Shifting the Genetic Furniture

 

Readers of these pages will know very well that cells are packets of magic. Of course, we often describe them in the simplest terms: ‘Sacs of gooey stuff with lots of molecules floating around.’ And it’s true that we know a lot about the protein pathways that capture energy from the food we eat and about the machinery that duplicates genetic material, makes new proteins and sustains life. Even so, although we’ve worked out much molecular detail, we have scarcely a clue about how ‘stuff’ in cells is organised. How do the tens of thousands of different types of proteins find their places in the seemingly chaotic jumble of a cell so that they can do their job? If that remains a mystery there’s an even more perplexing one in the form of the nucleus. That’s a smaller sac (i.e. a compartment surrounded by a membrane) that is home to most of our genetic material — i.e. DNA.

Sizing up the problem

It’s easy to see why evolution came up with the idea of a separate enclosure for DNA which only has to do two things: reproduce itself and enable regions of its four base code to be transcribed into molecules that can cross the nuclear membrane to be translated into proteins in the body of the cell. But here’s the puzzle. The nucleus is very small and there’s an awful lot of DNA — over 3,000 million bases in each of the two strands of human DNA (and, of course, two complete sets of chromosomes go to make up the human genome) — so 2 metres of it in every cell. A rather pointless exercise, unless you go in for pub quizzes, is to work out the length of all your DNA if you put it together in a single string. 1013 cells (i.e. 1 followed by 13 zeros) in your body: 2 metres per cell. Answer: your DNA would stretch to the sun and back 67 times.

Mmm. More relevantly, the nucleus of a cell is typically about 6 micrometres (µm) in diameter — that’s six millionths of a metre (6/1,000,000 metre), into which our 2 metres must squeeze.

Time for some serious packing to be done but it’s not just a matter of stuffing it in any old how and sitting on the lid. As we’ve just noted, every time cells divide all the DNA has to be replicated and regions (i.e. genes) are continually being “read” to make proteins. So the machinery in the nucleus has to be able to get at specific regions of DNA and disentangle them sufficiently for code reading. Part of evolution’s solution to these problems has been to add proteins called histones to DNA (the term chromosome refers to DNA together with histone packaging proteins and other proteins). To understand how this leads to “more being less”, consider DNA as a length of cotton. If you just scrunch the cotton up into a ball you get a tangled mess. But if you use cotton reels (aka histones — two or three hundred million per cell), you can reduce the great length to smaller, more organized blocks — which is just as well because they’re all that stands between life and a tangled mess.

Thinking of histones as cotton reels helps a bit in thinking about how the nucleus achieves the seemingly impossible but the fact of the matter is that we have no real idea about how DNA is unravelling is controlled so that the two strands can be unzipped and replicated, yet alone the way in which starting points for reading genes are found by proteins.

Undeterred by our profound ignorance Haifeng Wang and colleagues at Stanford University have just done something really amazing. They came up with a way of moving regions of DNA from the jumble of the nuclear interior to the membrane and they showed that this can change the activity of genes. They used CRISPR (that we described in Re-writing the Manual of Life) to insert a short piece of DNA next to a chosen gene. The insert was tagged with a protein designed to attach to a hormone that also binds to a protein (called emerin) that sits in the nuclear membrane. So the idea was that when the hormone is added to cells it can hook on to the DNA tag and, by attaching to emerin, can drag the chosen gene to the membrane. The coupling agent is a plant hormone (abscisic acid) although it also occurs in other species, including humans. Wang & Co christened their method CRISPR-GO for CRISPR-Genome Organizer.

Tagging a DNA insert with a protein so that a coupling molecule can pull a region of DNA to a protein in the membrane of the nucleus. From Wang et al., 2018.

Repositioning regions of DNA in the nucleus. DNA is labeled blue which defines the shape of the nucleus. Red dots are specific genes before (left) and after (right) adding the coupling agent. From Pennisi 2018.

How did CRISPR-GO go?

Astonishingly well. Not only could it shift tagged DNA from the interior to the membrane of the nucleus but the rearrangements could change the way cells behaved. Depending on which regions were moved and where to, cells grew more slowly or more rapidly.

So this is a really remarkable technical feat — but it’s not just molecular pyrotechnics for fun. It looks as though this approach may offer at long last a way of dissecting how cells go about getting a controlled response out of the mind-boggling complexity that is their genetic material.

References

Wang, H. et al. (2018). CRISPR-Mediated Programmable 3D Genome Positioning and Nuclear Organization. Cell 175, 1405-1417.

Pennisi, E. (2018). Moving DNA to a different part of the nucleus can change how it works. Science Oct. 11th.

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Dyslexic DNA

Writing in code

Did you notice a few months back that some boffins had written a book in DNA? No, that’s not a typo: what they did was to transcribe a 53,000 word book – plus pictures – into a synthetic DNA sequence. In essence, they re-wrote the book in binary by taking the four bases that make the genetic code of life and setting A and C to equal zero whilst G or T represented one. The result wasn’t without its typos: in the just over five million bits needed there were ten mistakes. So rather better than my touch-typing then. But there was a real commercial point behind this exercise, aside from showing, yet again, the astonishing coding capacity of our genetic material. One gram of DNA (you’ve got 500 grams) can store more than 100 billion DVDs, so not merely is it the ultimate in compacted data but it’s amazingly tough stuff – think of sequencing the woolly mammoth, in the freezer for thousands of years – by comparison with the latest software updates for my computer which usually mean I can’t read files 10 years old. And if I dig out my 20 year old 35 mm slides from the attic, chances are they’ll adorned by fungal growths.

Genetic switches

So DNA’s great for long-term information storage but this was by no means the first attempt to use biological molecules in ways we normally associate with electronic devices. When the code of DNA is ‘read’ to make an intermediate (RNA) from which, in turn, proteins can be made it’s acting as a biological transistor: a switch and amplifier that responds to an input signal. The DNA code ‘reader’ is a molecular machine called RNA polymerase (RNA pol) that moves step-wise along a strand of DNA, adding units one at a time to a growing molecule of RNA, complementary in sequence to the DNA template. This process is called ‘transcription’. In its wake another molecular machine can ‘translate’ the RNA codes into protein. RNA pol therefore ‘flows’ along a strand of DNA rather like a current of electrons through a transistor and, because RNA can makes lots of copies of a protein, the system has built-in amplification. Input control is via proteins that stick to segments of DNA called promoters and ‘switch on’ RNA pol (i.e., an analog input). After that the sequence of DNA itself can, in effect, say either ‘go’ or ‘stop’: short sequence motifs can wave RNA pol through or make it stall. The output signal is the protein made – and if you make green fluorescent protein (GFP) you can shine light on it and measure how much you’ve got from the fluorescence emitted.

Over the last few years a number of such gadgets have been made and inserted into bacterial cells to work as simple digital logic gates. In electronic-speak these have included DNA AND gates (giving a high output only if two inputs are high) and OR gates (a high output if one or both the inputs to the gate are high). They’re genetic transistors, processing signals like the logic gates built from transistors that, in combinations of billions, are the basis of computer memory and microprocessors.

Throwing a DNA switch

Throwing a DNA switch

So what’s new?

For biological gates the problem has been that each needs its own construct (a DNA plasmid) and to make more complicated bits {e.g., EXCLUSIVE OR (XOR) gates (high output only if the inputs are different) or EXCLUSIVE NOR (XNOR) gates (output high only if inputs equal)} lots of constructs are required, each having to be persuaded to enter bacteria and to work in a stable fashion.

Step forward Drew Endy and colleagues from Stanford who, by dint of some very clever molecular biology, have combined multiple logic elements into a single construct – which they call a ‘transcriptor’. The switching capacity of their devices comes from integrases – enzymes made by viruses that infect bacteria – that can invert (flip) short stretches of DNA. These can be designed as switchable ‘go’ or ‘stop’ signals for RNA pol. Back in the 1940s Barbara McClintock, working on maize, discovered that stretches of DNA can be shifted around within the genome – they’re called ‘transposons’ – and integrases do the same thing as the enzymes that switch transposons around. McClintock remains, incidentally, the only lady to win a Nobel Prize for Medicine on her own. The great thing about integrases is that they can be turned on simply by adding the appropriate activator to the medium surrounding the cells.

This remarkable advance means that essentially any kind of gate can be built into a single, synthetically made genetic transistor, regulated by a range of integrases. The potential is somewhat mind-boggling but includes being able to monitor in real time the effects of drugs on the behavior of individual cells.

When John Bardeen, Walter Brattain and William Shockley (a Brit by origin but really another Stanford man) invented the transistor (they got the 1956 Nobel Prize in Physics) they can have had little idea of the impact it would have on mankind. But they really would have been staggered to know that, 60 years on, their successors would be shaping our genetic material to act as semiconductors in living cells.

Anything else?

So, as far as I can see, Drew Endy and his chums have done pretty well everything except build an EOR gate that responds to any input with “Don’t blame me”. But they’re such smart guys I bet they’ve got one of those in the fridge too – it was just that the journal editor lacked a sense of humour and wouldn’t publish it. Science editors have form in this department – recall the tale of Albert Szent-Gyorgyi who, whilst a member of my department back in the 1920s, isolated ascorbic acid (the vitamin that stops you getting scurvy) and, convinced it was a sugar (so it should have the suffix -ose – it’s actually made from glucose by oxidation) but not knowing the exact structure, sent his results to the Biochemical Journal calling it ‘ignose’. When the editor said ignose was silly Albert suggested ‘godnose’, getting a predictable response!

References

http://www.guardian.co.uk/science/2012/aug/16/book-written-dna-codehttp://www.huffingtonpost.com/2013/03/29/biological-computer_n_2981753.html

Bonnet, J., Yin, P., Ortiz, M.E., Subsoontorn, P. and Endy, D. (2013). Amplifying Genetic Logic Gates. Science 28 March 2013 / Page 1/ 10.1126/science.1232758http://www.sciencemag.org/content/early/recent