Extended Data Figure 8 : Dynamic regulation of H3K9ac at LEC genes, and PROX1 does not interact with the KDR gene or affect H3K9 acetylation at the KDR gene.

From: The role of fatty acid β-oxidation in lymphangiogenesis

Extended Data Figure 8

a, Proportion of LEC versus non-LEC genes (refers to all annotated genes not included in LEC ontology) with enhanced H3K9ac (red portion) in pLECs versus VECs. The green portion denotes genes without significantly enhanced H3K9ac levels. b, Proportion of Prox1-target genes versus unrelated genes with enhanced H3K9ac (red portion) in pLECs versus VECs. The green portion denotes genes without significantly enhanced H3K9ac levels. cf, H3K9ac enrichment around the PROX1 (c) and SOX18 (e) genes in VECs (blue) and pLECs without (green) and with CPT1AKD (dark blue). Top line above each snapshot: scheme of the gene; grey blocks denote the exons of the gene of interest; black blocks denote exons of adjacent genes. Red arrowheads indicate sites of PROX1 enrichment in the PROX1 (c) or SOX18 (e) genes. Bottom line under each snapshot: chromosomal location of the gene (human genome build GRCh37/hg19). Grey boxes at the chromosomal locations denote regions with H3K9ac peak areas as determined using MACS. When the top half of the box is green, it denotes regions of significantly enhanced H3K9ac in pLECs versus VECs. When the bottom half of the box is red, it denotes regions of significantly reduced H3K9ac in pLECs with CPT1AKD versus pLECs. q value is the FDR corrected P value. Quantitative PCR verification of H3K9ac ChIP-seq results for the sequences of PROX1 (d) and SOX18 (f) (asterisks in c, e, respectively) on H3K9ac-ChIP samples from pLECs without (ctrl) and with CPT1A silencing (CPT1AKD) versus VECs, expressed as relative to input (n = 3). g, p300 enrichment around the VEGFR3 gene in VECs (blue) and pLECs (green). Top line: scheme of the gene; grey blocks denote the exons of the gene of interest; black blocks denote exons of adjacent genes. Red arrowheads indicate PROX1-binding sites. Bottom line, chromosomal location of the gene (human genome build GRCh37/hg19); grey boxes at the chromosomal locations, regions with H3K9ac peak areas. Asterisk denotes region confirmed by qPCR in h. h, Quantitative PCR verification of p300 ChIP-seq results for the sequence of VEGFR3 on p300 ChIP samples from pLECs versus VECs as a percentage of input, expressed as relative to VEC. i, PROX1–Flag enrichment around the human KDR (VEGFR2) gene (PROX1–Flag, green) versus empty-Flag control (empty–Flag, blue) in VECs transduced with a lentiviral vector expressing Flag-tagged PROX1 (PROX1–Flag) or empty-Flag, and PROX1 enrichment (using anti-PROX1 antibody ChIP) in pLECs (PROX1, green) and VECs (GFP, blue), as compared to the input chromatin (purple) and determined by ChIP-seq analysis. Top line, scheme of the KDR gene; grey blocks denote KDR exons and black blocks denote exons of adjacent genes. Bottom line: chromosomal location (human genome build GRCh37/hg19) (q < 0.1; q value is the FDR corrected P value). j, H3K9ac enrichment around the human KDR gene in VECs (blue) and pLECs (green). Top line, scheme of the KDR gene; grey blocks denote KDR exons and black blocks denote exons of adjacent genes. Bottom line: chromosomal location (human genome build GRCh37/hg19). q value is the FDR corrected P value. Mean ± s.e.m. of n independent experiments. Statistical test, Chi-square test was used to compare gene ontology subsets for presence of increased H3K9ac. ANOVA and Bonferroni post-hoc test were used in multiple group comparisons. Statistical analysis for ChIP-seq is described in Methods. *P < 0.05.