Pluripotent stem cells (PSCs) exist in the embryo during early development¹ and can develop into virtually any kind of cell in the body – pluripotent literally means “many-powered”.

In the transition from early to late pluripotency, networks of genes need to be expressed at the correct times and places in order for the cell to develop normally. The chromatome – in other words, the proteins bound to the genome – is crucial for this.²

However, biologists still lack a full understanding of how the chromatome evolves throughout stem cell differentiation: which proteins are driving development forward, and which are just along for the ride?

Heinrich Leonhardt’s group in Munich developed a technique called Chromatin Aggregation Capture followed by Data-Independent Acquisition (ChAC-DIA) to investigate this.

ChAC-DIA works by isolating chromatin fibres from cells, breaking them into many smaller fragments, and then isolating the chromatin-associated proteins using trypsin, an enzyme which “cuts off” the proteins from the DNA. These proteins are purified and, using a specialised type of mass spectrometry, separated by size. Each fragment was computationally analysed to find out which proteins are bound at each pluripotency stage.

Using ChAC-DIA, Leonhardt’s group successfully identified thousands of proteins in the mouse chromatome that drive forward stem cell pluripotency, and found that each pluripotency stage has distinct chromatome signatures. ChAC-DIA was also able to detect very sparsely-bound proteins in each pluripotency stage previously not observed via traditional techniques.

What were these proteins involved in?

Towards the mid- and late pluripotency stages in mouse PSCs, they observed increased binding of proteins involved in chromatin compaction, in particular DNA methyltransferases such as SUV39H1 and -2.²

SUV39H1/2 are involved in depositing H3K9 trimethylation (H3K9me3) marks to trigger condensation of chromatin, which is critical in late pluripotency. H3K9me3-mediated condensation allows the genome to, essentially, organise all its files and shut off lineage-inappropriate genes to allow it to embark on its new life as a specialised cell.

What’s in it for us?

The authors then compared these data to the human embryonic stem cell (hESC) chromatome. Curiously, ChAC-DIA in hESCs unveiled increased binding of proteins involved in the HIPPO signalling pathway, which has been involved in holding back stem cells from maturing³, while in (pluripotency stage-matched) mouse cells this was not identified.

This finding implies that cells from mice may be reliant on alternative pathways to human cells in order to progress through development.

ChAC-DIA is sensitive…

In comparison to commonly-used similar methods like ChIP-MS, which uses specific antibodies to isolate proteins before mass spectrometry, this new method uses almost 100-fold fewer cells, making it more sensitive, plus less time-consuming in the laboratory.

ChIP-based methods are falling out of favour amongst chromatin biologists because they require garguantuan amounts of cells (often upwards of 2 million per experiment) and are therefore not particularly sensitive. ChAC-DIA therefore offers a significant advantage in this case.⁴

…but does have limitations

The authors concluded that the increase in expression of certain proteins in the chromatome meant that they became increasingly involved in determining the cell’s identity. However, some proteins identified with ChAC-DIA may not have been fully active.

A recently-developed technique from Rahul Satija’s lab mapped phosphorylation (typically a mark of protein activation) in nuclear proteins, which may be useful in future when investigating catalytic activity of proteins across the chromatome.

Moreover, ChAC-DIA is performed in bulk, with thousands of cells being analysed at once. Bulk methods develop an average chromatome of the cell population, and therefore may miss out chromatome differences between individual stem cells, which would be important in understanding pluripotency in small structures like embryos. Again, we’re not quite there with doing mass spectrometry on individual cells though.

“Yeah, and?”

ChAC-DIA also has implications beyond development, and could be applied clinically to understand chromatome differences between, for instance, cancer cells and healthy cells. However, its reliance on big, bulky, specialised mass spectrometers may also hinder how widespread the technique will become outside of academic settings.

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Notes

1. And in the fully-developed adult too! There are PSCs in your bone marrow that specialise into all the different types of red blood cells throughout your life. ↩︎
2. As do enhancer-promoter contacts, which I discussed previously here. ↩︎
3. Though note that reports are conflicting. Hippo signalling in human PSCs may actually drive forward differentiation via its interactions with other signalling pathways, namely TGF-β. ↩︎
4. Methods like CUT&RUN, which can work in live cells unlike ChIP, are gaining popularity. So why didn’t the authors use this as a comparison? Well, firstly, we haven’t found a way to combine CUT&RUN with mass spectrometry yet, and also CUT&RUN looks at where in the genome proteins are bound, which was not the authors’ primary question (they cared more about which proteins were expressed and when). So it does appear that ChIP-MS is the most appropriate method to compare ChAC-DIA to. ↩︎

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Discussion point

Is more sensitivity in a biological technique always useful?

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