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Mechanistic and single-cell dissection of repressive 3D chromatin organization at facultative heterochromatin domains – Heterochromatin_In_3D

Heterochromatin_ln_3D

Mechanistic and single-cell dissection of repressive 3D chromatin organization at facultative heterochromatin domains

Objectives

Cell fate choice and maintenance require a precise balance of gene activation and repression. An essential component in<br />these processes is the selective formation and maintenance of repressive heterochromatin domains with long-term stability and minimal variation among individual cells. I and others have found that different types ofmammalian facultative heterochromatin adopt specific 3D organizations within the cell nucleus. At the global scale, H3K9me2-marked heterochromatin is found at the repressive nuclear periphery, whereas the H3K27me3 mark clusters in distinct foci within the active nuclear interior. A t the local scale, H3K9me2 and H3K27me3-marked domains form 3D compartments that are physically separated from their surroundings. A defining function of these types of facultative heterochromatin therefore is the formation o f repressive microenvironments at different positions in the nucleus. Currently though, a precise understanding of how 3D organization is mechanistically linked to the repressive function offacultative heterochromatin, if there are functional differences between different types o f facultative heterochromatin and if the 3D organization plays a role in minimizing repressive variation within the cell population remains poorly understood.

My long-term objectives are to dissect the function o f 3D genome organization in facultative heterochromatin function, stability and consistency and to determine how changes in 3D heterochromatin organization support correct cell fate choice and maintenance. T o address these questions, I propose two research projects:
[1] Mechanistic dissection o f 3D facultative heterochromatin organization using a newly developed cellular platform that allows targeted modification o f 3D genome organization at specific gene loci.
[2] High-resolution studies of the link between 3D facultative heterochromatin organization and consistency of repression in individual cells, using a novel nanopore-based single-cell 4C-seq assay.

Using the @RAction funding, a 48-months work-program will be established at the CNRS-CGM / future I2BC (Gif-sur- Yvette) in which a post-doc, a PhD student, a technician and I will use cutting edge techniques like 4C-seq, microfluidic transcript profiling and ChlP-seq to mechanistically link 3D genome organization and facultative heterochromatin function, both within the cell population and in individual cells. An important aspect of this program is the development of two novel technological platforms: a nanopore sequencing-based assay for high-resolution study o f 3D genome organization in individual cells and a cell system that allows the systematic determination of regulatory protein function in 3D genome organization at defined places in the genome.

The outcome ofthese studies will provide important insights into our understanding ofepigenetics, nuclear organization and transcriptional regulation. Moreover, the development oftwo technological platforms will open up opportunities for collaboration, with early access for the French research community. Additionally, the single-cell 4C-seq assay may be exploited for commercial use in a clinical diagnostics setting. As a result, this project will importantly strengthen the position of the CGM / I2BC and the French research community in the field of 3D genome organization.

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Cell fate choice and maintenance require a precise balance of gene activation and repression. An essential component in these processes is the selective formation and maintenance of repressive heterochromatin domains with long-term stability and minimal variation among individual cells. I and others have found that different types of mammalian facultative heterochromatin adopt specific 3D organizations within the cell nucleus. At the global scale, H3K9me2-marked heterochromatin is found at the repressive nuclear periphery, whereas the H3K27me3 mark clusters in distinct foci within the active nuclear interior. At the local scale, H3K9me2 and H3K27me3-marked domains form 3D compartments that are physically separated from their surroundings. A defining function of these types of facultative heterochromatin therefore is the formation of repressive microenvironments at different positions in the nucleus. Currently though, a precise understanding of how 3D organization is mechanistically linked to the repressive function of facultative heterochromatin, if there are functional differences between different types of facultative heterochromatin and if the 3D organization plays a role in minimizing repressive variation within the cell population remains poorly understood.

My long-term objectives are to dissect the function of 3D genome organization in facultative heterochromatin function, stability and consistency and to determine how changes in 3D heterochromatin organization support correct cell fate choice and maintenance. To address these questions, I propose two research projects:
[1] Mechanistic dissection of 3D facultative heterochromatin organization using a newly developed cellular platform that allows targeted modification of 3D genome organization at specific gene loci.
[2] High-resolution studies of the link between 3D facultative heterochromatin organization and consistency of repression in individual cells, using a novel nanopore-based single-cell 4C-seq assay.

Using the @RAction funding, a 48-months work-program will be established at the CNRS-CGM / future I2BC (Gif-sur-Yvette) in which a post-doc, a PhD student, a technician and I will use cutting edge techniques like 4C-seq, microfluidic transcript profiling and ChIP-seq to mechanistically link 3D genome organization and facultative heterochromatin function, both within the cell population and in individual cells. An important aspect of this program is the development of two novel technological platforms: a nanopore sequencing-based assay for high-resolution study of 3D genome organization in individual cells and a cell system that allows the systematic determination of regulatory protein function in 3D genome organization at defined places in the genome.

The outcome of these studies will provide important insights into our understanding of epigenetics, nuclear organization and transcriptional regulation. Moreover, the development of two technological platforms will open up opportunities for collaboration, with early access for the French research community. Additionally, the single-cell 4C-seq assay may be exploited for commercial use in a clinical diagnostics setting. As a result, this project will importantly strengthen the position of the CGM / I2BC and the French research community in the field of 3D genome organization.

Project coordination

Daan Noordermeer (CNRS - Institute for Integrative Biology of the Cell (I2BC) - UMR 9198)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS - Institute for Integrative Biology of the Cell (I2BC) - UMR 9198

Help of the ANR 449,996 euros
Beginning and duration of the scientific project: June 2015 - 48 Months

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