Novel CAGE clusters. (A) Histograms for genomic distance distributions of H3K27ac (top panels) and H3K4me1 tag counts (bottom panels) are shown for eRA+Treg centered across expression-binned CAGE clusters across a 5-kb genomic interval contingent on their location relative to annotated TSSs. CAGE clusters represent a merge of all T-cell subpopulations, and clusters with no associated CAGE tags in Tregs (none) were separated from the expressed ones, which were ranked according to CAGE cluster activity and binned into 5 equal-sized groups (quintiles). Expression bins are color coded as indicated. The average H3K27ac deposition peaks at the nucleosome immediately downstream of the CAGE cluster and shows a dip just upstream of CAGE clusters, indicating a nucleosome-free region. The average H3K4me1 deposition is broader. Marking of the nucleosome immediately downstream of the CAGE cluster decreases with increased TSS activity (as indicated by the red arrow), reflecting the higher degree of H3K4 methylation (trimethylation; me3) associated with highly active promoters. Of note, CAGE clusters distal from annotated promoters do show the highest H3K4me1 deposition at the nucleosome immediately downstream of the TSS, suggesting that many of them may represent enhancers. (B-C) 5′-RACE confirms the presence of spliced transcripts from novel CAGE TSSs. UCSC browser graphics are shown for the indicated genomic positions, including H3K27ac signal of expanded populations, GencodeV10 gene annotation, and CAGE signals for all 8 T-cell subpopulations. In the bottom of each panel, results from 5′-RACE-PCR of the indicated cell types are aligned (only products representing novel TSSs are shown). Numbers of sequenced clones are indicated in brackets. (D) Relative luciferase activity (RLU) of promoter construct for the annotated (prom) and novel FOXP3 and CTLA4 TSSs (new TSS) in unstimulated and phorbolmyristate acetate/ionomycin-stimulated (stim) Jurkat T cells.