 μm.