Suzanne de Bruijn

56 Chapter 1.2 A Active retinal enhancer element in UCSC genome browser 50 kb hg38 59,430,000 59,440,000 59,450,000 59,460,000 59,470,000 59,480,000 59,490,000 59,500,000 59,510,000 59,520,000 59,530,000 GeneHancer LINC01476 Scale chr17: B Binding of tissue-specific transcription factors CRX NRL Scale chr17: GH Reg Elems (DE) 50 kb hg38 59,430,000 59,440,000 59,450,000 59,460,000 59,470,000 59,480,000 59,490,000 59,500,000 59,510,000 59,520,000 59,530,000 GENCODE v32 Comprehensive Transcript Set (only Basic displayed by default) GeneHancer Regulatory Elements and Gene Interactions Hu1_ret_H3K27ac_R1_trimmed_sorted_fixchr_ext200_10Mnorm.bw LINC01476 55 _ 0 _ 55 _ 0 _ Hu5_ret_ATAC_R2 0 _ Hu1_ret_H3K27ac 55 _ 0 _ C Expression of enhancer element CAGE reads Monocytes CD14+ CTCF Histone Mods by ChIP-seq Signal from ENCODE/Broad Max counts of CAGE reads CD14+ CTCF 50 _ 1 _ Max counts 100 0 _ 100 _ 0 _ 50 D Open chromatin conformation ATAC E Enrichment of active histone modification marks H3K27Ac Scale chr17: GH Reg Elems (DE) 50 kb hg38 59,430,000 59,440,000 59,450,000 59,460,000 59,470,000 59,480,000 59,490,000 59,500,000 59,510,000 59,520,000 59,530,000 GENCODE v32 Comprehensive Transcript Set (only Basic displayed by default) GeneHancer Regulatory Elements and Gene Interactions Hu13_ret_Nrl_R1_trimmed_sorted_fixchr_ext200_10Mnorm.bw Hu6_ret_Crx_B11X_trimmed_sorted_fixchr_ext200_10Mnorm.bw LINC01476 Hu13_ret_Nrl_R1 55 _ 0 _ Hu6_ret_Crx_B11X 55 _ 0 _ 50_ 0_ Scale chr17: GH Reg Elems (DE) 50 kb hg38 59,430,000 59,440,000 59,450,000 59,460,000 59,470,000 59,480,000 59,490,000 59,500,000 59,510,000 59,520,000 59,530,000 GENCODE v32 Comprehensive Transcript Set (only Basic displayed by default) GeneHancer Regulatory Elements and Gene Interactions Hu13_ret_Nrl_R1_trimmed_sorted_fixchr_ext200_10Mnorm.bw Hu6_ret_Crx_B11X_trimmed_sorted_fixchr_ext200_10Mnorm.bw Hu5_ret_ATAC_R2_p_trimmed_repaired_sorted_fixchr_ext200_10Mnorm.bw LINC01476 Hu13_ret_Nrl_R1 55 _ 0 _ Hu6_ret_Crx_B11X 55 _ 0 _ Hu5_ret_ATAC_R2 55 _ 0 _ 50_ 0_ Figure 4. Interpretation of potential regulatory variants using the UCSC genome browser. To evaluate a potential regulatory variant, several publicly available datasets in the University of California Santa Cruz (UCSC) browser can be assessed to determine whether a variant is located within an active cis regulatory element based on the presence of active enhancer or promoter hallmarks. 164 (A) A known active retinal enhancer element described by de Bruijn et al. and included in the GeneHancer database is visualized in UCSC browser. 153,155 The enhancer element overlaps with a long non-coding gene LINC01476 , and is predicted to bind to the promoter region of the YPEL2 gene (GeneHancer). 153 The enhancer element is enriched for several active enhancer hallmarks. (B) Firstly, the element is bound by retina-specific transcription factors (NRL and CRX) as confirmed by ChIP-sequencing performed in a human retina sample (GEO database: GSE137311). 145 (C) Secondly, cap analysis gene expression (CAGE) that allows 5’ end sequencing of cDNAs confirms expression of the enhancer element, as shown by the FANTOM5 CAGE human dataset (Data available from: https://fantom.gsc.riken.jp/data/ ). 165 (D) Thirdly, an open chromatin conformation of the enhancer element is confirmed by ATAC-sequencing in a human retina sample (GSE137311). 145 (E) Lastly, the enhancer element is enriched for histone-modification marks that are associated with enhancer activity such as H3K27Ac as determined using ChIP-sequencing performed in human retina (GSE137311). 145