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Telomeres, RNA and nucleosomes - five great papers from 2011

This fall we successfully finished studies on three important topics of our lab: (1) For the alternative telomere lengthening (ALT) pathway that is active in some tumors we could show how telomere extension is induced via a DNA repair mechanism (Chung et al., J. Cell Sci.; Osterwald et al., Biotechnol. J.). (2) A previously unknown structural function of RNA for stabilizing active transcription compartments in the cell nucleus was discovered (Caudron-Herger et al, Nucleus), and nuclear architecture by RNA in general was reviewed (Caudron-Herger & Rippe, Curr. Opin. Genet. Dev.). (3) The unwrapping of DNA from the nucleosome was modelled in the context of transcription factor binding to DNA (Ettig et al., Biophys. J.; Teif & Rippe, Briefings Bioinf.).

Alternate ending - living on without telomerase

The ends of the chromosomes, the telomeres, are repetitive DNA sequences that shorten every time a cell divides during the process of duplicating its genome. Once the telomeres become very short the cell stops dividing. Thus, telomeres work like a cellular clock that keeps an eye on the number of cell divisions. And once the cell's time is over it can no longer divide. Circumventing this control mechanism is crucial for tumor cells in order to proliferate without limits. In the majority of tumors this is accomplished by reactivating telomerase, an enzyme that normally extends the telomeres only in embryonic cells, and thus resets the cellular clock during development. However, a 10-15% fraction of tumors keeps on dividing without telomerase by making use of what is called the ALT-mechanism for "Alternative Lengthening of Telomeres". The hallmark of ALT cancer cells is a special type of complexes of promyelocytic leukemia (PML) protein at the telomeres that are termed ALT-associated PML nuclear bodies or APBs.ALT-tumors can be identified by the presence of APBs on fluorescence microscopy images since normal cells do not have these structures. However, the function of APBs has remained mysterious. In a recent study, Inn Chung and Karsten Rippe from the German Cancer Research Center together with Heinrich Leonhard from the LMU in Munich applied a novel approach to study APBs. They succeeded in artificially making APBs in living cells by tethering PML and other APB proteins to the telomeres. In this manner they could not only trace the assembly of APBs but were able to investigate what happens after APB formation. They could show that the de novo formed APBs induced the extension of the telomere repeat sequence by a DNA repair synthesis mechanism. This demonstrates for the first time that APBs have an important function for the alternative telomere lengthening mechanism, and suggests that disrupting APBs would stop proliferation of ALT-positive tumor cells once their telomeres become too short. This makes APBs a promising new target of cancer cells, in which the ALT mechanism is active.

Chung, I., Leonhardt, H. & Rippe, K. (2011). De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation. J. Cell Sci. 124, 3603-3618. doi: 10.1242/jcs.084681 | Abstract | Reprint (9.4 MB) | Comment

Osterwald, S., Wörz, S., Reymann, J., Sieckmann, F., Rohr, K., Erfle, H. & Rippe, K. (2012). A three-dimensional colocalization RNA interference screening platform to elucidate the alternative lengthening of telomeres pathway. Biotechnol. J. 7, 103-116. doi: 10.1002/biot.201000474 | Abstract | Reprint (1.8 MB)

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The return of the coding RNAs

The non-coding RNA "dark side" of the genome has generated a lot of buzz over the last years. What started off with small RNAs in control of protein translation in the cytoplasm has now spread into the rest of the cell. Judging from a slew of high-profile papers, the non-coding RNA Empire is gearing up for another strike to take over the nucleus, too, with its lncRNA and lincRNA troops claiming supremacy of all things chromatin and epigenetics. However, it appears that the messenger RNA Alliance is not ready to surrender yet, and is preparing for a return from the outer territories to become more than just a boring protein production template.

Via a combination of high-resolution fluorescence microscopy, biochemical RNA purification and a genome-wide analysis by deep sequencing the study by Caudron-Herger et al. reveals that some coding RNAs have an unexpected function for maintaining the structure of active transcription compartments via their 3'-untranslated regions. Admittedly, these parts of the transcript make a certain amount of "non-codingness" hard to avoid even for pure mRNAs. But hey, have a closer look at the dark side: Didn't some short open reading frames found in "non-coding" RNAs secretly turn out to be translated into peptides?

Within the RNA universe, it seems the lines between Jedi knights and the dark side of the Force are blurring. As master Yoda might have said: "Meditate on this, I will." (This text was adapted from the Comment in EpiGenie on our paper.)

Caudron-Herger, M., Müller-Ott, K., Mallm, J.-P., Marth, C., Schmidt, U., Fejes-Tóth, K. & Rippe, K. (2011). Coding RNAs with a non-coding function: maintenance of open chromatin structure, Nucleus 2, 410-424. doi: 10.4161/nucl.2.5.17736 | Abstract | Reprint (4.4 MB)

Caudron-Herger, M. & Rippe, K. (2012). Nuclear architecture by RNA. Curr. Opin. Genet. Dev. 22, doi: 10.1016/j.gde.2011.12.005,

 

Accessing DNA in the nucleosome

The nucleosome complex of DNA wrapped around a histone protein octamer organizes the genome of eukaryotes and regulates the access of protein factors to the DNA. We performed molecular dynamics simulations of the nucleosome in explicit water to study the dynamics of its histone-DNA interactions as described in Ettig et al. A high-resolution histone-DNA interaction map was derived that revealed a five-nucleotide periodicity, in which the two DNA strands of the double helix made alternating contacts. On the 100-ns timescale, the histone tails mostly maintained their initial positions relative to the DNA, and the spontaneous unwrapping of DNA was limited to 1-2 basepairs. In steered molecular dynamics simulations, external forces were applied to the linker DNA to investigate the unwrapping pathway of the nucleosomal DNA. This detailed analysis of DNA-histone interactions revealed molecular mechanisms for modulating access to nucleosomal DNA via conformational rearrangements of its structure.

 

Computer simulations of the dynamic structure of a nucleosome. The nucleosome consists of an octamer histone protein complex with DNA wrapped around it in almost two turns. It represents the basic building block of chromatin. In a human cell about 30 million nucleosomes organize the genome of 6 billion DNA base pairs. In the image nucleosome conformations are overlayed in 0.2 nanosecond time intervals. The DNA is color coded with increasing simulation time from red to white to blue. The core histone proteins are shown in white. Already during the very short simulation time period of 2 nanoseconds the nucleosome conformation is very dynamic. For further details of investigating nucleosome and chromatin features in computer simulations see Ettig et al. 2011,

 

How the unwrapping of nucleosomal DNA affects the access of transcription factor binding to the genome in a competive binding equilibrium was investigated in the second paper on this topic (Teif & Rippe, 2011).

Ettig, R., Kepper, N., Stehr, R., Wedemann, G. & Rippe, K. (2011). Dissecting DNA-histone interactions by molecular dynamics simulations of unwrapping DNA from the nucleosome, Biophys. J. 101, 1999-2008. doi: 10.1016/j.bpj.2011.07.057 | Abstract | Reprint (5.8 MB)

Teif, V. & Rippe, K. (2012). Calculating transcription factor binding to nucleosomal DNA for large genomic regions, Briefings Bioinf. 12, doi: 10.1093/bib/bbr037 | Abstract | Reprint (0.4 Mb)

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