Journal: bioRxiv

Article Title: A basement membrane discovery pipeline uncovers network complexity, new regulators, and human disease associations

doi: 10.1101/2021.10.25.465762

Figure Lengend Snippet: a , Dot plot depicts conserved and enriched Interpro protein domains in BM zone components. Dots are sized according to number of domain occurrences within BM genes (count ratio, Supplementary table 3 ). b , A domain-based interactome for human BM zone genes. Nodes represent BM and CSI proteins and are sized according to network degree score. Lines connecting nodes are color-coded to indicate the number of shared domains. Black and red arrowheads highlight hub proteins with the highest degree and betweenness centrality scores respectively ( Methods and Supplementary table 11 ). c , Domain structure of the hub protein HSPG2/perlecan. Bar chart shows the number of BM zone proteins sharing specific domains with HSPG2. d , Left, confocal sum projections of C. elegans gonadal BM LAMC/LAM-2::mNG in control vs. HSPG2 /unc-52 RNAi-treated 72-h adult animals (n = 10 animals examined each). Top right, middle-plane confocal z-slice of HSPG2/UNC-52::mNG in an adult animal, yellow arrowhead indicates fluorescence signal in the gonadal BM; bottom right, quantification of gonadal BM UNC-52::mNG levels throughout post-embryonic development below (n ≥ 13 for each developmental stage; fluorescence was not detected between early L1 and early L4 stages). Scale bar represents 25 μm. For boxplots, edges indicate the 25 th and 75 th percentiles, the line in the box represents the median, and whiskers mark the minimum and maximum values.

Article Snippet: Boxplots were prepared in GraphPad Prism 7.

Techniques: Control, Fluorescence

Journal: bioRxiv

Article Title: A basement membrane discovery pipeline uncovers network complexity, new regulators, and human disease associations

doi: 10.1101/2021.10.25.465762

Figure Lengend Snippet: a , Heatmap summarizing changes in LAMC/LAM-2::mNG and COL4A1/EMB- 9::mRuby2 fluorescence upon target gene knockdown in C. elegans . b , Confocal middle-plane z-slices of gonadal BM LAMC/LAM-2::mNG and COL4A1/EMB-9::mRuby2 in control and RNAi-targeted adt-2 , dbl-1 , and sax-3 72-h adult animals (boxed regions magnified in insets) with quantifications of fluorescence intensity shown below (n ≥ 20 each). **** p -value < 0.0001, ns—not significant; one-way ANOVA with post-hoc Dunnett’s test. Scale bars: 25 μm. c , Left, brightfield images of 2-day post fertilization (dpf) zebrafish embryos injected with indicated gRNAs (boxed regions magnified in insets). Right, observed frequency of intracerebral haemorrhage (arrowheads). Scale bar represents 600 μm. d , Top left, schematic cross-section of a 5-dpf zebrafish embryo ( Biorender ); red box indicates imaged region. Bottom, confocal images of type IV collagen immunofluorescence in control gRNA , adamts3 gRNA , and robo1 gRNA -injected 5-dpf embryos; fluorescence intensity within dashed boxes is quantified on the top right (n = 5 for each treatment). *** p -value < 0.001, ns—not significant; one-way ANOVA with post-hoc Dunnett’s test. Scale bar represents 30 μm. e , Collagen IV immunofluorescence in 5-dpf embryos treated with DMSO (control) or SB431542 (TGFBR1 inhibitor) on the left; quantification of fluorescence intensity within dashed boxes on the right (n = 5 each). *** p- value < 0.001, unpaired Student’s t test. Scale bar represents 30 μm. For boxplots, edges indicate the 25 th and 75 th percentiles, the line in the box represents the median, and whiskers mark the minimum and maximum values.

Article Snippet: Boxplots were prepared in GraphPad Prism 7.

Techniques: Fluorescence, Knockdown, Control, Injection, Immunofluorescence

Journal: bioRxiv

Article Title: A basement membrane discovery pipeline uncovers network complexity, new regulators, and human disease associations

doi: 10.1101/2021.10.25.465762

Figure Lengend Snippet: a , MATN2 genomic structure indicating four pLoF variants (top) and associated phenotypic abnormalities represented as HPO terms (bottom, bolded). b , Western blots of MATN2 in lysate and ECM fractions derived from endogenous MATN2-depleted human podocytes over-expressing V5-tagged wild-type or variant MATN2. c , In vitro minigene splicing assay ( Methods ) demonstrating altered splicing of MATN2 variant 1. d , Fold change in BM component levels upon MATN2 knockdown as determined by fractional proteomic analysis of podocyte-derived ECM. e , MPZL2 genomic structure indicating two 100KGP pLoF variants (top) and associated phenotypic abnormalities (bottom, bolded). f , Brightfield images of tail regions (dashed lines) in control gRNA and mpzl2b gRNA -injected 4-dpf zebrafish embryos. Arrowheads highlight reduced fin fold extension in mpzl2b crispants. Scale bar represents 100 µm. g , Transmission electron microscopy (TEM) of gut and kidney BMs (yellow arrowheads) in control and mpzl2b crispant embryos. Red arrows indicate BM irregularities. Scale bar represents 2 µm (gut), 1 µm (kidney). h , Quantification of gut (n = 39 each) and kidney (n = 100 each) BM thickness . **** p -value < 0.0001, ns—not significant; unpaired Student’s t test. i , Assessment of proteinuria (NL-D3 levels) in mpzl2b crispants injected with wild-type human MPZL2 mRNA or MPZL2 c.72del variant mRNA (n = 24 each). *** p -value < 0.001, ** p -value < 0.01; ns—not significant; one-way ANOVA with post-hoc Dunnett’s test. For boxplots, edges indicate the 25 th and 75 th percentiles, the line in the box represents the median, and whiskers mark the minimum and maximum values.

Article Snippet: Boxplots were prepared in GraphPad Prism 7.

Techniques: Western Blot, Derivative Assay, Expressing, Variant Assay, In Vitro, Splicing Assay, Knockdown, Control, Injection, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: A basement membrane discovery pipeline uncovers network complexity, new regulators, and human disease associations

doi: 10.1101/2021.10.25.465762

Figure Lengend Snippet: a , Top, genetic pedigree for a 100KGP family carrying two LAMA5 pLoF variants. Bottom, phenotypic abnormalities observed in two LAMA5 variant-carrying fetuses (19 and 20 week gestation) represented as bolded HPO terms. b , Top, LAMA5 genomic structure and variant locations. Bottom, in vitro minigene splicing assay demonstrating altered splicing of LAMA5 variant 1b. c , Left, Picrosirius red/fast green staining of the 19-week dysplastic kidney; asterisks indicate cysts. Boxed area is magnified on the right; arrowhead and arrows denote abnormal glomerulus and tubules, respectively. Scale bars represent 5 mm (left), 100 µm (right). d , Pan-laminin (LAM, magenta) and laminin β2 (LAMB2, green) immunofluorescence in wild-type (left) and dysplastic (right) fetal kidney sections. Asterisk indicates laminin surrounding a cyst. Boxed regions are magnified below. Arrowheads, see text. Scale bar represents 50 µm. e , Brightfield images of zebrafish tail regions (dashed lines) in control gRNA and lama5 gRNA -injected 4-dpf embryos. Arrowheads highlight reduced fin fold extension in lama5 crispants. Scale bar represents 100 µm. f , TEM of gut and kidney BMs (yellow arrowheads) in control and lama5 crispants. Boxed regions are magnified in insets. Red arrows indicate BM morphological irregularities. Scale bars represent 2 µm (gut), 1 µm (kidney). g , Proteinuria in control versus lama5 crispants (n = 24 each). *** p -value < 0.001, unpaired Student’s t test. For boxplots, edges indicate the 25 th and 75 th percentiles, the line in the box represents the median, and whiskers mark the minimum and maximum values.

Article Snippet: Boxplots were prepared in GraphPad Prism 7.

Techniques: Variant Assay, In Vitro, Splicing Assay, Staining, Immunofluorescence, Control, Injection

Journal: Epigenetics & Chromatin

Article Title: Impact of 3D genome organization, guided by cohesin and CTCF looping, on sex-biased chromatin interactions and gene expression in mouse liver

doi: 10.1186/s13072-020-00350-y

Figure Lengend Snippet: Cohesin and CTCF ChIP-seq binding strength and proximity to genes. a Box plots of normalized ChIP-seq signal for the peak sets indicated on the x -axis. Peaks with sex differential binding for cohesin ( top graph ) and CTCF (b ottom graph ) are shown. Each pair of boxplots represents the male and female ChIP-seq signal for the same set of peaks, defined by their sex bias and peak type (CAC or CNC, for ΔCohesin peaks; and CAC or Lone CTCF, for ΔCTCF peaks), as indicated below the x -axis. Peak scores were calculated by average intra-peak ChIP signal, normalized by total sequence reads per million in peak (RIPM; see “ ”). Female-biased peaks were, on average, stronger than male-biased peaks by M–W test: p ≤ 0.001 for female vs male CAC(ΔCoh), CAC(ΔCTCF), and for CNC, but not for Lone CTCF peaks. b Distance from each indicated set of cohesin and CTCF peaks to the nearest enhancer DHS. Cumulative frequency curves indicate the fraction of each group on the y -axis, within the distance in kb to the nearest enhancer DHS indicated on the x -axis. Enhancer DHS were defined based on their high ratio of the enhancer histone mark H3K4me1 over the promoter mark H3K4me3 at DHS . Sex-biased CNC peaks are closer to enhancer DHS (median distance to eDHS of 0.22 kb for male-biased CNCs and 0.12 kb for female-biased CNCs; KS pval < 0.0001 for all comparisons) than the other CTCF and cohesin peak classes (M CAC(ΔCTCF): 14.98 kb; F CAC(ΔCTCF) 13.76 kb; M Lone ΔCTCF: 13.88 kb; F Lone ΔCTCF: 7.17 kb). Female-biased CNC peaks are significantly closer to enhancer DHS than are male-biased CNC peaks ( p = 0.0351; KS t -test). Male-biased CAC(ΔCohesin) peaks were closer to enhancers than female-biased CAC(ΔCohesin) peaks ( p = 0.002; KS t -test), however, the reverse was found for CAC(ΔCTCF) peaks ( p = 0.0052; KS t -test). Distance to nearest enhancer was not significantly different between male-biased and female-biased Lone CTCF peaks ( p = 0.1068; KS t -test). P values for comparisons between male-biased and female-biased peaks of the same class are shown for each plot (KS t -test). c Distance from each indicated set of cohesin and CTCF peaks to the nearest TSS. Cumulative frequency curves indicate the fraction of each group on the y -axis within the distance in kb to the nearest TSS indicated on the x -axis. TSS for protein coding (RefSeq) and liver lncRNA genes were considered . Female-biased cohesin and CTCF peaks are closer to TSS than male-biased CTCF and cohesin peaks of the same class (significance by KS t-test is indicated at top left of each plot). Distance to the TSS was not significantly different for male-biased versus female-biased CNC peaks ( p = 0.1458; KS t -test). d Proximity of sex-biased cohesin and CTCF binding sites to sex-biased genes. Peak designations were as follows: Proximal, peaks < 20 kb from a sex-biased gene TSS; Intra-TAD, peaks within the same intra-TAD loop as a sex-biased gene; or TAD, peaks in the same TAD as a sex-biased gene. Each of these groups is mutually exclusive. TAD loop and intra-TAD loop coordinates were from the indicated references. A set of 983 sex-biased biased protein-coding genes was used in this analysis (see Additional file : Table S1 of ). e Cumulative frequency curves show the fraction of each group ( y -axis) within the distance in kb to the nearest sex-biased DHS or H3K27ac genomic region ( x -axis), based on a merged list of published sex-biased DHS and sex-biased H3K27ac ChIP-seq peaks for male and female mouse liver. For this analysis, CAC peaks with sex-biased binding of CTCF and cohesin were combined and presented as a single group [CAC (Both)]. Male-biased and female-biased CNC peaks are significantly closer to sex-biased DHS/H3K27ac than the four other peak classes ( p < 0.001; KS t -test). Female-biased CNC peaks were significantly closer to sex-biased DHS/H3K27ac than male-biased CNC peaks ( p = 0.0094; KS)

Article Snippet: Boxplots, cumulative distribution plots, and statistical analyses were implemented using GraphPad Prism 7.

Techniques: ChIP-sequencing, Binding Assay, Sequencing