Periodic Reporting for period 1 - REPLICHROM4D (Chromatin organization and mitotic inheritance of epigenetic states: genome-wide dynamics of H3.1 and H3.3 histone variants during DNA replication)
Periodo di rendicontazione: 2018-03-01 al 2020-02-29
In each cell of an organism, the genome is read in a distinct manner which defines its identity. The organization of the genome into chromatin is crucial in this respect. Indeed, chromatin packages the genome in the nucleus of eukaryotic cells by wrapping DNA around histones. As the building blocks of chromatin, histones can thus modulate all DNA-based transactions in the nucleus. This capacity to modulate genome function takes advantage of their versatility. In most organisms, histones exist as distinct variants that can each harbor different modifications and interact with specific partners. Histone variants, together with their modifications and binding partners, produce a variety of chromatin states that mark the genome to distinguish active or repressed regions according to cell identity. Each cell thus harbors a distinct chromatin signature.
It is still unclear whether this signature can be transmitted to daughter cells after division. Throughout the cell cycle, chromatin states are constantly challenged by the disassembly, mobilization and turnover of histones at thousands of different sites involved in gene transcription or chromosome organization. Most striking is the challenge of DNA replication during S phase. Each time the genome is copied, chromatin is disassembled and doubled, and parental histones are displaced. Both old and new histones must be hence deposited in a coordinate manner to restore the existing chromatin landscape. In this context, different histone variants rely on the partnership with specific chaperones that handle their recycling or de novo deposition.
A major unresolved issue is how these different variants are deposited throughout the genome as DNA is replicated. This is particularly relevant for cancer cells, which divide and replicate indefinitely disregarding their original set of instructions.
Our objective was thus to characterize the de novo deposition of selected variants on replicating chromatin at genome-wide resolution. We focused on two highly conserved variants of the histone H3 family that are frequently mutated in pediatric brain tumors, namely H3.1 and H3.3. We combined novel sequencing assays with in-depth bioinformatics analyses to investigate their deposition patterns during S phase. Our findings reveal an unanticipated layer of chromatin organization in different functional states, that depends on the distinct deposition pathways of these variants.
It is still unclear whether this signature can be transmitted to daughter cells after division. Throughout the cell cycle, chromatin states are constantly challenged by the disassembly, mobilization and turnover of histones at thousands of different sites involved in gene transcription or chromosome organization. Most striking is the challenge of DNA replication during S phase. Each time the genome is copied, chromatin is disassembled and doubled, and parental histones are displaced. Both old and new histones must be hence deposited in a coordinate manner to restore the existing chromatin landscape. In this context, different histone variants rely on the partnership with specific chaperones that handle their recycling or de novo deposition.
A major unresolved issue is how these different variants are deposited throughout the genome as DNA is replicated. This is particularly relevant for cancer cells, which divide and replicate indefinitely disregarding their original set of instructions.
Our objective was thus to characterize the de novo deposition of selected variants on replicating chromatin at genome-wide resolution. We focused on two highly conserved variants of the histone H3 family that are frequently mutated in pediatric brain tumors, namely H3.1 and H3.3. We combined novel sequencing assays with in-depth bioinformatics analyses to investigate their deposition patterns during S phase. Our findings reveal an unanticipated layer of chromatin organization in different functional states, that depends on the distinct deposition pathways of these variants.
We developed a new method to selectively follow the deposition of H3.1 and H3.3 at genome-wide resolution combining specific capture of newly synthesized variants with deep sequencing. In parallel, we implemented a set of bioinformatics tools and machine learning approaches to study their dynamics, investigate their deposition at specific sites, and explore associated properties.
An original manuscript describing the main findings is currently in preparation, and will be submitted for publication in a peer-reviewed journal. A review summarizing the state-of-the-art was published in the Journal of Cell Biology (Mendiratta et al., 2018). Preliminary results were presented at international conferences and public outreach events. In addition, we further used the tools that we developed in the framework of two collaborative projects that will be similarly submitted for publication.
An original manuscript describing the main findings is currently in preparation, and will be submitted for publication in a peer-reviewed journal. A review summarizing the state-of-the-art was published in the Journal of Cell Biology (Mendiratta et al., 2018). Preliminary results were presented at international conferences and public outreach events. In addition, we further used the tools that we developed in the framework of two collaborative projects that will be similarly submitted for publication.
Our work has been key in elucidating de novo deposition patterns of H3 variants in replicating cells. Our findings show how distinct deposition pathways enable to define a functional genome organization at a level that had not been appreciated before. The impact will be important for understanding how to coordinate replication timing and transcription in dividing cells to keep a distinct identity. Results are expected to provide significant insights into chromatin dynamics during DNA replication. This will not only further our understanding of how epigenetic states can be transmitted through cell division but also identify mechanisms that can go awry during tumorigenesis.