Journal: Genes, Chromosomes & Cancer

Article Title: Sister chromatid cohesion defects are associated with chromosomal copy number heterogeneity in high hyperdiploid childhood acute lymphoblastic leukemia

doi: 10.1002/gcc.22933

Figure Lengend Snippet: Copy number in REH cells with low expression of RAD21 (RAD21‐KD cells) and controls, analyzed by interphase fluorescence in situ hybridization for chromosomes X, 2, 3 and 21. A, Overall copy number of chromosome X, monosomy expected; B, overall copy number of chromosome 21, trisomy expected; C, nucleus from replicate RAD21.1‐3, showing one copy of chromosome X and two copies of chromosome 21; D, nucleus from replicate RAD21.2‐3, showing two copies of chromosome X and three copies of chromosome 21; E, nucleus from replicate RAD21.2‐2, showing one copy of chromosome X and four copies of chromosome 21; F, nucleus from replicate RAD21.1‐3, showing one copy of chromosome X and five copies of chromosome 21; G, overall copy number of chromosome 2, disomy expected; H, overall copy number of chromosome 3, disomy expected; I, nucleus from replicate RAD21.2‐4, showing two copies of chromosome 2 and one copy of chromosome 3; J, nucleus from replicate RAD21.2‐5, showing one copy of chromosome 2 and three copies of chromosome 3; K, nucleus from replicate control 5, showing two copies of chromosome 2 and three copies of chromosome 3; L, nucleus from replicate control 5, showing two copies of chromosome 2 and two copies of chromosome 3

Article Snippet: Gene expression levels were determined 1 week after transduction by RT‐PCR (7500 Real‐Time PCR system; Applied Biosystems, Waltham, MS), using probes from Taqman (Life Technologies, Carlsbad, CA) for RAD21 (Hs00366721_mH) and HPRT1 (Hs02800695_m1).

Techniques: Expressing, Fluorescence, In Situ Hybridization

Journal: Journal of proteome research

Article Title: Proteomic Profile Identifies Dysregulated Pathways in Cornelia de Lange Syndrome Cells With Distinct Mutations in SMC1A and SMC3 Genes

doi: 10.1021/pr300760p

Figure Lengend Snippet: Analysis of cohesin binding at the human c-MYC locus. (A) Schematic of human c-MYC gene. Black solid boxes indicate translated regions. Cohesin binding to MINE and first exon is denoted by an asterisk. Arrows indicate transcriptional start sites of the P1 and P2 promoter. Location of primer pairs A-C are indicated, ChIP assays with antibody against RAD21 were analysed by real-time PCR with primers pairs B and C specific for the promoter region of the c-MYC locus. Binding at each site was determined relative to primer A, where no RAD21 binding was predicted. (B) Results showed that RAD21 co-localized to the promoter region of c-MYC in all SMC1A- and SMC3-mutated CdLS cell lines, with the exception of CdL057, whereas bound cohesin was dramatically reduced in exon 1 in CdLVH, CdLSS, CdL057, CdL060, CdL107 CdLS cell lines. CdL203, which shares the same mutation with CdL060, showed a decrease close to significant (p = 0.07). Since no difference was found in control cell lines, data was pooled. Results shown are the averages of three independent experiments. The graphs show the average and the standard error of the normalized values and *p < 0.05.

Article Snippet: Antibody against RAD21 (Bethyl Laboratories) was used.

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Mutagenesis, Control

Journal: Journal of proteome research

Article Title: Proteomic Profile Identifies Dysregulated Pathways in Cornelia de Lange Syndrome Cells With Distinct Mutations in SMC1A and SMC3 Genes

doi: 10.1021/pr300760p

Figure Lengend Snippet: Mutated SMC1A and SMC3 co-immunoprecipitate with RAD21 in the CdLS cell lines. (A) SMC1A (and SMC3) was found to be co-precipitated with RAD21, (B) whereas no RAD21 signal was detected in the IPs using IgG-coated beads. (C) In addition, RAD21co-precipitated with SMC1A (and SMC3) and (D) no SMC specific signal was detectable in the IPs using IgG-coated beads.

Article Snippet: Antibody against RAD21 (Bethyl Laboratories) was used.

Techniques:

Journal: Nature genetics

Article Title: Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes.

doi: 10.1038/s41588-020-0647-9

Figure Lengend Snippet: Fig. 2 | Variable interactions across boundaries occur independently from intra-domain compaction. a, Hi-C contact matrices and Oligopaint probe design for neighboring domains on chromosomes 12 (left) and 22 (right), with corresponding A/B compartment designations, CTCF and RAD21 binding profiles and Hi-C insulation scores. b, Representative 3D-STORM localizations (above) and 3D hull reconstructions (below) for neighboring domains on chr12:11.6–13.6 Mb. Scale bars, 500 nm. c, Domain volume quantified from 3D-STORM images. Chr12.D1 (median = 0.2629 μm3, n = 91); Chr12.D2 (median = 0.1830 μm3, n = 91); Chr22.D1 (median = 0.2749 μm3, n = 95); Chr22.D2 (median = 0.2320 μm3, n = 95). d, Spatial overlap between neighboring domains, normalized to the volume of the upstream domain. Chr12 (median = 0.1013 μm3, n = 91); Chr22 (median = 0.05466 μm3, n = 95). ***P < 0.001, two-tailed Mann–Whitney U-test. e, Volume of spatial overlap between neighboring domains D1 and D2 at chr12:11.6–13.6 Mb (x axis) versus the particle density of either domain (y axis). n = 91 chromosomes.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Immunofluorescence was performed using the following primary antibodies: RAD21 (Santa Cruz sc-166973, 1:100), PCNA (Santa Cruz sc-56, 1:100), CENPF (Novus Biologicals NB500-101, 1:100).

Techniques: Hi-C, Binding Assay, Insulation, Two Tailed Test, MANN-WHITNEY

Journal: Nature genetics

Article Title: Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes.

doi: 10.1038/s41588-020-0647-9

Figure Lengend Snippet: Fig. 3 | Cohesin promotes interactions within and across domain boundaries. a, Hi-C contact matrices and Oligopaint probe design of chr12:11.6–13.6 Mb and chr22:33.2–36.8 Mb in HCT-116-RAD21-AID cells before or after 6 h of auxin treatment. b, Representative FISH images of neighboring domains across chr12:11.6–13.6 Mb before and after auxin treatment. Dashed lines represent nuclear edges. Scale bar, 5 μm (left) or 1 μm (zoomed images, below). c, Locus-specific differences in the percentage of domain pairs in contact following auxin treatment. Each bar represents an average of two biological replicates. d, Fold change in contact frequency between neighboring domains following auxin treatment versus the IS of their intervening boundary. Each point represents an average of two biological replicates. n = 17 boundaries. e, Cumulative distribution of overlap between the neighboring domains at chr12:11.6–13.6 Mb before (n = 1,625) and after auxin treatment (n = 1,607). Overlap was normalized to the volume of the upstream domain. ***P < 0.001, two-tailed Mann–Whitney U-test. f, Cumulative distribution of overlap between the neighboring domains at chr22:33.2–36.8 Mb before (n = 2,835) and after auxin treatment (n = 2,803). Overlap was normalized to the volume of the upstream domain. P < 0.001, two-tailed Mann–Whitney U-test. g, Representative 3D hull reconstructions of 3D-STORM localizations for neighboring domains on chr12:11.6–13.6 Mb. Scale bar, 500 nm. h, Spatial overlap between domains on chr12:11.6–13.6 Mb and chr22:33.2–36.8 Mb, normalized to the volume of the upstream domain. *P = 0.044 for domains on chromosome 12 (before auxin: median = 0.1013 μm3, n = 91; after auxin: median = 0.08967 μm3, n = 76); *P = 0.029 for domains on chromosome 22 (before auxin median = 0. 05466 μm3, n = 95; after auxin median = 0. 03250 μm3, n = 105), two-tailed Mann–Whitney U-test.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Immunofluorescence was performed using the following primary antibodies: RAD21 (Santa Cruz sc-166973, 1:100), PCNA (Santa Cruz sc-56, 1:100), CENPF (Novus Biologicals NB500-101, 1:100).

Techniques: Hi-C, Two Tailed Test, MANN-WHITNEY

Journal: Nature genetics

Article Title: Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes.

doi: 10.1038/s41588-020-0647-9

Figure Lengend Snippet: Fig. 6 | Cohesin alters the transcriptional bursting frequency of boundary-proximal genes. a, Representative images of intronic RNA FISH to the HS3ST1 transcript before and after auxin treatment. Dashed lines represent nuclear edges. Scale bar, 5 μm. b, Scatterplot indicating the gene expression changes previously reported by PRO-seq8 and distance to nearest boundary for genes assayed by RNA FISH. The mean domain size denoted by a dashed line is 343.9 kb. c, Hi-C contact matrices of the loci surrounding GALNT5 and HS3ST1. Hi-C maps shown for HCT-116 cells before and after auxin treatment to degrade RAD21. d, Change in bursting frequency of each gene following auxin treatment by intronic RNA FISH. n > 227 chromosomes. An average of three biological replicates per gene is shown. e, Change in gene expression previously reported by PRO-seq8 versus change in bursting frequency detected by intronic RNA FISH (R2 = 0.9047, two-sided Pearson correlation). P < 0.0001, n = 14 boundaries. f, Model of single-cell variability in domain formation. Two architectural domains are depicted in green and magenta, with arrows indicating a boundary-proximal promoter in each domain. Colored rectangles represent the appropriate enhancer for each gene. Cohesin promotes variable boundary bypass such that the boundary-proximal chromatin is asymmetrically incorporated with the neighboring domains in a large fraction of cells. The boundary-proximal promoters thus alternate their contact with regulatory elements in their respective domains, which can result in a transcriptional burst. In the absence of cohesin, the boundary-proximal region is more often excluded from either domain, such that promoters in this region are less frequently exposed to their regulatory elements. This could explain both downregulation and upregulation of DEGs if a boundary-proximal gene were looped out away from a distal enhancer or silencer, respectively.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Immunofluorescence was performed using the following primary antibodies: RAD21 (Santa Cruz sc-166973, 1:100), PCNA (Santa Cruz sc-56, 1:100), CENPF (Novus Biologicals NB500-101, 1:100).

Techniques: Gene Expression, Hi-C

Journal: Science Advances

Article Title: ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

doi: 10.1126/sciadv.1601602

Figure Lengend Snippet: ( A ) Table of the most relevant proteins identified by LC-MS/MS in the affinity purification of ASXL1-associated proteins using FLAG-ASXL1 overexpressing HEK293T cells. Spectral counts (unique and total) for each interacting protein are shown. ( B to E ) Reciprocal IP and Western blotting confirmed interaction of ASXL1 with SMC1A, SMC3, and RAD21 in nuclear fraction derived from HEK293T cells transfected with pcDNA3.1 + (Vec) or FLAG-tagged ASXL1 (ASXL1). Nuclear extractions were subjected to IP using indicated antibodies against FLAG (B), SMC1A (C), SMC3 (D), or RAD21 (E). IB, immunoblot. ( F ) Western blot shows the endogenous interaction between ASXL1 and SMC1A, SMC3, and RAD21 in BM cells of WT mice. IgG, immunoglobulin G. ( G ) Gel filtration analysis of nuclear extracts from FLAG-ASXL1 overexpressing cells. ASXL1 and the cohesin complex were coeluted from a Superose 6 HR gel filtration column, as analyzed by Western blotting. The numbers over the lanes represent the eluted fraction numbers.

Article Snippet: The shRNA plasmids of ASXL1 (TG306527), SMC1A (TL513033), and RAD21 (TL501846) were purchased from OriGene.

Techniques: Liquid Chromatography with Mass Spectroscopy, Affinity Purification, Western Blot, Derivative Assay, Transfection, Filtration

Journal: Science Advances

Article Title: ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

doi: 10.1126/sciadv.1601602

Figure Lengend Snippet: ( A ) Schematic diagram of the full-length (FL) ASXL1 and the truncated variants of Asxl1 [amino acids (aa) 1 to 1010, 1 to 420, 1 to 587, and 401 to 587]. Binding affinity was determined by the pull-down efficiency of IP with anti-FLAG and Western blotting with cohesin antibodies. NLS, nuclear localization signal. ( B to E ) Western blotting analysis of nuclear fractions and anti-FLAG immunoprecipitates from pcDNA3.1 + , or each truncated ASXL1 transfected HEK293T cells using antibodies against FLAG, SMC1A, SMC3, or RAD21.

Article Snippet: The shRNA plasmids of ASXL1 (TG306527), SMC1A (TL513033), and RAD21 (TL501846) were purchased from OriGene.

Techniques: Binding Assay, Western Blot, Transfection

Journal: Science Advances

Article Title: ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

doi: 10.1126/sciadv.1601602

Figure Lengend Snippet: ( A and B ) The myeloid cells with premature sister chromatid separation are frequently seen in PB smears (A) and BM (B) of Asxl1 +/− and Asxl1 −/− mice with MDS. Red arrows indicate the abnormal nuclear bridging. Scale bars, 5 μm (A) and 10 μm (B). ( C and D ) Representative cells with premature sister chromatid separation in cultured WT, Asxl1 +/− , and Asxl1 −/− LK cells. Red arrows indicate the premature sister chromatid separation. The frequency of cells with premature sister chromatid separation is shown in (C). Y axis shows the percentage of cells with premature sister chromatid separation within all binucleated cells. Data are represented as means ± SEM from three independent experiments. *** P < 0.001 and ** P < 0.01. Scale bars, 5 μm. ( E ) The frequency of cells with premature sister chromatid separation in the HeLa GFP-H2B cells with hASXL1 KD and hASXL1 KD plus mAsxl1 rescues. KD of ASXL1 leads to increased frequency of cells with premature sister chromatid separation in HeLa GFP-H2B cells. Reintroducing full-length mAsxl1 rescued the premature sister chromatid separation in HeLa cells with ASXL1 KD. Data are represented as means ± SEM from three independent experiments. *** P < 0.001 and ** P < 0.01. ( F and G ) SMC1A or RAD21 KD leads to premature sister chromatid separation in HeLa GFP-H2B cells. Representative photomicrographs show the cells with premature sister chromatid separation, as indicated by red arrowheads (G). The frequency of cells with premature sister chromatid separation is shown in (F). Y axis shows the percentage of cells with premature sister chromatid separation within all binucleated cells. Data are represented as means ± SEM from three independent experiments. *** P < 0.001 and ** P < 0.01. Scale bars, 5 μm. ( H ) Western blotting shows the expression of full-length ASXL1 and ASXL1 amino acids 401 to 587 in HeLa GFP-H2B cells transfected with vector only, full-length ASXL1, or ASXL1 amino acids 401 to 587. β-Actin serves as loading control. ( I and J ) ASXL1 amino acids 401 to 587 induce chromatin bridging in HeLa GFP-H2B cells. Quantification of the frequency of cells with premature sister chromatid separation in HeLa GFP-H2B cells transfected with pcDNA3.1 + , full-length ASXL1, or ASXL1 amino acids 401 to 587 (J). Data are represented as means ± SEM from three independent experiments. ** P < 0.01 for ASXL1 amino acids 401 to 587 fragment versus pcDNA3.1 + or full-length ASXL1. Red arrows indicate the premature sister chromatid separation. Scale bars, 5 μm.

Article Snippet: The shRNA plasmids of ASXL1 (TG306527), SMC1A (TL513033), and RAD21 (TL501846) were purchased from OriGene.

Techniques: Cell Culture, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Science Advances

Article Title: ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

doi: 10.1126/sciadv.1601602

Figure Lengend Snippet: ( A ) Venn diagram showing overlapping peaks between ASXL1, SMC1A, and RAD21 ChIP-seq in WT LK cells. ( B ) Genomic distribution of ASXL1/SMC1A/RAD21 triple overlapping ChIP peaks in WT LK cells. ( C ) The overlap analysis shows the peak reads in WT and Asxl1 −/− LK cells based on ASXL1/SMC1A/RAD21 overlapping peaks from WT LK cells. Zero base pair (bp) is defined as the peak of ASXL1 binding sites on the genome of WT LK cells. Decreased genomic cohesin complex occupancy is seen in Asxl1 −/− LK cells. The overlap peaks of SMC1A and RAD21 represent the cohesin occupancy on the genome. Comparison of the SMC1A/RAD21 overlapping peaks between WT and Asxl1 −/− LK cells identified 7833-peak loss and 1175-peak gain in the Asxl1 −/− LK cells. TSS, transcription start site. ( D ) The pie chart represents the percentage of genes with no cohesin occupancy change (remain) or cohesin loss (SMC1A and/or RAD21 peak loss) in Asxl1 −/− BM LK cells based on all ASXL1/SMC1A/RAD21 triple overlapping peaks of WT LK cells. ( E ) DNA recognition sequence of SMC1A and RAD21 in WT or Asxl1 −/− LK cells. The SMC1A and RAD21 recognized identical DNA motif as CTCF.

Article Snippet: The shRNA plasmids of ASXL1 (TG306527), SMC1A (TL513033), and RAD21 (TL501846) were purchased from OriGene.

Techniques: ChIP-sequencing, Binding Assay, Sequencing

Journal: Science Advances

Article Title: ASXL1 interacts with the cohesin complex to maintain chromatid separation and gene expression for normal hematopoiesis

doi: 10.1126/sciadv.1601602

Figure Lengend Snippet: ( A ) The heatmap shows the differentially expressed genes associated with loci of no changes in SMC1A and RAD21 occupancy in Asxl1 −/− BM LK cells. ( B ) The heatmap shows the differentially expressed genes associated with loss of SMC1A and/or RAD21 in Asxl1 −/− BM LK cells. ( C ) The GO analysis of the 237 up-regulated genes (of the ~1600 genes with loss of RAD21 and/or SMC1A occupancy in Asxl1 −/− LK cells) in Asxl1 −/− LK cells compared to the WT LK cells. ( D ) GO analysis of the 65 down-regulated genes (of ~1600 genes with loss of RAD21 and/or SMC1A occupancy in Asxl1 −/− LK cells) in Asxl1 −/− LK cells compared to the WT LK cells. The P values of each GO term are represented by the red dots, and the gene set counts are represented by the bars. ( E ) Heatmap of differentially expressed genes of myeloid malignancy relevance within Asxl1/SMC1/RAD21 overlapping loci in Asxl1 −/− LK cells. ( F and G ) Genome browser tracks of the Cbfb and Fus locus with overlapping ASXL1, SMC1A, or RAD21 peaks. ( H ) Relative RNA level of Cbfb, Fus, and Stat3 in LK cells as determined by qPCR. Data are represented as means ± SEM from three independent experiments. *** P < 0.001 and ** P < 0.01.

Article Snippet: The shRNA plasmids of ASXL1 (TG306527), SMC1A (TL513033), and RAD21 (TL501846) were purchased from OriGene.

Techniques:

Journal: BMC Microbiology

Article Title: Alterations of the vaginal microbiome in healthy pregnant women positive for group B Streptococcus colonization during the third trimester

doi: 10.1186/s12866-022-02730-8

Figure Lengend Snippet: Distribution of samples by GBS status across community states (CST)

Article Snippet: L. iners dominant CST-III was the dominant community state representing 52.2% (23/44), this was followed by L. crispatus dominant CST-I (6/44, 13.6%), L. johnsonii dominant CST-Other (5/44, 11.3%), CST-VII (4/44, 9%), L. jensenii dominant CST-V (2/44, 4.5%), and finally L. gasseri dominant CST-II (1/44, 2.2%) and Lactobacillus -poor CST-IV (1/44, 2.2%).

Techniques: