Understanding how genes are turned on and off in our cells is a fundamental question in biology. A significant part of this regulation happens at the chromatin level. Chromatin is a complex of DNA and proteins found in the nucleus of our cells, and it plays a key role in packaging our DNA and controlling gene expression. Various “marks” on chromatin can indicate whether a gene is turned on or off. But how do these marks interact with RNA, the molecules that help turn genetic instructions into actions?
The Challenge of Studying Chromatin-Associated RNA
Long non-coding RNAs (lncRNAs) are a type of RNA that don’t code for proteins but have important roles in regulating gene expression. Some of these lncRNAs can influence chromatin marks, which in turn affects gene regulation. However, identifying which lncRNAs interact with specific chromatin marks has been difficult due to technical limitations. Current methods often rely on antibodies to detect these interactions, but this approach can be noisy and imprecise.
Introducing Chrom-seq: A New Technique
Researchers at the Northwest University School of Medicine have developed a new method called Chrom-seq to tackle this problem. Chrom-seq is designed to efficiently capture RNAs that are associated with various chromatin marks in living cells. This method uses a highly specific chromatin mark reader combined with a protein called APEX2, which acts like a tiny biochemist inside the cell.
Identification of RNAs associated with H3K27me3 with Chrom-seq
(A) Schematic description of the Chrom-seq. Reader (CBX7 and dPC) is used to recognize H3K27me3-modified chromatin. Black stars indicate Btn-An. (B) Volcano plot shows the enrichment of ncRNAs in HEK293T cells. Enriched ncRNAs are labeled in red dots (n = 542), and the validated RNAs are highlighted in blue. (C) 3D pie chart shows the number and percentage of coding and noncoding genes. (D) Donut chart shows the proportion of Chrom-seq reads aligned to feature regions classified as exonic, intronic, and intergenic. (E and F) Validation of candidate genes by RIP-qPCR. RNAs were enriched using H3K27me3 (E) and EZH2 (F) antibodies, respectively. NC, negative control. Statistic P values by two-tailed Student’s t test are shown. *P < 0.05, ***P < 0.001, and ****P < 0.0001. ns, not significant. (G) Western blot of H3K27me3 and H3 in total protein from cells depleted of SETD5-AS1 (∆SETD5-AS1) and LINC00641 (∆LINC00641) using ASOs. (H) Confocal fluorescent imaging shows the distribution of H3K27me3 in nucleus (DAPI) in ∆SETD5-AS1, ∆LINC00641, and ASO control (NC) cells. Scale bar on the lower left corner represents a length of 10 μm and 5 μm in the white box inset. (I) Profile plots (upper) and heatmaps (lower) represent ChIP-seq signals upon ∆LINC00641 (left) and ∆SETD5-AS1 (right) around wild-type H3K27me3 peaks. (J) Box plots show the change of gene expression levels within the ±50 kb range of the H3K27me3 peaks upon ∆LINC00641 (left) and ∆SETD5-AS1 (right). Wilcoxon signed-rank test was performed. (K) Genome browser shots show the distribution of ChIP-seq (green) and RNA sequencing (RNA-seq) (yellow) signals from samples of NC, ∆LINC00641, and ∆SETD5-AS1. PGBD5 gene locus was used as an example. The bar plot shows the quantification of ChIP-seq signal in the designated red box.
Here’s how it works:
Chromatin Mark Readers: These are special proteins that can specifically bind to certain chromatin marks. For example, mCBX7/dPC binds to the H3K27me3 mark, mCBX1 binds to H3K9me3, and mTAF3 binds to H3K4me3.
APEX2 Enzyme: This enzyme can catalyze a reaction that tags nearby RNAs with a biotin molecule, a kind of molecular “hook.”
Biotin Labeling: When the chromatin mark readers bind to their specific marks, APEX2 labels the surrounding RNAs with biotin.
RNA Isolation: Using streptavidin-coated beads (which have a strong affinity for biotin), the labeled RNAs can be isolated for analysis.
The Benefits of Chrom-seq
Chrom-seq offers several advantages over traditional methods:
Antibody-Free: It doesn’t rely on antibodies, which can often produce unreliable results.
High Sensitivity and Efficiency: Chrom-seq is better at detecting RNAs associated with chromatin marks, making it more sensitive and efficient.
Cost-Effective: This method is less expensive compared to other techniques.
Discoveries with Chrom-seq
Using Chrom-seq, researchers detected RNA species significantly associated with the chromatin marks H3K27me3, H3K9me3, and H3K4me3. These marks are associated with gene repression, gene silencing, and gene activation, respectively. By mapping which RNAs are associated with these marks, scientists can begin to understand how lncRNAs might regulate gene expression through chromatin modification.
Conclusion
Chrom-seq represents a significant step forward in our ability to study the interactions between RNA and chromatin marks. By providing a more sensitive, efficient, and cost-effective method, it opens up new possibilities for exploring the regulatory roles of lncRNAs in epigenetic events. This could ultimately lead to a deeper understanding of gene regulation and potentially new avenues for therapeutic interventions in diseases where gene expression goes awry.
Fan L, Sun W, Lyu Y, Ju F, Sun W, Chen J, Ma H, Yang S, Zhou X, Wu N, Yi W, Chen E, Villaseñor R, Baubec T, Yan J. (2024) Chrom-seq identifies RNAs at chromatin marks. Sci Adv 10(31):eadn1397. [article]