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: 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: