Yeast Isw1p forms two separable complexes in vivo - Supplementary Materials Only

There are several classes of ATP-dependent chromatin remodeling complexes, which modulate the structure of chromatin to regulate a variety of cellular processes. The budding yeast, Saccharomyces cerevisiae, encodes two ATPases of the ISWI class, Isw1p and Isw2p. Previously Isw1p was shown to copurify with three other proteins. Here we identify these associated proteins and show that Isw1p forms two separable complexes in vivo (designated Isw1a and Isw1b). Biochemical assays revealed that while both have equivalent nucleosome-stimulated ATPase activities, Isw1a and Isw1b differ in their abilities to bind to DNA and nucleosomal substrates, which possibly accounts for differences in specific activities in nucleosomal spacing and sliding. In vivo, the two Isw1 complexes have overlapping functions in transcriptional regulation of some genes yet distinct functions at others. In addition, these complexes show different contributions to cell growth at elevated temperatures

Dependency of ISW1a Chromatin Remodeling on Extranucleosomal DNA▿

The nucleosome remodeling activity of ISW1a was dependent on whether ISW1a was bound to one or both extranucleosomal DNAs. ISW1a preferentially bound nucleosomes with an optimal length of ∼33 to 35 bp of extranucleosomal DNA at both the entry and exit sites over nucleosomes with extranucleosomal DNA at only one entry or exit site. Nucleosomes with extranucleosomal DNA at one of the entry/exit sites were readily remodeled by ISW1a and stimulated the ATPase activity of ISW1a, while conversely, nucleosomes with extranucleosomal DNA at both entry/exit sites were unable either to stimulate the ATPase activity of ISW1a or to be mobilized. DNA footprinting revealed that a major conformational difference between the nucleosomes was the lack of ISW1a binding to nucleosomal DNA two helical turns from the dyad axis in nucleosomes with extranucleosomal DNA at both entry/exit sites. The Ioc3 subunit of ISW1a was found to be the predominant subunit associated with extranucleosomal DNA when ISW1a is bound either to one or to both extranucleosomal DNAs. These two conformations of the ISW1a-nucleosome complex are suggested to be the molecular basis for the nucleosome spacing activity of ISW1a on nucleosomal arrays. ISW1b, the other isoform of ISW1, does not have the same dependency for extranucleosomal DNA as ISW1a and, likewise, is not able to space nucleosomes

Tudor-SN Interacts with Piwi Antagonistically in Regulating Spermatogenesis but Synergistically in Silencing Transposons in <i>Drosophila</i>

<div><p>Piwi proteins associate with piRNAs and functions in epigenetic programming, post-transcriptional regulation, transposon silencing, and germline development. However, it is not known whether the diverse functions of these proteins are molecularly separable. Here we report that Piwi interacts with Tudor-SN (Tudor staphylococcal nuclease, TSN) antagonistically in regulating spermatogenesis but synergistically in silencing transposons. However, it is not required for piRNA biogenesis. TSN is known to participate in diverse molecular functions such as RNAi, degradation of hyper-edited miRNAs, and spliceosome assembly. We show that TSN colocalizes with Piwi in primordial germ cells (PGCs) and embryonic somatic cells. In adult ovaries and testes, TSN is ubiquitously expressed and enriched in the cytoplasm of both germline and somatic cells. The <i>tsn</i> mutants display a higher mitotic index of spermatogonia, accumulation of spermatocytes, defects in meiotic cytokinesis, a decreased number of spermatids, and eventually reduced male fertility. Germline-specific TSN-expression analysis demonstrates that this function is germline-dependent. Different from other known Piwi interters, TSN represses Piwi expression at both protein and mRNA levels. Furthermore, reducing <i>piwi</i> expression in the germline rescues <i>tsn</i> mutant phenotype in a dosage-dependent manner, demonstrating that Piwi and TSN interact antagonistically in germ cells to regulate spermatogenesis. However, the <i>tsn</i> deficiency has little, if any, impact on piRNA biogenesis but displays a synergistic effect with <i>piwi</i> mutants in transposon de-silencing. Our results reveal the biological function of TSN and its contrasting modes of interaction with Piwi in spermatogenesis, transposon silencing, and piRNA biogenesis.</p></div

Yeast Isw1p Forms Two Separable Complexes In Vivo

There are several classes of ATP-dependent chromatin remodeling complexes, which modulate the structure of chromatin to regulate a variety of cellular processes. The budding yeast, Saccharomyces cerevisiae, encodes two ATPases of the ISWI class, Isw1p and Isw2p. Previously Isw1p was shown to copurify with three other proteins. Here we identify these associated proteins and show that Isw1p forms two separable complexes in vivo (designated Isw1a and Isw1b). Biochemical assays revealed that while both have equivalent nucleosome-stimulated ATPase activities, Isw1a and Isw1b differ in their abilities to bind to DNA and nucleosomal substrates, which possibly accounts for differences in specific activities in nucleosomal spacing and sliding. In vivo, the two Isw1 complexes have overlapping functions in transcriptional regulation of some genes yet distinct functions at others. In addition, these complexes show different contributions to cell growth at elevated temperatures

<i>tsn</i> mutations cause spermatogenic cell overproliferation, cytokinetic defects during meiosis, fewer spermatid bundles, and reduced male fertility.

<p>(<b>A</b>) The apical tip region of 2-day-old adult <i>tsn</i>^<i>1120</i><i>/tsn</i>^<i>0614</i> mutant testes. Testes were stained with anti-VASA (germ cell, red) and anti-Hts (fusome, green) antibodies. DNA was labeled by DAPI (blue). The swollen apical tip of the mutant testis was filled with VASA-positive germ cells containing branched fusomes (arrow head) and faint DNA staining, indicating these cells are spermatocytes. A few bundles of elongated spermatids with their heads (arrow) resided at the apical tip of the mutant testis, in contrast to their normal localization at the basal region of the testis. Asterisk: the hub. (<b>B</b>) Phase contrast images of round spermatids from adult WT (left) and <i>tsn</i>^<i>1120</i><i>/tsn</i>^<i>0614</i> mutant (right) testes. In mutant testes, round spermatids containing a single Nebenkern (mitochondrial derivates) associated with two haploid nuclei were observed, indicating there were meiotic cytokinesis defects (see text). (<b>C</b>) The quantification of spermatid bundle number in 2-week-old adult testes revealed that <i>tsn</i>^<i>1120</i><i>/tsn</i>^<i>0614</i> mutants had fewer spermatid bundles compared to the WT. (<b>D-E</b>) The third instar larval testes of WT and <i>tsn</i>^<i>1120</i><i>/tsn</i>^<i>0614</i> mutant were immunostained with anti-phospho-histone3 (pH3, mitotic index, red) and anti-Hts (fusome, green) antibodies. DNA was labeled by DAPI (blue). Asterisks indicate the hubs. The <i>tsn</i> mutant testis had more pH3-positive spermatogonia and was larger in size. (<b>F</b>) The quantification of pH3-positive spermatogonia per testis. The WT testes showed an average of 2.56 pH3-positive cells per testis (n = 27) and the <i>tsn</i>^<i>1120</i><i>/tsn</i>^<i>0614</i> mutant had an average of 3.44 pH3-positive cells per testis (n = 27) (see text). (<b>G</b>) The fertility of <i>tsn</i> mutant (red line) and WT (blue line) males. Initially, the <i>tsn</i>^<i>1120</i><i>/tsn</i>^<i>0614</i> mutant males showed a similar fertility as the WT controls, but the fertility was decreased faster over time compared to the WT males. Error bars represent mean ± standard error of the mean ((C) and (F) N ≧ 27; (G) N≧ 3). ** denotes P<0.01. The differences between WT and <i>tsn</i> mutant males In (G), <i>p</i> = 0.0000437 (by t-test) for the differences between WT and <i>tsn</i> mutant males.</p

TSN is highly expressed in embryos and adult gonads.

<p>(<b>A</b>) Expression pattern of TSN protein in different key developmental stages and tissues. Western blot analysis using mouse anti-TSN antibody revealed that TSN was highly expressed in the embryonic stage as well as adult ovaries and testes. (<b>B-D”</b>) Immunostaining of TSN (using mouse anti-TSN antibody, green) in adult WT ovaries. DNA was labeled by DAPI (blue). TSN was localized to the cytoplasm of both germline and somatic cells in adult ovaries. G, germarium; S1-S5, stage 1–5 egg chambers, respectively; TF, terminal filament. (<b>C-C”</b>) Magnified images of a germarium including a S1 egg chamber. (<b>D-D”</b>) Magnified images of B-B” showing the S3 egg chamber. (<b>E-F””</b>) Immunostaining TSN (using mouse anti-TSN antibody, green) in WT adult testes. Germ cells were labeled with anti-VASA antibody (red) and somatic cells were labeled with anti-Tj antibody (purple). DNA was labeled by DAPI (blue). TSN was localized to the cytoplasm of germline and somatic cells in adult testes. Magnified images of the apical region of the testis are shown in F-F””. (G-G‴) Immunostaining of TSN (green) and Piwi (red) in WT adult testes. DNA was labeled by DAPI (blue). Piwi was localized in the nuclei of hub cells, early germ cells and somatic cyst cells. Asterisk: the hub.</p

TSN antagonizes the germline function of Piwi.

<p>The testes were stained with anti-Piwi (green), anti-VASA (germ cell, red), and anti-Tj (somatic cells, purple) antibodies. DNA was labeled by DAPI (blue). (<b>A-A‴</b>) Germline knockdown of Piwi using <i>nosVP16-Gal4</i> driver. (<b>B-B‴</b>) The <i>tsn</i>^<i>1120</i><i>/tsn</i>^<i>0614</i> mutant displayed a swollen apical tip of the testis. (<b>C-C‴</b>) The depletion of Piwi in germ cells rescued <i>tsn</i> mutant phenotype, suggesting TSN antagonizes the germline function of Piwi in regulating spermatogenesis.</p

TSN is a novel Piwi-interacting protein.

<p>(<b>A</b>) Schematic depiction of protein domain structures of TSN. TSN contains five staphylococcal nuclease-like domains (SN1-SN5) and a methyl lysine/arginine recognizing Tudor domain. (<b>B</b>) Co-immunoprecipitations of Piwi and TSN with anti-Piwi antibody. Input was the cytoplasmic fraction of the lysates from 0-12h WT embryos. 5% of input was used in the Western blot analysis. IgG was used as the control. F.L., flow-through; IP, immunoprecipitates. (<b>C</b>) Reciprocal co-immunoprecipitations of TSN and Piwi with mouse anti-TSN antibody. Input was the cytoplasmic fraction of lysates from 0–12 h WT embryos. 10% of input was used in the Western blot analysis. IgG was used as controls for the co-immunoprecipitations. F.L., flow-through; IP, immunoprecipitates. (<b>D-F‴</b>) Immunostaining of Piwi (green), TSN (using rabbit anti-TSN-N antibody, red), and the germ cell marker VASA (purple) in 0-1h WT embryos. DNA was labeled by DAPI (blue). (<b>D-D‴</b>) The whole embryo. PGCs, primordial germ cells. (<b>E-E‴</b>) Magnified images of E-E‴ showing the region of PGCs. Piwi and TSN were both highly enriched in the cytoplasmic region of PGCs. (<b>F-F‴</b>) Magnified images of D-D‴ showing the somatic cells. Piwi and TSN were both localized to the somatic nuclei. <b>(G)</b> Co-immunostaining of Piwi and TSN in PGCs, merged image of E and E’. <b>(H)</b> Co-immunostaining of Piwi and TSN in somatic cells, merged image of F and F’.</p

Comparative analysis of antibodies to SARS-CoV-2 between asymptomatic and convalescent patients

Summary: The SARS-CoV-2 viral pandemic has induced a global health crisis, which requires more in-depth investigation into immunological responses to develop effective treatments and vaccines. To understand protective immunity against COVID-19, we screened over 60,000 asymptomatic individuals in the Southeastern United States for IgG antibody positivity against the viral Spike protein, and approximately 3% were positive. Of these 3%, individuals with the highest anti-S or anti-RBD IgG level showed a strong correlation with inhibition of ACE2 binding and cross-reactivity against non-SARS-CoV-2 coronavirus S-proteins. We also analyzed samples from 94 SARS-CoV-2 patients and compared them with those of asymptomatic individuals. SARS-CoV-2 symptomatic patients had decreased antibody responses, ACE2 binding inhibition, and antibody cross-reactivity. Our study shows that healthy individuals can mount robust immune responses against SARS-CoV-2 without symptoms. Furthermore, IgG antibody responses against S and RBD may correlate with high inhibition of ACE2 binding in individuals tested for SARS-CoV-2 infection or post vaccination