Periodic Reporting for period 3 - DYMOCHRO (Dynamics of modified chromatin domains)
Reporting period: 2022-07-01 to 2023-12-31
The cells of our body contain nearly the same genetic information but greatly differ in function. Accordingly, each cell interprets its genetic information in a distinct way. This involves chemical modifications of the DNA and the histone proteins, which are in close contact with the DNA. These modifications, which are often called “epigenetic”, are linked to the cellular gene expression program and the cellular identity. Deregulated modifications are often found in diseased cells, indicating that their faithful regulation is important for our health.
DNA and histone modifications are on the one hand dynamic, as they can change in response to different signals from outside. On the other hand, they are stable enough so that the cellular identity they encode can be maintained over time. How this balance between dynamic plasticity and stability is mechanistically regulated is largely unclear. It is also elusive how histone modifications contribute to the formation and maintenance of broad domains and subcompartments that are found across our genome. As many cellular proteins are involved in the regulation of DNA and histone modifications, which have multiple functions and are partly redundant, it is difficult to study this system using loss-of-function approaches in living cells.
The objective of this project is to reconstitute a minimal system to investigate the formation and maintenance of domains of modified histones, both in vitro and in living cells. On the one hand, this entails the development of single-molecule assays to visualize histone modifications on individual chromatinized templates in vitro and to assess their biophysical properties. On the other hand, this comprises the implementation of a CRISPR/dCas9-based system to generate ectopic modified domains in living cells.
DNA and histone modifications are on the one hand dynamic, as they can change in response to different signals from outside. On the other hand, they are stable enough so that the cellular identity they encode can be maintained over time. How this balance between dynamic plasticity and stability is mechanistically regulated is largely unclear. It is also elusive how histone modifications contribute to the formation and maintenance of broad domains and subcompartments that are found across our genome. As many cellular proteins are involved in the regulation of DNA and histone modifications, which have multiple functions and are partly redundant, it is difficult to study this system using loss-of-function approaches in living cells.
The objective of this project is to reconstitute a minimal system to investigate the formation and maintenance of domains of modified histones, both in vitro and in living cells. On the one hand, this entails the development of single-molecule assays to visualize histone modifications on individual chromatinized templates in vitro and to assess their biophysical properties. On the other hand, this comprises the implementation of a CRISPR/dCas9-based system to generate ectopic modified domains in living cells.
We have started to set up both systems mentioned above. For the in vitro system, we have purified recombinant human histones, including cysteine mutants that can be labeled with organic dyes. We have prepared histone octamers and have reconstituted nucleosomes on different DNA templates, which were either derived from phage lambda DNA or from arrays of strong “601” nucleosome positioning sequences. We have characterized the reconstituted material using nuclease digestion and gel shift assays. Furthermore, we have expressed and purified fusions of dCas9 and histone methyltransferase domains to deposit histone modifications on the reconstituted material, and we have expressed and purified recombinant heterochromatin protein 1 (HP1). We have built a prism-based TIRF microscope to visualize single DNA molecules with or without labeled nucleosomes in a microfluidic flow chamber, and we have started to compare different strategies to passivate these flow chambers in order to study different properties of the reconstituted material. The components to form model heterochromatin domains in vitro and study them by single-molecule microscopy are therefore available. For the live-cell system, we have prepared different versions of labeled dCas9-fused histone modifiers, and we have successfully recruited them to distinct target loci. We have started to compare different strategies to make the recruitment reversible.
Having established the minimal systems as planned, the next step will be to use them to investigate the dynamic regulation of modified chromatin domains.