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Need for speed – measuring genome interactions in living cells

A second is only a blink of an eye in our daily life. However, a lot is happening within one second to a protein in the nucleus of a living cell. For following its binding to the genome one has to conduct measurements with a resolution of 1/1000 second. To do so scientists from the DKFZ in Heidelberg have developed a new method that allowed them to trace the binding of certain molecular machines, the chromatin remodelers. These protein complexes can reorganize the genome to direct the readout of the DNA sequence information to certain parts..

The human genome consists of DNA wrapped around small histone proteins. These globular "nucleosome" complexes are connected by segments of protein free linker DNA into a chain with a pearl necklace like structure. Gene activation requires freely accessible DNA, and genes can be inactivated by occluding DNA parts within a nucleosome. Hence, the nucleosome positions determine a readout pattern: Information in the DNA linker region between nucleosomes is more easily accessible as opposed to DNA sequences that are located within a nucleosome. Making DNA accessible, or to cover it in a nucleosome, is done by a specific class of proteins in the cell nucleus, the chromatin remodelers. These energy-consuming molecular machines can move nucleosomes along the DNA chain to establish accessibility patterns on newly synthesized DNA during cell division, and to switch between "on" and "off" states of a gene. They are an essential part of a regulatory network that allows the cell to select specific programs to differentiate into a brain cell or a skin cell from the identical genetic information.

How do chromatin remodelers rearrange the genome to control the readout of the DNA information in the cell nucleus? Scientists at the DKFZ in the group of Karsten Rippe were trying to figure this out for quite some time. By pushing their advanced fluorescence microscopy equipment to the limit, they learnt that under normal conditions about 1 million chromatin remodelers continuously bind and dissociate to nucleosomes to test whether all of the 30 million nucleosomes found in a single cell are at the "right" position. "You have to be fast to follow chromatin remodelers in living cells down to the 1/1000 second time frame" says Karsten Rippe "but at the same time you also need to track them if they bind for seconds or minutes, and that was impossible with the techniques we have been using." Luckily, Fabian Erdel, a PhD student in the group, came up with an idea on how to measure the binding of proteins to the genome in the time regime that could not be studied so far. The graduate student conducted experiments, in which he extinguished a fluorescent tag attached to chromatin remodelers by illuminating a part of the sample with a high-intensity laser beam. He noticed that a "shadow" appeared from bleached proteins that had moved while the fluorescence microscopy image was still being recorded. And the shape of this shadow changed if proteins moved faster or were slowed down because they were binding to nucleosomes.

 

Experimental microscopy images after bleaching the fluorescence in a circular or rectangular region. The dark regions represent bleached proteins that have moved while the image was recorded.

"It was challenging to figure out a way to calculate how long a chromatin remodeler binds to a nucleosome from the shadow image", he says, "but now the method, which we call Pixel-wise Photobleaching Profile Evolution Analysis or 3PEA, turns out to have new exciting applications, since it has great time resolution and also the measurement itself can be done within a second." With their new 3PEA method, the DKFZ researchers measured that a chromatin remodeler translocates within one second through almost the whole cell nucleus in a random walk and tests more than 300 nucleosomes without becoming active. And only occasionally a nucleosome that carries specific signals would keep the remodeler for seconds or even minutes to become shifted to a new position on the DNA. Thus, nucleosome positioning by chromatin remodelers seems to be a highly regulated process that requires specific signals to move a nucleosome. The misinterpretation of these signals due to aberrant chromatin remodeler activity is linked to several types of cancers. Thus, the next challenge will be to decipher the signals the "chromatin remodeling code" that tells the remodelers to move one nucleosome but not others.

Erdel, F. & Rippe, K. (2012). Quantifying transient binding of ISWI chromatin remodelers in living cells by pixel-wise photobleaching profile evolution analysis. Proc. Natl. Acad. Sci. USA, published online 5 November 2012. doi: 10.1073/pnas.1209579109 | Abstract | Reprint (3.4 MB)

Press release from the University of Heidelberg (English)

Press release from the University of Heidelberg (German)

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