To see clearly, sometimes you just need a good pair of glasses or contacts. Now, Dong Xing’s lab (Peking University) has combined single cell Hi-C and RNA-seq to get a “Hi-RES” picture of chromatin and gene expression from the same cells simultaneously.
Sure, separate Hi-C and RNA-seq data from different cells have been combined before, but you could get the wrong picture with this view. So, the Xing lab developed “Hi-C and RNA-seq employed simultaneously” (cleverly called HiRES). Here’s how this multi-omic method brings things into focus:
- Reverse transcribe genomic DNA, then perform in situ Hi-C
- Sort single cells with flow cytometry into each well of a microtiter plate and amplify
- Prepare barcoded single cell libraries and sequence
Throughout the procedure, the genome and transcriptome are kept together. And the team could analyze thousands of single cells in just a few days.
When HiRES was used on cells from embryonic mouse embryos, four cell populations were identified with the RNA data. Hi-C analyses showed that cells diverged into distinct ectoderm and mesoderm populations by embryonic day 11.5 (E11.5). And it turned out that A/B compartments and TAD boundaries were similar for cell types close in developmental age, but then diverged gradually as development progressed, so these chromatin features didn’t only depend on cell type.
Some components of RNA clusters, however, were in different Hi-C clusters, which could be due to cells undergoing a lot of division early in embryogenesis. So, the team used HiRES and assigned cells to seven different cell cycle stages. Here’s what they insight they captured:
- Cell-cycle stages are one of the most important factors determining the genome architecture
- Chromosome unfolding after mitotic exit [DNA synthesis during S phase] determined chromatin organization of interphase cells, but when a cell had a short G1 phase, these processes overlapped
Xing’s lab also peered into differential interactions (DIs) with SimpleDiff, a computational pipeline they developed. Early neurons and mixed late mesenchyme had the highest DIs, which made sense since they were distinctly clustered on the structural maps. But many early-stage cell types had DIs that were not so clear-cut from the previous analyses. Even though the chromatin structures had been similar, some cell-type-specific rewiring of chromatin interactions was going on.
Combining DI and transcription data enabled the team to link DIs to genes, and they called these gene-associated DIs (GADIs). A lot of GADIs were at transcription start sites, and they could provide information on enhancer-promoter interactions. GADI analyses also showed that:
- When chromatin changes happened before expression, the chromatin had been active
- When genes were in a repressed chromatin state, the chromatin relaxed first, then transcription occurred
The bottom line? The 3D chromatin genome is linked to gene activation and function, though more experiments are needed to pin down the exact mechanism.
Bring this into focus by reading more at Science, June 2023.