Reading glasses, or “readers,” help you see text and other fine details up close. Inspired by our desire for increased resolution, a talented team is using histone readers to more clearly see which long noncoding RNAs (lncRNAs) interact with particular histone modifications. Their method, called Chrom-seq, doesn’t require highly pure antibodies or crosslinking, making it less expensive and more specific.
lncRNAs can place histone writers, readers, and erasers at certain loci to help deposit or remove histone modifications. When these nucleic acids are not expressed properly, diseases like colon cancer can develop.
Only a few of the predicted 172,112 human lncRNAs are characterized, so Jian Yan’s clear-sighted team at Northwest University and City University of Hong Kong teamed up with collaborators in Europe to hunt down more. They developed Chrom-seq, which combines well-established reader proteins with a proximity biotinylation strategy to grab onto and isolate lncRNAs that are close to histone modifications. Here are the fine details:
- Histone readers for specific modifications are each fused to 10 SunTag epitopes
- Single-chain Fvs (scFvs) are fused to APEX2, an enzyme that adds a biotin onto nearby molecules
- The scFVs bind to the SunTag epitopes, so that each histone reader carries 10 APEX2 molecules to each modification
- Labeling takes less than a minute, and biotinylated RNA is isolated with streptavidin-coated beads
Using Chrom-Seq with the CBX7 reader specific for H3K27me3 in HEK293T cells, the team identified more than 1,400 RNAs that included over 500 ncRNAs.
To see whether Chrom-seq-enriched lncRNAs were really involved in recruiting PRC2, the enzyme that methylates H3K27, the team immunoprecipitated RNAs that bound to the EZH2 subunit of PRC2. SETD5-AS1 and LINC00641—two lncRNAs isolated by Chrom-seq—were found in immunoprecipitations using either HEK293T or HCT116 cells. Next, they knocked down SETD5-AS1 or LINC00641 with antisense oligonucleotides. Here’s what they found:
- The knock-downs slightly reduce the amount of H3K27me3, as seen by Western blotting and immunofluorescence
- ChIP-Seq showed that the knock-downs affect most of the H3K27me3 sites in wild-type cells, so the lncRNAs are needed to preserve this modification
Similar ChIP-Seq results were found for H3K9me3 when knocking down an lncRNA specific for that modification, though no changes in H3K9me3 levels were seen via Western blotting or immunofluorescence. That could be due to the significant overlap of these lncRNAs with those identified with readers for H3K27me3, suggesting there could be an interaction between molecules associated with these modifications. Finally, the detail-oriented team took a magnifying glass to similar methods and compared them against Chrom-seq using data from the literature.
Overall, the team says that Chrom-seq requires fewer cells, takes less time, and is less expensive than similar methods and could be applied to the study of epigenetic modifications of DNA and RNA in the future.
See the data up close at Science Advances, July 2024.