grna sequences targeting hif1a  (Addgene inc)

Journal: bioRxiv

Article Title: Hypoxia promotes airway differentiation in the human lung epithelium

doi: 10.1101/2024.08.09.607336

Figure Lengend Snippet: (A) Diagram of HRE-ODD-GFP reporter construct and oxygen-dependent GFP turnover. (B) Microscope images of merged brightfield and GFP channels. The HRE-ODD-GFP reporter progenitors were cultured under normoxia for 6 days to form organoids, then treated with normoxia or hypoxia for 10 days without passaging. Control cells were cultured under normoxia with routine passaging. Scale bars = 600 µm. (C) The percentage of organoids containing ≥ 1 GFP + cell(s). Data shown as mean ± SD, n = 7 (normoxia), 8 (hypoxia) experimental replicates from 2 biological donors. (D) Roxadustat (FG-4592) inhibits PHD enzymes under normoxia and stabilises HIFα subunits. (E) HIF1α and HIF2α were stabilised under normoxia by Roxadustat in 4 organoid lines with β-actin as loading control. (F) Roxadustat treatment activated the HIF pathway under normoxia and recapitulated hypoxia-induced differentiation. RT-qPCR detection of organoids cultured in SRM ± Roxadustat for 30 days. Fold changes were normalised to the mean of SRM – Roxadustat (with DMSO) condition. Data shown as Log 2 (fold change), n = 6 biological donors. Statistical comparisons by two-way ANOVA with Bonferroni’s multiple comparisons test. (G) Design of targeted DamID-seq for HIF1α and HIF2α. Dam-HIFα fusion proteins are expressed at a low level due to rare translation reinitiation events. The fusion proteins methylate adenines in the GATC sequences near their DNA binding sites. (H) Global distributions of HIF1α and HIF2α binding sites relative to the Transcriptional Start Site (TSS). (I) Quantification of HIF1α and HIF2α binding signals surrounding the TSS. HIF1α and HIF2α signals were normalised to Dam-only control. (J) Gene ontology analysis of HIF1a and HIF2a common target genes. (K) Venn diagram comparing HIF1α and HIF2α target genes with highlighted gene lists. (L) Gene track views showing averaged DamID signals and consensus peaks from three biological replicates over selected HIF1α and HIF2α binding and no binding genes. Gene expression was normalised to ACTB in RT-qPCR. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also .

Article Snippet: For mutated HIF1α and HIF2α overexpression, the HIF1A and HIF2A CDS were cloned from plasmids gifted from William Kaelin (Addgene, #87261, #25956) and inserted into Tet-ON vectors with EF1a-TagRFP-2A-tet3G., For CRISPRi, the gRNA sequences targeting HIF1A , HIF2A , KLF4 , KLF5 were selected from published dataset and inserted into U6-gRNA-EF1a-EGFP-CAAX lentiviral vectors (Addgene, #167936)., For targeted DamID, the wild-type HIF1A and HIF2A CDS were inserted into DamID vectors with SFFV-mNeonGreen as upstream open reading frame.

Techniques: Construct, Microscopy, Cell Culture, Passaging, Control, Quantitative RT-PCR, Binding Assay, Gene Expression

Journal: bioRxiv

Article Title: Hypoxia promotes airway differentiation in the human lung epithelium

doi: 10.1101/2024.08.09.607336

Figure Lengend Snippet: (A) HIF1α is inhibited by CRISPRi. The dCas9-KRAB effector was tagged with DHFR and stabilised in presence of TMP (Trimethoprim) to reduce leaky expression from the Tet-on promoter. HIF1α protein levels decreased after 4-9 day knock down under hypoxia as shown by Western blot. (B) Upper panel: experimental design. The non-targeting control (NTC) or HIF1A- knock down ( HIF1A- KD) organoids were cultured under normoxia or hypoxia for 30 days. The dCas9 was induced 2 days ahead of hypoxia treatment. Lower panel: RT-qPCR results. Fold changes were normalised to the average of NTC + normoxia condition (not shown). Bars represent mean Log 2 (fold change) ± SD, n = 4 experimental replicates from 3 biological donors. 2 gRNAs tested. (C) Immunostaining of organoids with NTC or HIF1A- KD induction for 30 days showed changes in organoid shape (Ecad: E-cadherin) and differentiation (ASCL1, KRT5). Representative images from 3 organoid lines. (D) The NTC and HIF1A -KD organoids were cultured under normoxia or hypoxia for 9 days and used for bulk RNA-seq with 2 gRNAs and 3 biological donors for each condition. Heatmap showing 7309 differentially expressed genes (DEGs) (|Log2(fold change)| > 0.5, Padj < 0.05, merged from DEGs in comparisons of hypoxia + NTC vs normoxia + NTC, and hypoxia + HIF1αKD vs hypoxia + NTC) across all samples, representative genes labelled. (E) and (F) GSEA results of 9621 DEGs ( Padj < 0.05) between hypoxia and normoxia NTC organoids (E), 5904 DEGs ( Padj < 0.05) between HIF1A- KD and NTC hypoxic organoids (F). (G) Stabilised form of HIF1α was induced by Tet-On system under normoxia with GFP as control. (H) Immunostaining of organoids overexpressing GFP or HIF1α for 30 days under normoxia. Representative images of 2 organoid lines. (I) and (J) HIF1α overexpression under normoxia induced HIF pathway genes (I) and differentiation genes (J). The fold changes normalised to the average of GFP-overexpression organoids. Data shown as Log 2 (fold change), n = 3 biological donors. Scale bars=100 µm. For RT-qPCR, Gene expression was normalised to ACTB . Statistical test: two-way ANOVA with Bonferroni’s multiple comparisons test. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also .

Article Snippet: For mutated HIF1α and HIF2α overexpression, the HIF1A and HIF2A CDS were cloned from plasmids gifted from William Kaelin (Addgene, #87261, #25956) and inserted into Tet-ON vectors with EF1a-TagRFP-2A-tet3G., For CRISPRi, the gRNA sequences targeting HIF1A , HIF2A , KLF4 , KLF5 were selected from published dataset and inserted into U6-gRNA-EF1a-EGFP-CAAX lentiviral vectors (Addgene, #167936)., For targeted DamID, the wild-type HIF1A and HIF2A CDS were inserted into DamID vectors with SFFV-mNeonGreen as upstream open reading frame.

Techniques: Expressing, Knockdown, Western Blot, Control, Cell Culture, Quantitative RT-PCR, Immunostaining, RNA Sequencing, Over Expression, Gene Expression

Journal: bioRxiv

Article Title: Hypoxia promotes airway differentiation in the human lung epithelium

doi: 10.1101/2024.08.09.607336

Figure Lengend Snippet: (A) RT-qPCR control of samples used for bulk RNA-seq. NTC and HIF1A -knock down ( HIF1A- KD) organoids were cultured under normoxia or hypoxia for 9 days. Fold changes were normalised to the mean of NTC + normoxia condition. Bars represent mean Log 2 (fold change) ± SD, n = 8 experimental replicates from 4 biological donors with 2 gRNAs. For bulk RNA-seq, 6 replicates (3 biological donors with 2 gRNAs) for each condition were selected. Statistical test: two-way ANOVA with Tukey’s multiple comparisons test. (B) Heatmap of 655 DEGs related to GSEA terms enriched in hypoxia compared to normoxia NTC organoids. Every 1 in 4 genes are labelled due to space limitations. (C) Heatmap of 345 DEGs related to GSEA terms enriched in HIF1A- KD compared to NTC hypoxic organoids. Every 1 in 2 genes were labelled due to space limitations. (D) RT-qPCR of NTC and HIF1A- KD organoids cultured in airway differentiation medium (AWDM) under normoxia for 15 days. The fold changes were normalised to the mean of normoxia + SRM condition (not shown). Bars represent Log 2 (fold change) ± SD, n = 5 (NTC), 6 ( HIF1A- KD) experimental replicates from 3 biological donors with 2 gRNAs. Statistical test: two-way ANOVA with Bonferroni’s multiple comparisons test. Gene expression was normalised by ACTB in RT-qPCR. Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For mutated HIF1α and HIF2α overexpression, the HIF1A and HIF2A CDS were cloned from plasmids gifted from William Kaelin (Addgene, #87261, #25956) and inserted into Tet-ON vectors with EF1a-TagRFP-2A-tet3G., For CRISPRi, the gRNA sequences targeting HIF1A , HIF2A , KLF4 , KLF5 were selected from published dataset and inserted into U6-gRNA-EF1a-EGFP-CAAX lentiviral vectors (Addgene, #167936)., For targeted DamID, the wild-type HIF1A and HIF2A CDS were inserted into DamID vectors with SFFV-mNeonGreen as upstream open reading frame.

Techniques: Quantitative RT-PCR, Control, RNA Sequencing, Knockdown, Cell Culture, Gene Expression

Journal: bioRxiv

Article Title: Hypoxia promotes airway differentiation in the human lung epithelium

doi: 10.1101/2024.08.09.607336

Figure Lengend Snippet: (A) and (B) KLF4 and KL5 expression level from bulk RNA-seq data (as described in ) comparing Normoxia + NTC to Hypoxia + NTC (A), or Hypoxia + NTC to Hypoxia + HIF1A -knock down (KD) (B), n = 6 (2 gRNAs and 3 donors) for each condition. Each line indicates the change of the average counts of the two gRNA replicates from the same biological donor. (C) HIF2A knock down decreased KLF4 but not KLF5 expression under hypoxia. Data shown as Log 2 (fold change), n = 5 experimental replicates from 4 biological donors. 2 gRNAs used. (D) and (E) Stabilised HIF1α and HIF2α (as described in , ) overexpression under normoxia increased KLF4 expression. Data shown as Log 2 (fold change), n = 3 (HIF1α), 5 (HIF2α) biological donors. (F) KLF4 and KLF5 expression in organoid scRNA-seq dataset (as described in ). (G) KLF4 and KLF5 expression in epithelial cells of the human fetal lung atlas. (H) Immunostaining of 10 pcw human fetal lung section showing KLF4 and TP63 expression. Arrows indicate KLF4 + TP63 + cells. Scale bars = 100 µm. RT-qPCR gene expression was normalised to ACTB . Statistical test: two-way ANOVA with Bonferroni’s multiple comparisons test. Significance levels: *p < 0.05, **p < 0.01.

Article Snippet: For mutated HIF1α and HIF2α overexpression, the HIF1A and HIF2A CDS were cloned from plasmids gifted from William Kaelin (Addgene, #87261, #25956) and inserted into Tet-ON vectors with EF1a-TagRFP-2A-tet3G., For CRISPRi, the gRNA sequences targeting HIF1A , HIF2A , KLF4 , KLF5 were selected from published dataset and inserted into U6-gRNA-EF1a-EGFP-CAAX lentiviral vectors (Addgene, #167936)., For targeted DamID, the wild-type HIF1A and HIF2A CDS were inserted into DamID vectors with SFFV-mNeonGreen as upstream open reading frame.

Techniques: Expressing, RNA Sequencing, Knockdown, Over Expression, Immunostaining, Quantitative RT-PCR, Gene Expression