Thank you @stefanschoenfelder.bsky.social - far too modest!

Many thanks especially to Colm Doyle (now at @embl.org), who began this work with his beta globin locus study and @seanflynn.bsky.social (now of sites.google.com/view/flynnlab/ at @exeter.ac.uk - great things to come) for an incredible journey, as well as all authors at @cruk-ci.bsky.social! 9/9

....then edited them out to show how the regulation of genes in 3D contact was affected. 8/9

For the first part of this journey, don't forget to check out genomebiology.biomedcentral.com/articles/10.... to find out how we developed a technique, ViCAR, for mapping 3D interactions specific to #epigenetic features (such as G4s)... 7/9

How do G4s function at enhancers? In our #proteomics pulldowns, RNA polymerase II subunits were enriched, together with known G4 binding proteins - but not some common architectural factors (e.g. YY1, CTCF) - we confirmed that some RNAPII subunits specifically bind G4s (Fig. 4A, F). 6/9

Inserting an unrelated G4 structure (Pu27 from the #Myc promoter - as featured in @isabelesain.bsky.social's brilliant work www.pnas.org/doi/abs/10.1...) recovers original globin expression and enhancer function (Figure 3D) 5/9

For the beta HS2 enhancer, this effect is masked by hyper activation of the neighbouring enhancer, HS3 - removing HS3 unveils a similar effect (Figure 3A-D for alpha, Figure 3E-H for beta). 4/9

What happened? In the alpha HS-40 enhancer, such a mutation (seven G-to-T mutations) was sufficient to reduce globin gene expression to levels observed when the whole enhancer region (of around 350 bases) is deleted. Enhancer-promoter contacts and local #chromatin accessibility is also reduced. 3/9

Using the classic #globin #superenhancer systems, we discovered two G4 structures in dominant enhancers in both the alpha globin and beta globin locus control systems - and replaced them with non-G4-forming sequences using CRISPR. 2/9

…and in the meantime, for anyone who missed it, here’s one we did earlier on mapping #epigenetic features together with their 3D #genomic contacts with ViCAR: genomebiology.biomedcentral.com/articles/10....

Thank you @seanflynn.bsky.social - looking forward to sharing science (mostly #epigenetics, #G4, gene regulation and #chromatin) with everyone, especially great work from the Flynn Lab!