Journal: Animals : an Open Access Journal from MDPI

Article Title: The Germline-Restricted Chromosome of Male Zebra Finches in Meiotic Prophase I: A Proteinaceous Scaffold and Chromatin Modifications

doi: 10.3390/ani14223246

Figure Lengend Snippet: HORMAD1 localization in a zebra finch pachytene spermatocyte ( A – D ). Locations of proteins SYCP3 (green) and HORMAD1 (white) are shown. Chromatin is stained with DAPI (blue). GRC: germline-restricted chromosome. The white circles point to GRCs. Scale bar = 5 µm.

Article Snippet: Immunostaining was performed using an anti-SYCP3 (synaptonemal complex protein 3) rabbit polyclonal antibody (Abcam, Cambridge, UK, ab15093), rabbit polyclonal anti-HORMAD1 antibody (Proteintech, Rosemont, IL, USA, 13917-1-AP), rabbit polyclonal anti-RPA32/RPA2 antibody (RPA; Abcam, ab10359), mouse polyclonal anti-RAD51 antibody (Abcam, ab88572), rabbit polyclonal anti-histone H3 antibody (tri methyl K27) (H3K27me3; Abcam, ab195477), mouse monoclonal anti-RNA polymerase II antibody (RNAP II; Abcam, ab5408), mouse monoclonal anti-gammaH2A.X antibody (phospho S139) [9F3] (γH2AFX; Abcam, ab26350), mouse monoclonal anti-SUMO-1 antibody (Zymed, San Francisco, CA, USA #33-2400), mouse monoclonal anti-ubiquityl histone H2A antibody (ubiH2A; Millipore, Billerica, MA, USA, #05-678), rabbit polyclonal anti-H3K9me3 antibody (Abcam, ab8898), rat monoclonal anti-histone H3 antibody (phospho S28) (H3S28ph; Abcam, ab10543), and a human anti-centromere antibody (CREST [calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia]; #90C-CS1058, Fitzgerald Industries International, Concord, MA, USA).

Techniques: Staining

Journal: Animals : an Open Access Journal from MDPI

Article Title: The Germline-Restricted Chromosome of Male Zebra Finches in Meiotic Prophase I: A Proteinaceous Scaffold and Chromatin Modifications

doi: 10.3390/ani14223246

Figure Lengend Snippet: Dynamics of GRC structure at prophase I of zebra finch meiosis. Schematic representations of the structure and behavior of meiotic chromosomes are depicted. As the scheme is simplified, it does not capture the chromatin’s loop organization. Abbreviations: GRC, germline-restricted chromosome; SC, synaptonemal complex; AE, axial element; LE, lateral element of SC; TF, transverse filament of central space of SC; as, asynapsis area; ds, desynapsis area. In AEs/LEs of SCs: HORMAD1 (blue dots), cohesins (red dots), and SYCP3 (green dots). Typically, the GRC was found to create a distinct chromatin domain at the nuclear periphery during prophase I ( A – D ). ( A ) Leptotene. At this stage, core proteins (cohesins, HORMAD1 and SYCP3) ( A′ ) are loaded into the chromosome axis; therefore, the nascent proteinaceous scaffolds of the AEs of autosomes and GRC can be observed. ( B ) Zygotene. Chromosomes form a bouquet. Autosomal AEs arise. Two AEs of homologous chromosomes align with each other and begin to form SC segments. SC regions either lack HORMAD1 or contain rare stand-alone signals. Unlike autosomes, where protein assembly of AEs was completed, loading of core proteins continues into the AE of the GRC (protein loading delay) ( B′ ). Asynaptic regions and some regions of the GRC univalent contain a large amount of HORMAD1. ( C ) Pachytene. Autosomes are fully synapsed. Loading of core proteins into the GRC AE is almost complete. Sometimes, some regions of the GRC remain without loaded core proteins (fragmented GRC AEs or GRC with gaps) ( C′ ). Unlike autosomes, the GRC univalent has a lot of HORMAD1. ( D ) Diplotene. Chromosomes are desynapsed. The GRC and desynapsing axes of autosomes undergo protein disassembly. Desynapsing segments of autosomes and GRC are enriched with HORMAD1. ( E ) Explanatory diagram.

Article Snippet: Immunostaining was performed using an anti-SYCP3 (synaptonemal complex protein 3) rabbit polyclonal antibody (Abcam, Cambridge, UK, ab15093), rabbit polyclonal anti-HORMAD1 antibody (Proteintech, Rosemont, IL, USA, 13917-1-AP), rabbit polyclonal anti-RPA32/RPA2 antibody (RPA; Abcam, ab10359), mouse polyclonal anti-RAD51 antibody (Abcam, ab88572), rabbit polyclonal anti-histone H3 antibody (tri methyl K27) (H3K27me3; Abcam, ab195477), mouse monoclonal anti-RNA polymerase II antibody (RNAP II; Abcam, ab5408), mouse monoclonal anti-gammaH2A.X antibody (phospho S139) [9F3] (γH2AFX; Abcam, ab26350), mouse monoclonal anti-SUMO-1 antibody (Zymed, San Francisco, CA, USA #33-2400), mouse monoclonal anti-ubiquityl histone H2A antibody (ubiH2A; Millipore, Billerica, MA, USA, #05-678), rabbit polyclonal anti-H3K9me3 antibody (Abcam, ab8898), rat monoclonal anti-histone H3 antibody (phospho S28) (H3S28ph; Abcam, ab10543), and a human anti-centromere antibody (CREST [calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia]; #90C-CS1058, Fitzgerald Industries International, Concord, MA, USA).

Techniques:

Journal: eLife

Article Title: TRIP13 localizes to synapsed chromosomes and functions as a dosage-sensitive regulator of meiosis

doi: 10.7554/elife.92195

Figure Lengend Snippet: Figure 5. HORMAD1 and HORMAD2 accumulate on the lateral elements of synapsed autosomes in Trip13-

Article Snippet: or resource Designation Source or reference Identifiers Additional information Gene (Mus musculus) Trip13 GenBank Gene ID: 69716 Genetic reagent (M. musculus) Trip13tm1.1(KOMP)Vlcg/JMmucd MMRCC MMRRC_050223- UCD Genetic reagent (M. musculus) Hormad1 knockout PMID:21079677; Shin et al., 2010 Rajkovic lab Genetic reagent (M. musculus) Sycp2 knockout PMID:16717126; Yang et al., 2006 Wang lab Genetic reagent (M. musculus) Rec8 knockout PMID:32232159; Guan et al., 2020 Wang lab Genetic reagent (M. musculus) 3×FLAG-Trip13 This paper Wang Lab Genetic reagent (M. musculus) Trip13-3×FLAG This paper Wang Lab Antibody Anti- ACTB (mouse monoclonal) Sigma Cat# A5441, RRID:AB_476744 WB (1:2000) Antibody Anti- centromere (CREST) (human polyclonal) Antibodies Incorporated Cat# 15- 234, RRID:AB_2687472 IF (1:500) Antibody Anti- SYCP1 (rabbit polyclonal) Abcam Cat# ab15090, RRID:AB_301636 IF (1:300) Antibody Anti- SYCP2 (guinea pig polyclonal) PMID:16717126 Custom made IF (1:150) Antibody Anti- SYCP3 (mouse monoclonal) Abcam Cat# ab97672, RRID:AB_10678841 IF (1:500) Antibody Anti- SYCP3 (rabbit polyclonal) ProteinTech Group Cat# 23024- 1- AP, RRID:AB_11232426 IF (1:500), WB (1:2000) Antibody Anti- HORMAD1 (rabbit polyclonal) ProteinTech Group Cat# 13917- 1- AP, RRID:AB_2120844 IF (1:300) Antibody Anti- HORMAD2 (rabbit polyclonal) PMID:19851446; Wojtasz et al., 2009 A gift from Toth lab IF (1:500) Antibody Anti- REC114 (rabbit polyclonal) PMID:31003867; Boekhout et al., 2019 A gift from Keeney Lab IF (1:100) Antibody Anti- REC8 (rabbit polyclonal) Custom made A gift from Mengcheng Luo lab IF (1:200) Antibody Anti- FLAG (mouse monoclonal) Sigma Cat# F3165, RRID:AB_259529 IF (1:300), WB (1:4000) Antibody Anti- SKP1 (rabbit polyclonal) Cell Signaling Cat# 12248S, RRID:AB_2754993 IF (1:200), WB (1:1000) Antibody Anti- TRIP13 (rabbit polyclonal) ProteinTech Group Cat# 19602- 1- AP IF (1:150), WB (1:1000) Chotiner et al. eLife 2023;12:RP92195.

Techniques:

Journal: eLife

Article Title: TRIP13 localizes to synapsed chromosomes and functions as a dosage-sensitive regulator of meiosis

doi: 10.7554/elife.92195

Figure Lengend Snippet: Figure 6. Localization of TRIP13 to the synaptonemal complex (SC) is independent of individual axial element components. (A) Immunofluorescent analysis of TRIP13 in Hormad1-/- spermatocytes from 2-month-old mice. (B) Immunofluorescent analysis of TRIP13 in Rec8-/- spermatocytes from 2-month- old mice. (C) Immunofluorescent analysis of TRIP13 in Sycp2-/- spermatocytes from 2-month-old mice. (D) Immunofluorescent analysis of TRIP13 in Skp1cKO spermatocytes from 2-month-old mice. Scale bars, 10 µm.

Article Snippet: or resource Designation Source or reference Identifiers Additional information Gene (Mus musculus) Trip13 GenBank Gene ID: 69716 Genetic reagent (M. musculus) Trip13tm1.1(KOMP)Vlcg/JMmucd MMRCC MMRRC_050223- UCD Genetic reagent (M. musculus) Hormad1 knockout PMID:21079677; Shin et al., 2010 Rajkovic lab Genetic reagent (M. musculus) Sycp2 knockout PMID:16717126; Yang et al., 2006 Wang lab Genetic reagent (M. musculus) Rec8 knockout PMID:32232159; Guan et al., 2020 Wang lab Genetic reagent (M. musculus) 3×FLAG-Trip13 This paper Wang Lab Genetic reagent (M. musculus) Trip13-3×FLAG This paper Wang Lab Antibody Anti- ACTB (mouse monoclonal) Sigma Cat# A5441, RRID:AB_476744 WB (1:2000) Antibody Anti- centromere (CREST) (human polyclonal) Antibodies Incorporated Cat# 15- 234, RRID:AB_2687472 IF (1:500) Antibody Anti- SYCP1 (rabbit polyclonal) Abcam Cat# ab15090, RRID:AB_301636 IF (1:300) Antibody Anti- SYCP2 (guinea pig polyclonal) PMID:16717126 Custom made IF (1:150) Antibody Anti- SYCP3 (mouse monoclonal) Abcam Cat# ab97672, RRID:AB_10678841 IF (1:500) Antibody Anti- SYCP3 (rabbit polyclonal) ProteinTech Group Cat# 23024- 1- AP, RRID:AB_11232426 IF (1:500), WB (1:2000) Antibody Anti- HORMAD1 (rabbit polyclonal) ProteinTech Group Cat# 13917- 1- AP, RRID:AB_2120844 IF (1:300) Antibody Anti- HORMAD2 (rabbit polyclonal) PMID:19851446; Wojtasz et al., 2009 A gift from Toth lab IF (1:500) Antibody Anti- REC114 (rabbit polyclonal) PMID:31003867; Boekhout et al., 2019 A gift from Keeney Lab IF (1:100) Antibody Anti- REC8 (rabbit polyclonal) Custom made A gift from Mengcheng Luo lab IF (1:200) Antibody Anti- FLAG (mouse monoclonal) Sigma Cat# F3165, RRID:AB_259529 IF (1:300), WB (1:4000) Antibody Anti- SKP1 (rabbit polyclonal) Cell Signaling Cat# 12248S, RRID:AB_2754993 IF (1:200), WB (1:1000) Antibody Anti- TRIP13 (rabbit polyclonal) ProteinTech Group Cat# 19602- 1- AP IF (1:150), WB (1:1000) Chotiner et al. eLife 2023;12:RP92195.

Techniques:

Journal: eLife

Article Title: TRIP13 localizes to synapsed chromosomes and functions as a dosage-sensitive regulator of meiosis

doi: 10.7554/elife.92195

Figure Lengend Snippet: Figure 7. FLAG-tagged TRIP13 proteins localize correctly and are functional. (A) Western blot analysis of tagged and untagged TRIP13 proteins in testes from P20 wild type (no tag), heterozygous-tagged, and homozygous-tagged males. (B) Immunofluorescence of FLAG-tagged TRIP13 in pachytene spermatocytes from P20 homozygous testes. N-terminal tag, 3×FLAG-Trip13; C-terminal tag, Trip13−3×FLAG. Scale bar, 10 μm. (C) A schematic illustration of TRIP13, SKP1, HORMAD1/2, and the synaptonemal complex. Relative locations of TRIP13 and SKP1 within the synaptonemal complex (SC) are depicted. HORAMD1/2 are retained in synapsed regions in Trip13-deficient or Skp1-deficient spermatocytes.

Article Snippet: or resource Designation Source or reference Identifiers Additional information Gene (Mus musculus) Trip13 GenBank Gene ID: 69716 Genetic reagent (M. musculus) Trip13tm1.1(KOMP)Vlcg/JMmucd MMRCC MMRRC_050223- UCD Genetic reagent (M. musculus) Hormad1 knockout PMID:21079677; Shin et al., 2010 Rajkovic lab Genetic reagent (M. musculus) Sycp2 knockout PMID:16717126; Yang et al., 2006 Wang lab Genetic reagent (M. musculus) Rec8 knockout PMID:32232159; Guan et al., 2020 Wang lab Genetic reagent (M. musculus) 3×FLAG-Trip13 This paper Wang Lab Genetic reagent (M. musculus) Trip13-3×FLAG This paper Wang Lab Antibody Anti- ACTB (mouse monoclonal) Sigma Cat# A5441, RRID:AB_476744 WB (1:2000) Antibody Anti- centromere (CREST) (human polyclonal) Antibodies Incorporated Cat# 15- 234, RRID:AB_2687472 IF (1:500) Antibody Anti- SYCP1 (rabbit polyclonal) Abcam Cat# ab15090, RRID:AB_301636 IF (1:300) Antibody Anti- SYCP2 (guinea pig polyclonal) PMID:16717126 Custom made IF (1:150) Antibody Anti- SYCP3 (mouse monoclonal) Abcam Cat# ab97672, RRID:AB_10678841 IF (1:500) Antibody Anti- SYCP3 (rabbit polyclonal) ProteinTech Group Cat# 23024- 1- AP, RRID:AB_11232426 IF (1:500), WB (1:2000) Antibody Anti- HORMAD1 (rabbit polyclonal) ProteinTech Group Cat# 13917- 1- AP, RRID:AB_2120844 IF (1:300) Antibody Anti- HORMAD2 (rabbit polyclonal) PMID:19851446; Wojtasz et al., 2009 A gift from Toth lab IF (1:500) Antibody Anti- REC114 (rabbit polyclonal) PMID:31003867; Boekhout et al., 2019 A gift from Keeney Lab IF (1:100) Antibody Anti- REC8 (rabbit polyclonal) Custom made A gift from Mengcheng Luo lab IF (1:200) Antibody Anti- FLAG (mouse monoclonal) Sigma Cat# F3165, RRID:AB_259529 IF (1:300), WB (1:4000) Antibody Anti- SKP1 (rabbit polyclonal) Cell Signaling Cat# 12248S, RRID:AB_2754993 IF (1:200), WB (1:1000) Antibody Anti- TRIP13 (rabbit polyclonal) ProteinTech Group Cat# 19602- 1- AP IF (1:150), WB (1:1000) Chotiner et al. eLife 2023;12:RP92195.

Techniques: Functional Assay, Western Blot, Immunofluorescence