Wiping out a cell’s identity shifts cellular reprogramming into high gear

Blog CAF-1 chromatin

The packaging of DNA into chromatin (image credit: Felsenfeld and Groudine, Nature 2013

If stretched out end to end, the DNA in just one cell of your body would reach a whopping six feet in length. A complex cellular structure called chromatin – made up of coils upon coils of DNA and protein – makes it possible to fit all that DNA into a single cell nucleus that’s only 0.0002 inches in diameter.

Chromatin: more than meets the eye
Once thought to merely play a structural role, mounds of data have shown that chromatin is also a critical regulator of gene activity. In fact, it’s a key component to maintaining a cell’s identity. So, for example, in the nucleus of a skin cell, genes related to skin function tend to lie within stretches of DNA having a loosely coiled chromatin structure. This placement makes the skin-related genes physically more accessible to become activated. But genes related to, say, heart, liver or brain cell function in that same skin cell tend to remain silent within tightly packaged, inaccessible chromatin.

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Depiction of (a) loosely packaged, accessible chromatin (red is DNA; blue is protein) vs (b) tightly packaged inaccessible chromatin. (Image credit: Interface Focus (2012) 2, 546–554)

As that skin cell divides and its DNA is replicated, there are various proteins that assemble and maintain the same chromatin positioning in their daughter cells, which helps them know they are skin cells. This cellular memory isn’t easy to erase, and it’s one of the reasons for the low efficiency when reprogramming a skin cell back into an embryonic stem cell-like state, also known as the induced pluripotent stem cell (iPSC) technique.

Blocking a DNA roadblock increases iPSC efficiency
So researchers at Harvard and in Vienna asked what if you blocked proteins responsible for arranging the chromatin – would it make it easier to generate iPSCs? The answer is a resounding “yes” based on data reported last Thursday in Nature. While previous studies asking the very same question have shown decent increases in iPSC reprogramming efficiency, this current research achieved orders of magnitude higher efficiency.

Using two independent screening methods, the research team systematically blocked the activity of hundreds of genes that play a role in the packaging of chromatin structure and maintaining cellular memory. These inhibition experiments were carried out in skin cells that were in the process of being reprogrammed into iPSCs. In both screening approaches, the inhibition of two proteins, collectively called chromatin assembly factor 1 (CAF-1), led to large increases in reprogramming efficiency.

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Induced pluripotent stem cell (iPS cell) colonies were generated after researchers at Harvard Stem Cell Institute suppressed the CAF1 gene. (Image credit: Sihem Chaloufi)

Inhibiting CAF-1 potently erases cell memory
While the inhibition of genes previously identified to block reprogramming led to a three to four-fold increase in iPSC generation, inhibition of CAF-1 dramatically increased efficiency 50 to 200 fold. Also, compared to a typical reprogramming time of nine days, in skin cells with CAF-1 inhibition, the first iPSCs were observed in just four days.

The increased ease of manipulating cells also applies to direct reprogramming. This alternative reprogramming method skips the iPSC process altogether and instead directly converts one adult cell type into another. In this case, the researchers were able to convert skin cells into neurons and one immune system cell type (B cells) into another (macrophages).

In a Harvard press release posted on Monday, co-first author Sihem Chaloufi, a postdoc in Konrad Hochedlinger’s lab at Harvard, succinctly described the overall finding:

“The cells forget who they are, making it easier to trick them into becoming another type of cell.”

This potent erasing of cell memory via CAF-1 inhibition could make it easier to derive many different cells types from iPSCs or direct reprogramming for use in drug testing, modeling human disease in a lab dish as well as scaling up production of future cell therapies.

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