Multiple Fra-1-bound enhancers showing different molecular and functional features can cooperate to repress gene transcription

Table 1 Putative functions borne by TGFB2 PIRs

PIR center position	PIR coordinates	Fra-1 peak	ATAC- seq	p300/CBP	CTCF	Putative function
− 46	chr1:218,471,651-218,473,869	–	–	–	− 46	Chromatin organization
+ 32	chr1:218,549,326-218,552,116	+ 32	✓	✓	–	Active enhancer
+ 116	chr1:218,630,568-218,638,881	+ 115	✓	✓	–	Active enhancer
+ 116	chr1:218,630,568-218,638,881	+ 118	✓	✓	–	Active enhancer
+ 136	chr1:218,651,100-218,656,338	+ 136	✓	✓	–	Active enhancer
+ 151	chr1:218,666,260-218,672,881	+ 151	✓	✓	–	Active enhancer
+ 240	chr1:218,753,682-218,767,687	+ 240	✓	✓	–	Active enhancer
+ 314	chr1:218,826,456-218,839,841	+ 314	✓	✓	–	Active enhancer
+ 314	chr1:218,826,456-218,839,841	+ 315	✓	✓	–	Active enhancer
+ 360	chr1:218,867,620-218,891,396	+ 360	✓	✓	–	Active enhancer
+ 729	chr1:219,232,878-219,269,584	+ 744	✓	✓		Active enhancer
+ 729	chr1:219,232,878-219,269,584	–	–	–	+ 722	Chromatin organization
+ 836	chr1:219,339,009-219,372,344	–	✓	–	–	Epromoter
+ 980	chr1:219,489,115-219,510,682	980	✓	✓	–	Active enhancer
+ 1041	chr1:219,553,878-219,563,787	–	–	–	+ 1041	Chromatin organization
+ 1222	chr1:219,730,289-219,752,737	–	–	–	+ 1222	Chromatin organization
+ 1316	chr1:219,822,398-219,844,303	–	–	+ 1314	+ 1316	Chromatin organization
+ 1449	chr1:219,939,514-220,000,504	+ 1426	✓	✓	–	Active enhancer
			–	–	+ 1460	Chromatin organization
			–	–	+ 1468	Chromatin organization

For more details, the molecular characteristics of TGFB2 PIRs are extensively presented in Additional file 3: Data S3. Not all PIRs are bound by Fra-1, suggesting that they do not all have the same function(s). At all Fra-1-bound PIRs, Fra-1 ChIP-seq signals overlap with p300/CBP ChIP-seq- and ATAC-seq signals and are surrounded by H3K4me1 and H3K27ac ChIP-seq signals (Additional file 3: Data S3). This suggests that these PIRs bear active TGFB2 enhancers. Besides this, certain PIRs show binding of CTCF at places neither bound by Fra-1 nor showing any detectable signals in ATAC-seq- and p300/CBP ChIP-seq experiments. As they are also poorly, or not, marked by H3K4me1, H3K4me3 and H3K27ac (Additional file 3: Data S3), they are likely to constitute 3D chromatin-organizing elements. Of note, PIRs + 729 and + 1449 are composite PIRs where a candidate Fra-1-bound active enhancer and one or two putative CTCF-bound structural elements are easily distinguished. Finally, PIR + 836 is not bound by Fra-1 but bears an annotated gene TSS and presents marks specifying active promoters, i.e. high Pol II-, H3K4me3- and H3K27ac signals, as well as ATAC-seq signal (Additional file 3: Data S3) and might be a TGFB2 Epromoter

ISSN: 2045-3701