Final Report Summary - CHROMDECON (analysis of postmitotic chromatin decondensation)
The nucleus undergoes amazing structural and functional reorganizations during cell division. In most animal cells the nuclear envelope breaks down at the beginning of mitosis, the chromatin massively condenses to rod-shaped chromosomes and the mitotic spindle assembles in order to segregate the hereditary information equally to both emerging daughter cells – processes which have been and still are intensively studied in many laboratories. Much less is known how at the end of mitosis the interphase state of the nucleus is re-established so that the DNA can be transcribed and replicated. Cytological, the segregated chromatin decondenses and a nuclear envelope reassembles around the chromatin in order to establish the separation between cytoplasm and nuclear interior but the underlying molecular mechanisms of this essential cellular process have been not defined.
In this project we set out to study mitotic chromatin decondensation using and optimizing a novel, in our lab developed, cell free assay. Here, using Xenopus egg extracts and mitotic chromatin the decondensation leading to a functional nucleus can be faithfully reconstituted. We showed that chromatin decondensation requires ATP- and GTP-hydrolysis and is thus an active cellular process. By a biochemical fractionation approach we identified the first chromatin decondensation factors, a dodecameric complex formed by two ATPases, RuvBL1 and RuvBL2, also known as pontin and reptin. Depletion of this complex blocks chromatin decondensation in the in vitro assay and downregulation of these essential ATPases delays chromatin decondensation and extends telophase in living cells as analyzed by life cell imaging. Employing different cell free assays we could show that RuvBL1 and RuvBL2 are specifically involved in chromatin decondensation but are not crucially required in other events happening at the same time in cells like reformation of the nuclear envelope and pore complexes. Current efforts concentrate on identifying relevant RuvBL1/RuvBL2 interactors during decondensation and define how these factors contribute to the process.
The necessity of GTP-hydrolysis in chromatin decondensation suggests that GTPases are critically involved. By a biochemical fractionation approach we identified a pair of GTPase, DRG1 and DRG2 as decondensation GTPases: Depletion of the proteins blocks chromatin decondensation in the biochemical assay and, accordingly, downregulation of the proteins delays decondensation. We could show that these ill-defined GTPase function as microtubule regulators linking this function and spindle dynamics in general to chromatin decondensation.
Our work also revealed that mitotic chromatin deondensation is mechanistically different from sperm DNA deondensation despite the fact that both result in massive compaction of highly condensed chromatin. They require different factors: mitotic chromatin decondensation requires RuvBL1/2, a factor not needed for sperm decompaction. Vice versa, nucleoplasmin (NPM2) is crucially needed for sperm decompaction and mediates the exchange of protamines to normal histones. This exchange and hence nucleoplasmin is not needed for mitotic chromatin decondensation.
Entering an unchartered research territory the project required significant method development. We have, in the course of the project, refined the cell free assay, where chromatin decondensation can be analyzed and developed a computer software to automatically analyze the decondensation state. Using electron microscopy, we analyzed the chromatin changes during mitotic exit on the structural level showing how the highly compacted individual chromosomes decompact, merge to a single chromatin body and form a nucleus. We also established an assay where chromatin decondensation can be followed in living cells by life cell imaging. Here, relevant factors can be downregulated. We implemented a automated image analysis in this pipeline so that analysis of large numbers of mitotic events and cells for robust readouts and medium-sized screening approaches could be used. In addition to the screening possibility this setup also enabled us testing the hypothesis we generated from the biochemical approaches in living cells. These methodological advances have been also published and made accessible to the field and, accordingly, are now used by a number of research teams.
In this project we set out to study mitotic chromatin decondensation using and optimizing a novel, in our lab developed, cell free assay. Here, using Xenopus egg extracts and mitotic chromatin the decondensation leading to a functional nucleus can be faithfully reconstituted. We showed that chromatin decondensation requires ATP- and GTP-hydrolysis and is thus an active cellular process. By a biochemical fractionation approach we identified the first chromatin decondensation factors, a dodecameric complex formed by two ATPases, RuvBL1 and RuvBL2, also known as pontin and reptin. Depletion of this complex blocks chromatin decondensation in the in vitro assay and downregulation of these essential ATPases delays chromatin decondensation and extends telophase in living cells as analyzed by life cell imaging. Employing different cell free assays we could show that RuvBL1 and RuvBL2 are specifically involved in chromatin decondensation but are not crucially required in other events happening at the same time in cells like reformation of the nuclear envelope and pore complexes. Current efforts concentrate on identifying relevant RuvBL1/RuvBL2 interactors during decondensation and define how these factors contribute to the process.
The necessity of GTP-hydrolysis in chromatin decondensation suggests that GTPases are critically involved. By a biochemical fractionation approach we identified a pair of GTPase, DRG1 and DRG2 as decondensation GTPases: Depletion of the proteins blocks chromatin decondensation in the biochemical assay and, accordingly, downregulation of the proteins delays decondensation. We could show that these ill-defined GTPase function as microtubule regulators linking this function and spindle dynamics in general to chromatin decondensation.
Our work also revealed that mitotic chromatin deondensation is mechanistically different from sperm DNA deondensation despite the fact that both result in massive compaction of highly condensed chromatin. They require different factors: mitotic chromatin decondensation requires RuvBL1/2, a factor not needed for sperm decompaction. Vice versa, nucleoplasmin (NPM2) is crucially needed for sperm decompaction and mediates the exchange of protamines to normal histones. This exchange and hence nucleoplasmin is not needed for mitotic chromatin decondensation.
Entering an unchartered research territory the project required significant method development. We have, in the course of the project, refined the cell free assay, where chromatin decondensation can be analyzed and developed a computer software to automatically analyze the decondensation state. Using electron microscopy, we analyzed the chromatin changes during mitotic exit on the structural level showing how the highly compacted individual chromosomes decompact, merge to a single chromatin body and form a nucleus. We also established an assay where chromatin decondensation can be followed in living cells by life cell imaging. Here, relevant factors can be downregulated. We implemented a automated image analysis in this pipeline so that analysis of large numbers of mitotic events and cells for robust readouts and medium-sized screening approaches could be used. In addition to the screening possibility this setup also enabled us testing the hypothesis we generated from the biochemical approaches in living cells. These methodological advances have been also published and made accessible to the field and, accordingly, are now used by a number of research teams.