Move over Oliver Twist, there’s a new orphan in town to satisfy our hunger for stories of DNA methylation. Two new tales of orphan CpGs help them find a home in our hearts and epigenomes through the application of cutting-edge epigenomics techniques and genetic engineering to embryonic stem cells and neuronal cells.
Making the Right Connections: Orphan CpG Islands Boost Poised Enhancer Power
Our story starts and ends with CpG islands. Half are fortunate enough to find a home close to a gene and the other half are orphans. But what are these mischievous orphan CpG islands (oCGis) up to? Thankfully, as the lab of Lab of Alvaro Rada-Iglesias (University of Cologne, Germany) shows, it’s not a life of crime as a pickpocket.
The talented team noticed that oCGIs are genetically distinct from promoter CGIs (pCGIs) and that ~60-80% of poised enhancers (PEs) associate with oCGIs. So, to see what makes these oCGIs so special, they turned to mouse embryonic stem cells (mESCs) and assayed them through molecular (ChIP-qPCR, FAIRE-qCPR, and NOMe-PCR), sequencing (bisulfite sequencing and 4C-seq), and genetic engineering (CRISPR-Cas9) approaches. They used these approaches to dissect PEs by inserting their transcription factor binding sites (TFBS), oCGI, or the combination of two into topologically associated domains (TADs) containing genes that aren’t active in the stages assayed. Upon neural differentiation they found that:
- The combination of oCGI and TFBS greatly increases target gene expression and produces a stronger effect than TFBS alone
- oCGIs don’t function on their own: there is no cis-regulatory effect as there is no local activation such as increased H3K27ac
- Rather, oCGIs function as boosters of PEs by facilitating physical and functional communication between PEs and target genes
- This is accomplished through an entourage of enhancer RNA (eRNA) and mRNA production, recruitment of PRC1 and PRC2, and the recruitment of proteins that could lead to phase-separated transcriptional condensates
- The boosting effect of oCGIs only occurs if the target gene promoter is CGI rich, it doesn’t happen in target genes with CGI poor promoters
So, as the story goes, the CpG rich get richer, all thanks to the help of our beloved oCGIs. This rags to riches story also shows the importance of making the right connections by demonstrating that CGIs are a key feature in understanding the compatibility of enhancer and gene pairs.
Check out this twitter thread for the full serial:
An Orphan’s New Home: BANP Binds the Orphan CGCG Motif in Promoter CpG Islands
You may be left thinking that those fancy pCGIs have all the privilege with their role in initiating the expression of the majority of mammalian transcripts generated by RNA polymerase II. But fear not, as even they too are orphans in their own way. After all, pCGIs contain the orphan CGCG motif and what’s a motif if it doesn’t have a warm and cozy transcription factor to call home? Thankfully, the charitable lab of Dirk Schübeler (Friedrich Miescher Institute for Biomedical Research, Switzerland) found a home for the orphan CGCG motif that was hiding in plain sight. They discovered it by:
- Inserting the CGCG motif into a specific locus within mESCs using recombinase-mediated cassette exchange (RMCE)
- Employing their previously developed genome-wide single-molecule footprinting method that utilizes methyltransferase footprinting followed by a bisulfite sequencing readout to demonstrate that the orphan CGCG motif is occupied
- Using affinity purification, with an oligonucleotide containing the CGCG motif as bait, followed by mass spectrometry, which detected BTG3-associated nuclear protein (BANP) as the only enriched protein
Then, they just had to be sure that this home was worthy of an orphan so near and dear. Their functional genomics studies revealed that:
- BANP is methylation sensitive and binds to the non-methylated CGCG motif in both mouse mESCs and human cancer cells
- This means it generally binds CGIs, as the motif is very frequently methylated in the rest of the genome
- BANP regulates the expression of metabolic genes in mESCs and neuronal cells
- Once BANP binds its motif, it opens chromatin (as determined by ATAC-seq) and phases nucleosomes (as determined by MNase-seq)
So, how did this orphanage hide in plain sight? Senior author Dirk Schübeler shares, “We believe that is because BANP is so essential: you touch it, and the cell dies. This made it hard to identify it by any kind of genetic screening approach, which makes us wonder whether there are more of these factors out there that are invisible to us for the same reasons.”
Breaking Out of the Orphanage: Fostering New Insight
Both oCGIs and the orphan CGCG motif in pCGIs help us redefine what the term orphan means by providing fundamental insight into one of the most well traversed features of the epigenetic landscape. What else could be hiding in plain sight for all these years? Perhaps it’s time we adopt more studies of orphan features.
If these stores have you saying, “Please sir may I have some more”, then feast on oCGIs in Nature Genetics, June 2021 and BANP in Nature, July 2021