Chromosome dynamics and genome stability

The genetic information stored in our DNA must be accurately expressed, duplicated and maintained to allow cellular proliferation, differentiation and development to a multicellular organism. Eukaryotic DNA replication starts at multiple sites throughout the genome and is necessarily coordinated with other chromosomal processes including transcription, chromatin assembly and maturation, recombination and DNA repair. Notably, chromosomes provide the fundamental scaffold for all these dynamic and in part simultaneously occurring processes. How are these molecular activities coordinated on our genome? Can some of the complexes interfere with each other at certain regions of the genome? Our ultimate goal is to understand the genetic and epigenetic principles how these fundamental processes are regulated and coordinated to work together on the genome of eukaryotic cells.

We use innovative cell biological, genetic and proteomic approaches in yeast and human cells as model organisms. We are particularly interested to identify the molecular players and characterize the sequence of events that allow DNA replication and transcription to occur simultaneously on our chromosomes - without major accidents leading to DNA damage and genome instability, a hallmark of cancer and many other human diseases.

What are the genetic and epigenetic principles to avoid transcription-replication interference?

DNA replication and transcription complexes initiate synthesis of complementary DNA or RNA strands from distinct genomic locations, termed origins and promoters, respectively. Importantly, chromatin presents the natural substrate of these DNA-templated processes. Eukaryotic chromatin is associated, interpreted and modified by numerous constituents, including histone posttranslational modifications, transcription factors, DNA and RNA metabolizing machineries, architectural proteins, chromatin remodeling and modifying enzymes, as well as chromatin-associated RNA molecules. Thus, to understand the molecular basis of these DNA transactions, it is critical to define the collective changes of the chromatin structure at the genomic regions where the transcription and replication machineries assemble and drive their biological reactions.

We further developed a method based on site-specific recombination to purify specific native chromosomal domains from yeast. Using this technique we can enrich a single copy locus at least 150,000-fold over every other genomic locus. Most importantly, the chromatin structure of the isolated material conserves important features of the chromatin in vivo. We use a combination of compositional, structural and functional analyses to identify the molecular players and the sequence of events that allow replication and transcription initiation. These studies will not only give deep insights into the chromatin-based rules of origin and promoter activation, but also contribute to further our understanding of the coordination of the replication timing and gene expression programs in eukaryotic cells, deregulated in a myriad of human disease states.

Figure 1:

A. System Conditionally expressed recombinase excises a genomic locus of interest (e.g. transcription or replication initiation site) flanked by RS elements (RS, boxed arrowheads) in form of a chromatin ring. After cell lysis, the soluble chromatin circles are purified via a recombinant LexA-TAP fusion protein (LexA-TAP, bracket connected to a line), binding to the LEXA DNA binding sites (LEXA, grey box) as well as to an affinity support (filled rectangle). Filled ovals represent chromatin components. B. Composition Cartoons show the N- and C-terminal tails of H3, H2A, H2B and H4 with relevant amino acids and their known modifications including acetylation, methylation, phosphorylation and ubiquitylation. C. Structure Isolated chromatin rings are subjected to psoralen crosslinking (black crosses). After DNA isolation, crosslinked molecules are denatured and analyzed by electron microscopy. Representative electron micrographs for circular and linearized single molecules are shown. D. Function The isolated native chromatin domains can be used for in vitro transcription and replication assays.

Further Information:

Brown, C.R., Eskin, J.A., Hamperl, S., Griesenbeck, J., Jurica, M.S., and Boeger, H. (2015). Chromatin structure analysis of single gene molecules by psoralen cross-linking and electron microscopy. Methods Mol. Biol. 1228, 93–121.

Hamperl, S., Brown, C.R., Perez-Fernandez, J., Huber, K., Wittner, M., Babl, V., Stöckl, U., Boeger, H., Tschochner, H., Milkereit, P., Griesenbeck J., (2014). Purification of specific chromatin domains from single-copy gene loci in Saccharomyces cerevisiae. Methods Mol. Biol. 1094, 329–341.

Hamperl, S., Brown, C.R., Garea, A.V., Perez-Fernandez, J., Bruckmann, A., Huber, K., Wittner, M., Babl, V., Stoeckl, U., Deutzmann, R., Boeger, H., Tschochner H., Milkereit P., Griesenbeck J. (2014). Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae. Nucleic Acids Res. 42, e2.

What are the genetic and epigenetic principles to tolerate and resolve transcription-replication interference?

Once initiated, transcription and replication machineries translocate along the same DNA template, often in opposing directions and at different rates. Mounting evidence suggests that transcription complexes can encounter replication forks on eukaryotic chromosomes and cells rely on numerous mechanisms to tolerate and resolve such transcription-replication conflicts. The absence of these mechanisms can lead to catastrophic effects on genome stability and cell viability.

We recently established an in vivo system to reconstitute and analyze encounters between the replication fork and a specific type of transcriptional barrier named R-loop in an inducible and localized fashion. In particular, we studied the influence of conflict orientation (head-on vs. co-directional, Figure 2) on R-loop formation and DNA damage during transcription-replication conflicts. Interestingly, a co-directional conflict can resolve R-loops, whereas a confrontation in head-on orientation stabilizes these structures. Thus, the co-orientation bias of transcription and replication in the human genome may help to minimize deleterious R-loops and maintain genomic stability. Since replication profiles are altered in cancer cells, this co-orientation bias may be perturbed and lead to an increased frequency of head-on conflicts, R-loop accumulation and genome instability in cancer genomes.

Using this system and other cell biological, genetic and proteomic approaches, we aim to elucidate the genetic and epigenetic mechanisms how cells respond, tolerate and resolve different types of transcription-replication conflicts. These studies could provide insights into the many emerging links between transcription-replication conflicts and genome instability, which are observed in development, cancer and many other physiological and disease contexts.

Further Information:

Hamperl, S., Bocek M., Saldivar, J. C., Swigut T., and Cimprich, K. A. (2017). Transcription-replication conflict orientation modulates R-loop levels and activates distinct DNA damage responses. Cell 170, 774–786. 

Hamperl, S., and Cimprich, K. A. (2016). Conflict Resolution in the Genome: How Transcription and Replication Make It Work. Cell 167, 1455–1467 (2016) 

Hamperl, S., and Cimprich, K.A. (2014). The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability. DNA Repair (Amst.) 19, 84–94.