EMBO/soda-nel huggingface dataset:
Piwi-piRNA complexes induce stepwise changes in nuclear architecture at target loci (2021)
Figure 1

Figure 1. Piwi-depletion results in decreased chromatin-associated Lamins.

(A) ChEP analysis using EGFP (control)- or Piwi-KD OSCs. Signals with an adjusted p-value (Bonferroni test) of less than 0.05 are highlighted in red. The signals for Lam, LamC, and H1 are labelled within the plots.

(B) Gene ontology terms related to proteins with a significant change upon Piwi-KD using ChEP analysis (adjusted p-value <0.05). Gene ontology terms from the biological process (blue), cellular component (orange), and molecular function (green) categories are listed along with their p-values (Fisher's Exact test, -log10).

(C) Western blot showing Piwi, Lam, LamC, and Tubulin (loading control) protein levels upon EGFP (control)-, Piwi-, Lam-, or LamC-KD. Note that KD of Lam or LamC is not complete since their depletion causes serious cell damage.

(D) Immunofluorescence of OSCs transfected with indicated siRNAs, using Lam and LamC antibodies (green). DAPI staining (gray) shows the location of nuclei. Scale bar: 5μm.

(E) Lam- or LamC-KD followed by qRT-PCR of RNA levels for piRNA target (mdg1 and gypsy) or non-target (roo) TEs. Error bars indicate SD (n=3). * = p-value<0.05 (t-test). Note that the complete depletion of Lam and LamC is impossible since their KD causes severe damage to cells.
Piwi-piRNA complexes induce stepwise changes in nuclear architecture at target loci (2021)
Figure 3

Figure 3. Piwi-depletion causes a decrease in intra-TAD interactions at piRNA target TE-insertion sites.

(A) Hi-C interaction matrices of the Control (EGFP)-KD and Piwi-KD OSCs at Chromosome 3L (40k resolution).

(B) Differential interaction heatmap for chromosome 3L (40k resolution), showing increased (red) and decreased (blue) interacting bins in log2 fold change upon Piwi-KD.

(C) Differential interaction heatmap for 16-19Mbp of chr3L shown in log2 fold change. piRNA target TE insertions are indicated at the bottom (green filled triangles), along with TADs defined using Hi-C interactions of Control (EGFP)-KD OSCs (black triangles).

(D) Differential intra-TAD interaction density score upon Piwi-KD (upper), centering TE insertions. Heatmap showing changes in the intra-TAD interaction score of each piRNA target TE insertion sites (lower). The results are shown for piRNA target TE and non-piRNA target TE insertions.

(E) Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in intra-TAD interactions upon Piwi-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89).

(F) Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in inter-TAD interactions upon Piwi-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89).

(G) Oligo-FISH images for changes in the intra-TAD interaction at the TAD described in (C), upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, green and magenta indicates FISH signal at the different positions of TADs (TAD-1 and TAD-2). Details of TAD probe design is described in Fig EV2C. Scale bar, 5μm. Distance between two FISH signals is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.

(H) Diagram illustrating the possible loss of the intra-TAD interaction for TADs harboring piRNA target TEs.

(I) Differential interaction heatmap for 16-19Mbp of chr3L shown in log2 fold change. TADs defined using Hi-C interactions of EGFP-KD (control, upper right) or Piwi-KD (lower left) OSCs are overlaid on the heatmap.

(J) Venn diagram displaying the number of TAD boundaries detected using EGFP- or Piwi-KD Hi-C interactions.

(K) Dot plot showing the correlation between the change in coverage of LADs for each TAD (x-axis), and changes in intra-TAD interactions upon Piwi-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89).
Piwi-piRNA complexes induce stepwise changes in nuclear architecture at target loci (2021)
Figure 4

Figure 4. Chromatin state is associated with changes in LADs and TADs.

(A) Differential interaction heatmap for Piwi- and Control (EGFP)-KD shown in log2 fold change. Hi-C data is shown with RNA levels (RNA-seq signal density), H3K9me3 association (H3K9me3 ChIP-seq signal density), H3K27Ac association (H3K27Ac ChIP-seq signal density), H3K4me3 association (H3K4me3 ChIP-seq signal density), H1 association (H1 ChIP-seq signal density), and lamin association (Lamin DamID-seq signal density). The genomic region flanking the gypsy insertion (red dashed line) is shown for OSCs with EGFP- and Piwi-KD. FlyBase protein-coding genes are indicated, together with chromosome position.

(B) Boxplots showing fold changes in the histone mark association of piRNA target or non-target TEs based on ChIP-seq upon Piwi-KD (Definition of piRNA target and non-target TEs are described in Fig EV1E). Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated by Wilcoxon rank-sum test.

(C) Dot plot showing the correlation between changes in Lamin DamID signal upon Piwi-KD (x-axis) and changes of indicated ChIP-seq signal in each LADs upon Piwi-KD (y-axis). The trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89).

(D) Dot plot showing the correlation between changes in intra-TAD interactions upon Piwi-KD (x-axis) and changes of indicated ChIP-seq signal in each TADs upon Piwi-KD (y-axis). The trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89).

(E) ChIP‐qPCR analysis of H3K27Ac and H3K4me3 occupancy on the reporter gene upon transfection of λN-Nxf2 expression vector or control EGFP expression vector. Bar graph shows the occupancy relative to that of the sample transfected with a control plasmid. Error bars indicate SD (n = 3), * = p-value<0.01 (t-test)..
Piwi-piRNA complexes induce stepwise changes in nuclear architecture at target loci (2021)
Figure 6

Figure 6. Downstream factors, H1, Egg, and HP1a, cooperatively regulate chromatin conformation in the Piwi-piRNA pathway.

(A) Differential interaction heatmap for chromosome 3L (40k resolution) in log2 fold change, showing increased (red) and decreased (blue) interacting bins upon H1(left)-, Egg(middle)-, or HP1a(right)-KD.

(B) Relative contact probability (RCP) plot showing corrected contact counts at different genomic ranges, genome widely. RCP values are compared between EGFP-KD (blue) and H1(left)-, Egg(middle)-, or HP1a(right)-KD OSCs.

(C) Differential intra-TAD interaction density score upon H1(left)-, Egg(middle)-, or HP1a(right)-KD (upper), centering TE insertions. Heatmap showing changes in the intra-TAD interaction score of each piRNA target TE insertion sites (lower). The results are shown for piRNA target TE (left) and non-piRNA target TE (right) insertions.

(D) Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in intra-TAD interactions upon H1(left)-, Egg(middle)-, or HP1a(right)-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). n.s.; not significant (p > 0.05)

(E) Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in inter-TAD interactions upon H1(left)-, Egg(middle)-, or HP1a(right)-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). n.s.; not significant (p > 0.05)

(F) Schematic model of stepwise Piwi-piRNA mediated regulation of nuclear architecture and chromatin state.

Expanded View Figure Legends
ADAR1 promotes robust hypoxia signaling via distinct regulation of multiple HIF1α-inhibiting factors (2019)
Figure 1

Fig 1. Characterization of the HIF1A-AS2 transcript and associated RNA editing events

(A) Relative expression levels of the HIF1A-AS2 transcript in the indicated cell lines under normoxia condition were monitored by qRT-PCR analysis and represented as 39 minus Ct values. (B) Sanger sequencing validation of RNA editing events on HIF1A-AS2. The editing frequencies of the indicated sites were quantitated based on three independent experiments and shown in Table EV 1. Asterisks mark the sites undergoing statistically significant increases in RNA editing. (C) The distributions of the confirmed HIF1A-AS2 RNA editing sites (red ticks) are shown in UCSC Genome Browser representation, in relation to the HIF1A-AS2 gene locus. The transcript structure of the HIF-1A, HIF1A-AS2, and HIF1A-AS(m) (a splicing form of HIF1A-AS2) genes are also presented, with the identified exons indicated by blue boxes. (D) Secondary structure of the full-length HIF1A-AS2 transcript was predicted by using the online database RNAfold. The region encompassing the double-stranded structure is shown with the RNA editing sites, as indicated. (E) RNA-IP of ADAR1 in HUVECs, followed by real-time PCR analysis of the HIF-1A and HIF1A-AS2 RNAs. The relative enrichment of transcript association was normalized to the IgG. GAPDH was used as a non-binding control. (F) & (G) Cells were cultured under normal or hypoxic condition. Relative levels of the HIF1A-AS transcripts in total vs. poly(A)+ RNA fractions (F), or in subcellular fractions (G), were determined by qRT-PCR. (H) FISH analysis of the spatial distribution of HIF-1A mRNA (red) and HIF1A-AS2 transcripts (white) in HUVECs subjected to mock or CoCl2 treatment for 5 hrs. Nuclei were counterstained by DAPI. Scale bars, 10 μm. For graphs shown in this figure, means ± S.D. calculated from three independent experiments are shown. P-values were calculated by two-tailed Student's t-test (n.s., not significant or P>0.05; *P<0.05; **P<0.01; ***P<0.001).
ADAR1 promotes robust hypoxia signaling via distinct regulation of multiple HIF1α-inhibiting factors (2019)
Figure 2

Fig 2. Hypoxia-induced transcription of HIF1A-AS2 triggers HIF-1A transcriptional silencing Expression of HIF1A-AS2 was suppressed in HUVECs by oligo phosphodiester nucleotides (ODN)-based knockdown. Cells were then treated with CoCl2 for 5 hrs to mimic hypoxic condition, and subjected to gene expression analyses: The RNA abundance of HIF-1A and HIF1A-AS2 was analyzed by real-time PCR and normalized by 18s rRNA (A). HIF-1α protein level was monitored by western blot, with β-actin as the internal control (B). Nuclear run-on assays were performed to assess the extent of nascent transcription of target genes under mock vs. CoCl2 treatment (C) or in the HIF1A-AS2 knockdown cells (D). HIF-1A mRNA expression was detected in a region-specific manner (5' vs. 3') by primers corresponding to exons 1-2 (E1-2) and exon 14-15 (E14-15). To further monitor transcription activity, ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells under CoCl2 treatment (E), or in HIF1A-AS2-knockdown vs. control cells (F). The precipitated DNA fragments were quantitatively analyzed by real-time PCR using primers for the HIF1A-AS2 and HIF-1A promoters, and normalized to the values of IgG. HIF1A-AS2 corresponds to the transcribed region. (G) Schematic of the convergent mode of transcriptional regulation of the HIF-1A sense and antisense gene pair. For graphs shown in this figure, means ± S.D. calculated from three independent experiments are shown. P-values were calculated by two-tailed Student's t-test (n.s., not significant or P>0.05; *P<0.05; **P<0.01; ***P<0.001).
ADAR1 promotes robust hypoxia signaling via distinct regulation of multiple HIF1α-inhibiting factors (2019)
Figure 3

Fig 3. Roles of ADAR1 in the regulation of HIF-1A/HIF1A-AS2 expression under hypoxia

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. (A) The expression levels of ADAR1, HIF-1A and HIF1A-AS2 transcripts were analyzed by qRT-PCR. (B) Nuclear run-on assay was done to measure the newly synthesized HIF-1A and HIF1A-AS2 RNAs in ADAR1-knockdown cells, followed by real-time PCR analysis of the indicated transcripts. (C) ChIP analysis of RNA polII binding on chromatin region of the HIF1A-AS2 and HIF-1A promoters. The extent of occupancy was compared between control and ADAR1-knockdown cells. The precipitated DNA fragments were quantitatively analyzed by real-time PCR and normalized by the values of IgG. (D) Chromatin binding of ADAR1 to HIF1A-AS2 and HIF-1A promoters under nomoxia (20% O2) vs. hypoxia (1% O2, 5 hrs) conditions, as assessed by ChIP. (E) The 3'-end extension of HIF1A-AS2 RNA transcription in ADAR1 knockdown cells (GFP, control) was examined by PCR with primers corresponding to the depicted regions of HIF1A-AS2 locus. GAPDH serves as an internal control. Sequence locations are shown in kbp and relative to the transcription start site. (F) & (G) Expression levels of HIF-1α protein (F) and HIF-1α downstream genes (G) in the indicated experimental conditions were analyzed respectively by western blot and real-time PCR, as indicated. qRT-PCR results were normalized by the expression of 18s rRNA. For graphs shown in this figure, means ± S.D. calculated from three independent experiments are shown. P-values were calculated by two-tailed Student's t-test (n.s., not significant or P>0.05; *P<0.05; **P<0.01; ***P<0.001).
ADAR1 promotes robust hypoxia signaling via distinct regulation of multiple HIF1α-inhibiting factors (2019)
Figure 4

Fig 4. ADAR1 antagonizes HIF1A-AS2-dependent suppression of HIF-1A

(A) Transcriptome-wide changes in the cells subjected to CoCl2 treatment, HIF1A-AS2 or ADAR1 knockdown were profiled by RNA-Sequencing. For genes that undergo up-regulation upon CoCl2 treatment (n = 505), overall expression profiles of the different cell groups are represented by the heatmap. The expression values (represented by normalized read counts) were displayed in shades of red or blue (linear scale) relative to the means of all corresponding values within individual experimental groups. Clusters of genes (based on unsupervised hierarchical clustering) are indicated and denoted by their experimental types/conditions. (B) GSEA comparing the gene expression profile by gene up-regulated upon CoCl2 treatment and those down-regulated in ADAR1 knockdown. The curves correspond to the observed enrichment score profiles in the CoCl2 gene set to the ADAR1 knockdown gene sets. The bar‐code plot below enrichment score plot denote the rank of each member in the CoCl2 up-regulated gene list. (C) The Venn diagram shows the degree of overlap between genes up-regulated by CoCl2 treatment and those down-regulated in the ADAR1-depletion and CoCl2 -treated cells. (D) The 238 targets shared by the above two gene sets are enriched in genes implicated in apoptosis, cell growth, angiogenesis, and hypoxia response pathways, as revealed by bioinformatics analysis using PANTHER classification system (P-value <0 .05) and false discovery rate < 0.05). (E) & (F) HUVECs were transfected with the indicated combinations of siRNA (GFP as control versus ADAR1) and ODN (GFP as control versus HIF1A-AS2), followed by CoCl2 treatment for 5 hrs. The expression levels of ADAR1 and HIF-1α protein (E) were analyzed respectively by immunoblotting. (F) For cells in (E), RNA expression of heat shock protein-encoding genes (HSPA1A, HSPA1B, and HSPA6) with reported roles in angiogenesis were profiled by qRT-PCR. (G) Montage of representative images and quantification of 2D capillary network formation assays on HUVECs subjected to the above experimental manipulation. Scale bars, 2000 μm. For graphs shown in this figure, means ± S.D. calculated from three independent experiments are shown. P-values were calculated by two-tailed Student's t-test (n.s., not significant or P>0.05; *P<0.05; **P<0.01; ***P<0.001).
ADAR1 promotes robust hypoxia signaling via distinct regulation of multiple HIF1α-inhibiting factors (2019)
Figure 5

Fig 5. ADAR1 regulates LIMD1 mRNA subcellular distribution and consequently its protein translation

Sanger sequencing validation of RNA editing of the LIMD1 (A) and VHL (B) transcripts. Downregulation of ADAR1 levels in MCF7 cells was done using specific siRNAs. Cells were then subjected to hypoxia treatment for 5 hrs. The extent of knockdown and gene alterations was monitored by western blotting using antibodies targeting LIMD1, VHL, PHD1, PHD2, ADAR1 and HIF-1α, as indicated (C). β-actin was used as loading control. (D) To characterize subcellular distribution of target transcripts, real-time PCR analysis was done on the fractionated MCF7 cells with the indicated experimental treatments. (E) Translation efficiency was determined by quantifying transcript association with the polyribosome fraction, using ADAR1-depleted MCF7 cells with hypoxia treatment. The abundance of LIMD1 in polysomes was analyzed by real-time PCR and normalized by the eEF1α levels. (F) To inspect HIF-1α protein abundance and modification with ubiquitin, anti- HIF-1α, anti-ubiquitylation (Ub), and anti-VHL immunoprecipitation experiments were performed, coupled with immunoblotting detection of ubiquitylation and HIF-1α, as indicated. MCF7 cells transfected with ADAR1 siRNA and treated with hypoxia were used. Protein signals were quantified using the ImageJ software. The fold changes of protein levels were determined on the basis of immunoreactive signals normalized by the input (In), with the control (siGFP) group being represented as 1. For graphs shown in this figure, means ± S.D. calculated from three independent experiments are shown. P-values were calculated by two-tailed Student's t-test (n.s., not significant or P>0.05; *P<0.05; **P<0.01; ***P<0.001).
Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease (2021)
Figure 2

Figure 2: Microbiota-dependent alterations in the microglial genetic profile.

(A) UMAP plot illustrating the color-coded aggregate transcript counts of published core signature genes for microglia. The scale bar represents color coded sequencing depth-corrected log transformed transcript counts.

(B) UMAP representation of 828 individual microglial cells from brain parenchyma derived from SPF (green dots) and GF (yellow dots).

(C) UMAP representation of microglial cells of SPF and GF mice color-coded for the results of Seurat clustering. The legend on the right hand side of the plot shows the color-cluster mapping.

(D) Stacked bar plot (Marimekko chart) color-coded for the relative cluster-wise contribution of microglial cells derived from SPF (green) and GF (yellow) mice. Ranges of p-values are provided for significantly enriched regions per cluster, based on hypergeometric tests. Benjamini-Hochberg correction was applied to correct for multiple testing (*P < 0.05; **P < 0.01; ***P < 0.001).

(E) Volcano plot of the differential gene expression analysis of n=318 SPF and 510 GF cells based on Wilcoxon Rank Sum tests. The shown genes are filtered based on an average log2 fold change (avg. log2FC) > 0.2 and adjusted p-value < 0.05.

(F) Heatmap of the top 10 differentially expressed genes per cluster (adjusted p-value of <0.05 based on Wilcoxon Rank Sum tests). The scale bar represents color-coded Pearson residuals.

(G) UMAP maps color-coded for transcript counts of genes enriched in subsets of microglial cells. The scale bars represent color coded sequencing depth-corrected log transformed transcript counts.

(H) Representative immunofluorescence images of Iba1 (green) and APOE (red) on cortical sections from SPF and GF mice. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. White arrowheads indicate APOE+/Iba1+ microglia and arrows show APOE-/Iba1+ microglia. Quantification of APOE+/Iba1+ microglia. Each symbol represents one mouse. At least three slides were examined per individual mouse. Data are presented as mean ± s.e.m. Significant differences were determined by unpaired t-test and marked with asterisks (*P < 0.05). Data are representative of two independent experiments.
Commensal microbiota divergently affect myeloid subsets in the mammalian central nervous system during homeostasis and disease (2021)
Figure 3

Figure 3: Choroid plexus macrophages properties are controlled by complex microbiota.

(A) UMAP plot illustrating the color-coded aggregated transcript counts of published core signature genes for CAMs and microglia. The scale bars represent color coded sequencing depth-corrected log transformed transcript counts.

(B) UMAP representation of 487 individual choroid plexus cells from lateral and fourth ventricles of SPF (green dots) and GF (yellow dots) mice.

(C) UMAP representation of the choroid plexus cells of SPF and GF mice color-coded for the Seurat cluster assignment. The color-clusters mappings are indicated on the right hand side of the plot. The cell type annotations are based on published gene expression profiles.

(D) Stacked bar plot (Marimekko chart) presenting the color-coded relative contribution of choroid plexus cells derived from SPF (green) and GF (yellow) mice. Ranges of p-values are provided for significantly enriched regions per cluster, based on hypergeometric tests. Benjamini-Hochberg correction was applied to correct for multiple testing (*P < 0.05; **P < 0.01).

(E) Volcano plots of the differential gene expression analysis of n=51 SPF and 136 GF stromal cpMΦs (left) and n= 85 SPF and 134 GF Kolmer epiplexus cell (CPepi) cells (right) based on Wilcoxon Rank Sum tests. The shown genes are filtered based on an avg. log2FC > 0.2 and adjusted p-value < 0.05.

(F) Heatmap of the top 10 differentially expressed genes per cluster (adjusted p-value of <0.05 based on Wilcoxon Rank Sum tests). The scale bar represents color-coded Pearson residuals.

(G) UMAP maps color-coded for transcript counts of genes enriched in subsets of choroid plexus cells. The scale bars represent color coded sequencing depth-corrected log transformed transcript counts.

(H) Representative Iba1+ immunohistochemistry in the choroid plexus of SPF, GF, ABX-treated, ASF, recolonized ASF, sDMDMm2, TLR2,3,4,7,9-deficient, SCFA-treated GF mice or FFAR2-deficient mice. Scale bar = 50 µm.

(I-O) Quantification of stromal CPMΦ. Each symbol represents one mouse. Three to four sections per mouse were examined. Means ± s.e.m. are indicated. Significant differences were evaluated by unpaired t-test or one-way ANOVA followed by Tukey's post-hoc comparison test and marked with asterisks (*P < 0.05, **P < 0.01, ***P < 0.001). Data are representative of at least two independent experiments.