hek293t 17  (ATCC)

Journal: Nucleic Acids Research

Article Title: A novel dual histone mark reader ZCWPW2 regulates meiotic recombination through lactylation and transcriptional regulation in humans and mice

doi: 10.1093/nar/gkag049

Figure Lengend Snippet: ZCWPW2 is indispensable for meiotic progression in both humans and mice. ( A ) Immunofluorescence of chromosome spreads showing ZCWPW2 (green) and SYCP3 (red) in WT spermatocytes at different meiotic stages. L, Leptotene; Z, Zygotene; P, Pachytene; D, Diplotene; Di, Diakinesis; scale bar: 5 μm. ( B ) Schematic overview of PRDM9/ZCWPW1/ZCWPW2 expression during germ cell development in human and mouse testes and ovaries. Spg, spermatogonia; L, Leptotene; Z, Zygotene; P, Pachytene; D, Diplotene; Di, Diakinesis; RS, round spermatids; ES, elongating/elongated spermatids; MI, meiosis metaphase I; MII, meiosis metaphase II. ( C ) Schematic representation of a 17-bp deletion in exon 3 of Zcwpw2 generated via CRISPR–Cas9 genome editing to produce knockout mice. ( D ) Litter size was assessed by natural mating over a 6-month period ( n = 3 biologically independent adult WT mice and KO mice; Two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( E ) H&E staining of testis sections from adult WT and Zcwpw2 KO mice. Blue arrow, spermatogonia; Red arrow, spermatocytes; Blue arrowhead, round spermatids; Red arrowhead, elongating spermatids; Cyan arrowhead, apoptotic cells; Asterisk, lumen of the seminiferous tubule; scale bar: 50 μm. ( F ) TUNEL staining (green) and DAPI counterstaining (blue) of testis sections from adult WT and Zcwpw2 KO mice; scale bar: 50 μm. ( G ) H&E staining of ovarian sections from WT and Zcwpw2 KO females at the embryonic (E15.5), neonatal (P0), and adult (8-week) stages. Red arrowhead, follicles at different developmental stages. Cyan arrowhead, primordial follicles; scale bar: 50 μm. ( H ) Schematic illustrating the process of screening ZCWPW2 variants in infertile men with NOA using WES. ( I ) Pedigree analysis and Sanger sequencing of the patient carrying a homozygous ZCWPW2 frameshift variant (c.557_558del). ( J ) Summary of the ZCWPW2 frameshift variant (c.557_558del) detected in the NOA patient and its rarity in population databases (*The NCBI reference sequence for ZCWPW2 was NM_001324169.2 .). ( K ) Western blotting analysis of HEK293T cells transfected with WT or mutant Flag- ZCWPW2 (c.557_558del). ( L ) H&E staining of testis sections from control and the patient carrying the ZCWPW2 mutation; scale bar: 50 μm. Spg, spermatogonia; PS, spermatocytes; RS, round spermatids; ES, elongating/elongated spermatids. ( M ) TUNEL staining (green) with DAPI counterstaining (blue) of testicular sections from control and the patient; scale bar: 50 μm (two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.).

Article Snippet: HEK293T cells (CRL-11268; ATCC) and GC-2 cells (CRL-2196; ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS; Gibco, 12483020).

Techniques: Immunofluorescence, Expressing, Generated, CRISPR, Knock-Out, Two Tailed Test, Staining, TUNEL Assay, Sequencing, Variant Assay, Western Blot, Transfection, Mutagenesis, Control

Journal: Nucleic Acids Research

Article Title: A novel dual histone mark reader ZCWPW2 regulates meiotic recombination through lactylation and transcriptional regulation in humans and mice

doi: 10.1093/nar/gkag049

Figure Lengend Snippet: ZCWPW2 shows enhanced enrichment at sites co-marked by H3K4me3 and H3K36me3 in the presence of PRDM9 and is found in complex with ZCWPW1 at these sites. ( A ) Schematic overview of CUT&Tag profiling of ZCWPW2 genomic binding sites in HEK293T cells transfected with Flag- ZCWPW2 (W2) alone or co-transfected with Myc- PRDM9 B . ( B ) Pie charts showing the genomic annotation of ZCWPW2 (W2) binding peaks in HEK293T cells transfected with Flag- ZCWPW2 alone (left) or co-transfected with Myc- PRDM9 B (right). ( C ) Bar graph showing the percentage of the top 10 000 ZCWPW2 (W2) or ZCWPW1 (W1) peaks overlapping with PRDM9-binding sites in the presence or absence of PRDM9. ( D ) Line plot showing ZCWPW2 (W2) binding signal centered around PRDM9 binding motifs (±3 kb) in HEK293T cells expressing Flag-ZCWPW2 with or without Myc-PRDM9. ( E ) Heatmaps and average signal plots showing ZCWPW2 (W2) enrichment centered on PRDM9-binding peaks (±2 kb) in HEK293T cells transfected with Flag- ZCWPW2 alone, co-transfected with Myc- PRDM9 B , or IgG control. ( F ) Venn diagram showing the overlap among ZCWPW2 (W2), ZCWPW1 (W1), and PRDM9 peaks in HEK293T cells co-expressing Myc-PRDM9. ( G ) Genome tracks snapshot showing representative genomic regions with binding profiles of ZCWPW2 (W2), ZCWPW1 (W1), and PRDM9 in HEK293T cells. ( H ) Genome track snapshots showing ZCWPW2 (W2) signals (green) in HEK293T cells co-expressing Flag-ZCWPW2 and Myc-PRDM9, and ZCWPW2 (W2) signals (blue) in cells expressing Flag-ZCWPW2 alone, together with tracks for PRDM9-induced H3K4me3 (pink) and H3K36me3 (orange). ( I ) ZCWPW2 peaks at H3K4me3/H3K36me3 sites in HEK293T cells co-expressing Flag-ZCWPW2 and Myc-PRDM9, as well as in cells expressing Flag-ZCWPW2 alone. ( J ) Co-IP assays were performed in HEK293T cells co-transfected with HA- ZCWPW1 (W1) and Flag- ZCWPW2 (W2). (K and L) Co-IP assays were performed on human ( K ) and mouse ( L ) testis lysates to detect endogenous interactions between ZCWPW1 (W1) and ZCWPW2 (W2). ( M ) Schematic overview showing full-length ZCWPW2 ( W2 ) and ZCWPW1( W1 ), along with their domain-deletion variants lacking either the zf-CW domain (ΔC), the PWWP domain (ΔP), or both (ΔC-ΔP), were generated and were co-transfected in HEK293T cells. ( N ) Co-IP analysis showing the interaction between full-length ZCWPW1 (W1) and full-length or domain-deletion variants of ZCWPW2 (W2). ( O ) Co-IP analysis of ZCWPW2 containing the PWWP domain (W2-ΔC) and full-length or domain-deletion variants of ZCWPW1 (W1) in HEK293T cells. Data for ZCWPW1 ChIP-seq in HEK293T cells, either transfected alone or co-transfected with PRDM9, were obtained from the GEO repository under accession number GSE141516 . ChIP-seq data for PRDM9 in HEK293T cells were retrieved from GEO accession GSE99407 .

Article Snippet: HEK293T cells (CRL-11268; ATCC) and GC-2 cells (CRL-2196; ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS; Gibco, 12483020).

Techniques: Binding Assay, Transfection, Expressing, Control, Co-Immunoprecipitation Assay, Generated, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: A novel dual histone mark reader ZCWPW2 regulates meiotic recombination through lactylation and transcriptional regulation in humans and mice

doi: 10.1093/nar/gkag049

Figure Lengend Snippet: ZCWPW2 binds to H3K4me3-mediated promoter regions in independence of PRDM9. ( A ) Schematic summary illustrating that ZCWPW2 binds strongly to H3K4me3-marked promoter regions both in the absence and presence of PRDM9. ( B ) GO analysis of genes marked by ZCWPW2 peaks at promoter-associated H3K4me3 sites in HEK293T cells. ( C ) CUT&Tag tracks display ZCWPW2 (W2) binding peaks at promoter regions of genes implicated in DNA repair (e.g. RAD51AP1, RAD51C, MRE11, ATM, BARD1, ZCWPW1 ) and chromosome segregation (e.g. SMC2, RAD21, ANKRD31 ), as well as lactylation modification (e.g. EP300 ). IgG was used as a negative control. ( D ) Confirmation of ZCWPW2 (W2) binding at key genes’ promoter regions by PCR amplification using primers targeting the promoter regions. IgG served as a negative control. ( E ) Relative enrichment of ZCWPW2 binding chromatin fragments was measured by qPCR using primers targeting the promoter regions ( n = 3 independent experiments; Two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( F ) Relative luciferase activity driven by promoter fragments of RAD51C, RAD51AP1, RAD21, ZCWPW1, MRE11, BARD1, EP300, ANKRD31, SMC2 , and ATM in HEK293T cells co-transfected with increasing amounts of Flag- ZCWPW2 (ZCWPW2-OE, + and ++), compared to the control (empty vector) ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( G ) qPCR analysis showing significant downregulation of Rad51c1, Rad51ap1, Rad21, Zcwpw1, Mre11a, Brd1, Ep300, Ankrd31, Smc2 , and Atm mRNA in testes from adult Zcwpw2 KO mice compared to WT mice ( n = 3 biologically independent WT mice and KO mice; Two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( H ) Bar graphs showing relative mRNA expression levels of key meiosis-related genes in testicular biopsies from the ZCWPW2 mutant patient compared to controls ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.).

Article Snippet: HEK293T cells (CRL-11268; ATCC) and GC-2 cells (CRL-2196; ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS; Gibco, 12483020).

Techniques: Binding Assay, Modification, Negative Control, Amplification, Two Tailed Test, Luciferase, Activity Assay, Transfection, Control, Plasmid Preparation, Expressing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: A novel dual histone mark reader ZCWPW2 regulates meiotic recombination through lactylation and transcriptional regulation in humans and mice

doi: 10.1093/nar/gkag049

Figure Lengend Snippet: The ZCWPW1–ZCWPW2 complex interacts with recombination-associated proteins and stabilizes LDHA activity. ( A ) Schematic of IP-MS-based identification of ZCWPW2-interacting proteins in human testis tissue. ( B ) GO enrichment analysis of ZCWPW2-interacting proteins. ( C ) Mass spectrometry–based intensity plot depicting meiosis-associated proteins that interact with ZCWPW2. ( D and E ) Co-IP analysis of endogenous SYCP1, TEX11, HSPA2, MLH1, and MSH2 proteins interacting with ZCWPW2 from human (D) and mouse (E) testis lysates. ( F ) Co-IP analysis of exogenous SYCP1, TEX11, HSPA2, MLH1, and MSH2 proteins interacting with exogenous ZCWPW2 in HEK293T cells. ( G–L ) Co-IP assays demonstrating the impact of ZCWPW1 knockdown (siRNA- W1 ) or overexpression (Myc- W1 ) on the interactions of Flag-ZCWPW2 with HA-HSPA2 (G), SYCP1 (H), MLH1 (I), MSH2 (J), TEX11 (K), and MDC1 (L) in HEK293T cells. ( M ) Co-IP assays demonstrating the impact of Zcwpw1 knockdown (siRNA- w1 ) on the interactions of endogenous Zcwpw2 with Hspa2, Sycp1, Mlh1, Msh2, Tex11, Mdc1, and Ldha in GC-2 cells. ( N ) Table summarizing the LDH family proteins detected in ZCWPW2 immunoprecipitates from human testis by IP-MS. ( O ) Co-IP assays demonstrating the interaction between ZCWPW2 and LDHA in human and mouse testes, as well as transfected HEK293T cells. ( P ) Co-IP analysis examining the interaction between Flag-ZCWPW2 and Myc-LDHA in HEK293T cells under conditions of ZCWPW1 knockdown (siRNA- W1 ) or overexpression (HA- W1 ). ( Q ) Schematic overview (top) and quantitative analysis (bottom) of LDH enzymatic activity in testis lysates from WT and Zcwpw2 KO juvenile mice. Activity was measured by assessing formazan absorbance at 450 nm following the conversion of lactate using the WST-8 assay ( n = 3 biologically independent WT mice and KO mice; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( R and S ) LDH enzymatic activity measured by WST-8-based formazan absorbance in treated GC-2 cells (R) and HEK293T cells (S) ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.).

Article Snippet: HEK293T cells (CRL-11268; ATCC) and GC-2 cells (CRL-2196; ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS; Gibco, 12483020).

Techniques: Activity Assay, Protein-Protein interactions, Mass Spectrometry, Co-Immunoprecipitation Assay, Knockdown, Over Expression, Transfection, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: A novel dual histone mark reader ZCWPW2 regulates meiotic recombination through lactylation and transcriptional regulation in humans and mice

doi: 10.1093/nar/gkag049

Figure Lengend Snippet: ZCWPW2 promotes lactylation and stability of recombination-associated proteins through coordination with ZCWPW1 in cultured cells. ( A ) Co-IP analysis of HEK293T cells overexpressed with HA-recombination-associated proteins (HSPA2, SYCP1, and MDC1) and Flag-ZCWPW2, with or without Myc-ZCWPW1, and in the presence or absence of siRNA targeting ZCWPW1 . Bar graph quantifies Kla signal intensity for each condition ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( B ) Co-IP analysis (left) for Kla of endogenous Mdc1, Hspa2, and Sycp1 in GC-2 cells after individual or combined knockdown of Zcwpw1 or/and Zcwpw2 using siRNAs. Quantification of Kla levels (right) showed a significant decrease in Kla upon Zcwpw1 or Zcwpw2 knockdown, with the greatest reduction observed upon dual silencing ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( C and D ) Co-IP (left) and quantification analysis (right) of Kla on endogenous Mdc1, Hspa2, and Sycp1 in GC-2 cells treated with the EP300 inhibitor A-485, the LDHA inhibitor sodium oxamate, or both (C), and with EP300 activator CTPB or exogenous lactate (D) ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.).

Article Snippet: HEK293T cells (CRL-11268; ATCC) and GC-2 cells (CRL-2196; ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS; Gibco, 12483020).

Techniques: Cell Culture, Co-Immunoprecipitation Assay, Two Tailed Test, Knockdown