11 research outputs found
Drosophila Kismet Regulates Histone H3 Lysine 27 Methylation and Early Elongation by RNA Polymerase II
Polycomb and trithorax group proteins regulate cellular pluripotency and differentiation by maintaining hereditable states of transcription. Many Polycomb and trithorax group proteins have been implicated in the covalent modification or remodeling of chromatin, but how they interact with each other and the general transcription machinery to regulate transcription is not well understood. The trithorax group protein Kismet-L (KIS-L) is a member of the CHD subfamily of chromatin-remodeling factors that plays a global role in transcription by RNA polymerase II (Pol II). Mutations in CHD7, the human counterpart of kis, are associated with CHARGE syndrome, a developmental disorder affecting multiple tissues and organs. To clarify how KIS-L activates gene expression and counteracts Polycomb group silencing, we characterized defects resulting from the loss of KIS-L function in Drosophila. These studies revealed that KIS-L acts downstream of P-TEFb recruitment to stimulate elongation by Pol II. The presence of two chromodomains in KIS-L suggested that its recruitment or function might be regulated by the methylation of histone H3 lysine 4 by the trithorax group proteins ASH1 and TRX. Although we observed significant overlap between the distributions of KIS-L, ASH1, and TRX on polytene chromosomes, KIS-L did not bind methylated histone tails in vitro, and loss of TRX or ASH1 function did not alter the association of KIS-L with chromatin. By contrast, loss of kis function led to a dramatic reduction in the levels of TRX and ASH1 associated with chromatin and was accompanied by increased histone H3 lysine 27 methylation—a modification required for Polycomb group repression. A similar increase in H3 lysine 27 methylation was observed in ash1 and trx mutant larvae. Our findings suggest that KIS-L promotes early elongation and counteracts Polycomb group repression by recruiting the ASH1 and TRX histone methyltransferases to chromatin
The histone variant His2Av is required for adult stem cell maintenance in the Drosophila testis.
Many tissues are sustained by adult stem cells, which replace lost cells by differentiation and maintain their own population through self-renewal. The mechanisms through which adult stem cells maintain their identity are thus important for tissue homeostasis and repair throughout life. Here, we show that a histone variant, His2Av, is required cell autonomously for maintenance of germline and cyst stem cells in the Drosophila testis. The ATP-dependent chromatin-remodeling factor Domino is also required in this tissue for adult stem cell maintenance possibly by regulating the incorporation of His2Av into chromatin. Interestingly, although expression of His2Av was ubiquitous, its function was dispensable for germline and cyst cell differentiation, suggesting a specific role for this non-canonical histone in maintaining the stem cell state in these lineages
<i>His2Av</i> is required cell autonomously for CySC maintenance but not for somatic cyst cell differentiation.
<p>(<b>A, B</b>): Percentage of testes with <i>His2Av<sup>810</sup></i> mutant (red line) or FRT 82B control (blue line) CySCs (A) or cyst cells (B) scored at indicated time points PCI. Data shows average ± S.D. (<b>C–C″</b>): Apical tip of testes 3 days PCI immunostained with anti-GFP (C, green and C″), anti-Zfh-1 (C, red and C′), and anti-Fas3 and TJ (blue). <i>His2Av<sup>810</sup></i> mutant CySCs (arrow), <i>His2Av<sup>810</sup></i> heterozygous CySCs (arrowhead) and hub (yellow dashed line). Scale bar: 12.5 µm (<b>D, D′</b>): Testes from 8 day PCI immunostained with anti-GFP (green) and anti-Eya (red). <i>His2Av<sup>810</sup></i> mutant (arrow) and heterozygous (arrowhead) cyst cells. Scale bar: 10 µm (<b>E–G</b>): Analysis of <i>Drosophila</i> eyes derived from heterozygous GMR-<i>hid/+</i> (E), homozygous <i>His2Av<sup>810</sup></i> (F), and homozygous FRT control (G) precursor cells.</p
<i>His2Av</i> is required cell autonomously for GSC maintenance.
<p>(<b>A–A′</b>): Apical tip of wild-type testis immunostained with anti-His2Av (red), anti-Vasa (green), and anti-Fas3 (blue). GSCs (arrows) identified as Vasa positive cells adjacent to the hub (yellow dashed line). (<b>B–B′</b>): Testes expressing Sa-GFP (green) to mark spermatocytes, immunostained with anti-His2Av (red), anti-GFP (green), and anti-Fas3 (blue). Inset shows spermatocyte nucleus with Sa-GFP marking the nucleolus and His2Av localized to the chromosomes; hub (yellow dashed line). (<b>C–D′</b>): Apical tip of wild-type testes immunostained with anti-His2Av (red, C–D′), anti-Tj (green, C) to mark CySCs (arrows, C, C′) adjacent to the hub (yellow dashed line) or anti-Eya (green, D) to mark cyst cells (arrowheads, D, D′). (<b>E–E′″</b>): Testes day 3 PCI immunostained with anti-GFP (green) to identify homozygous <i>His2Av<sup>810</sup></i> mutant GSCs (arrows), anti-His2Av (red), anti- Fas3 and Tj (blue) and DAPI (E′″); hub (yellow dashed line). Scale bars: 10 µm (<b>F</b>): Percentage of testes with <i>His2Av<sup>810</sup></i> mutant (red) or FRT 82B control (blue) GSCs scored at indicated times PCI.(<b>G</b>): Percentage of testes with <i>His2Av<sup>810</sup></i> mutant spermatocyte cysts in a genetic background either with (blue line, rescue) or lacking (red line, no rescue) a <i>His2Av-mRFP</i> genomic rescue transgene. Data shows average ± S.D.</p
IFN-gamma bioassay: development of a sensitive method by measuring nitric oxide production by peritoneal exudate cells from C57BL/6 mice
Interferon-gamma (IFN-gamma) is an important immunomodulatory and pleiotropic cytokine produced, primarily, by activated T lymphocytes and natural killer (NK) cells. We have devised a nitric -oxide (NO)-based bioassay for mouse IFN-gamma using resident peritoneal exudate cells (PECs) from C57BL/6 mice. Comparison with three existing bioassays demonstrated that this assay was very sensitive and detected IFN-gamma in the linear range of similar to 0.03 -0.25 U/ml. Other cytokines, e.g. interleukin (IL)-2, IL-4, IL-6, IFN-alpha/beta and tumor necrosis factor-alpha (TNF-alpha), either alone or in combination with IFN-gamma, did not greatly modulate NO levels produced by resident peritoneal exudate cells. The presence of exogenous NO3- and H2O2 did not interfere with the IFN-gamma induced NO production and detection. We also showed that the effect of lipopolysaccharide (LPS), which may be present in samples, could be suppressed by the use of Polymyxin B in the bioassay. The high sensitivity of the bioassay permitted the detection of low amounts of IFN-gamma in 1% mouse serum. In addition, this assay reproducibly detected bioactive IFN-gamma amounts in supernatants of activated T cells. The increase in IFN-gamma production by activated T cells in response to CD28 costimulation was similar to3-fold by this bioassay and similar to5-fold by ELISA. In summary, we have devised a simple, sensitive, inexpensive and high throughput method for the reproducible detection of bioactive IFN-gamma
<i>His2Av</i> mutant GSCs do not exhibit dramatic changes in epigenetic markers of transcriptional state.
<p>(<b>A–B′″</b>): <i>His2Av<sup>810</sup></i>/+ testes 5 days PCI immunostained with anti- GFP (A, B, green and A′, B′), anti-H3K4me3 (A, red and A″) or anti-H3K27me3 (B, red and B″) and counterstained with DAPI (A, B, blue and A′″, B′″). <i>His2Av<sup>810</sup></i> mutant GSCs (arrows), <i>His2Av<sup>810</sup></i> heterozygous GSCs (arrowhead) and hub (yellow dashed line). Scale bar: 10 µm (A) and 12.5 µm (B). (<b>C–D′″</b>): <i>scny<sup>02331</sup></i>/+ testes 6 days PCI immunostained with anti- GFP (green, C, D and C′, D′), anti-His2Av (red, C and C″) or anti-H3K4me3 (red, D and D″) and counterstained with DAPI (blue, C, D and C′″, D′″). <i>scny<sup>02331</sup></i> mutant GSCs (arrows), <i>scny<sup>02331</sup></i> heterozygous GSCs (arrowheads) and hub (yellow dashed line). Scale bar: 25 µm (C) and 12.5 µm (D).</p
<i>Domino</i> function is required for the association of His2Av with chromatin.
<p>(<b>A, B</b>): Percentage of testes with <i>dom<sup>k08108</sup></i> mutant (red) or FRT 42D control (blue) GSCs (A) and CySCs (B) scored at indicated times after clonal induction. Data shows average ± S.D. (<b>C–C′″</b>): Apical tip of <i>dom<sup>k08108</sup></i>/+ testes at day 6 PCI immunostained with anti-GFP (C, green and C′) and anti-His2Av (C, red and C″) and counterstained with DAPI (blue, C and C′″). <i>dom<sup>k08108</sup></i> mutant GSCs (arrows), <i>dom<sup>k08108</sup></i> heterozygous GSCs (arrowheads) and hub (yellow dashed line). Scale bar: 12.5 µm (<b>D</b>): Average ratio of His2Av intensity per unit area in GSC homozygous for FRT 42D, <i>dom<sup>k08108</sup></i> or <i>ISWI<sup>2</sup></i> to neighboring GSCs heterozygous for FRT 42D, <i>dom<sup>k08108</sup></i> or <i>ISWI<sup>2</sup></i>, respectively. Data shows average ± S.D. P-values of Student's t-test (2-tailed) are shown. (<b>E</b>): Percentage of testes with <i>His2Av<sup>810</sup></i> (blue) or <i>His2Av<sup>810</sup></i> ;<i>dom<sup>k08108</sup></i>/+ (red) GSCs scored at indicated times after clonal induction. Data shows average ± S.D. (<b>F–G′″</b>): Apical tip of <i>ISWI<sup>2</sup></i>/+ (F–F′″) testes at day 6 PCI or <i>His2Av<sup>810</sup></i>/+ (G–G′″) testes at day 5 PCI immunostained with anti-GFP (F, G, green and F′, G′) and anti-His2Av (F, red and F″) or anti-ISWI (G, red and G″) and counterstained with DAPI (blue, F, G and F′″, G′″). <i>ISWI<sup>2</sup></i> (F–F′″) or <i>His2Av<sup>810</sup></i> (G–G′″) mutant GSCs (arrows), <i>ISWI<sup>2</sup></i> (F–F′″) or <i>His2Av<sup>810</sup></i> (G–G′″) heterozygous GSCs (arrowheads) and hub (yellow dashed line). Scale bars: 12.5 µm (<b>H</b>): Percentage of testes with <i>His2Av<sup>810</sup></i> (blue) or <i>His2Av<sup>810</sup></i> ;<i>ISWI<sup>2</sup></i>/+ (red) GSCs scored at indicated times after clonal induction. Data shows average ± S.D.</p
DREF Genetically Counteracts Mi-2 and Caf1 to Regulate Adult Stem Cell Maintenance.
Active adult stem cells maintain a bipotential state with progeny able to either self-renew or initiate differentiation depending on extrinsic signals from the surrounding microenvironment. However, the intrinsic gene regulatory networks and chromatin states that allow adult stem cells to make these cell fate choices are not entirely understood. Here we show that the transcription factor DNA Replication-related Element Factor (DREF) regulates adult stem cell maintenance in the Drosophila male germline. A temperature-sensitive allele of DREF described in this study genetically separated a role for DREF in germline stem cell self-renewal from the general roles of DREF in cell proliferation. The DREF temperature-sensitive allele caused defects in germline stem cell self-renewal but allowed viability and division of germline stem cells as well as cell viability, growth and division of somatic cyst stem cells in the testes and cells in the Drosophila eye. Germline stem cells mutant for the temperature sensitive DREF allele exhibited lower activation of a TGF-beta reporter, and their progeny turned on expression of the differentiation factor Bam prematurely. Results of genetic interaction analyses revealed that Mi-2 and Caf1/p55, components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, genetically antagonize the role of DREF in germline stem cell maintenance. Taken together, these data suggest that DREF contributes to intrinsic components of the germline stem cell regulatory network that maintains competence to self-renew