Chromatin and cell biology

Genome dynamics

The genome is more than a linear string of genes. It has secondary, tertiary and quaternary higher-order structures. Strings of genes organize in chromosomal domains that are characterized by one of relatively few different types of chromatin, either repressive or activating. In the 3D space, each of the elements in one domain makes frequent contacts with other elements in the same domain. These entities have been called physical- or topologically associated- domains (TADs).
At a chromosome level, individual TADs form contacts with other TADs, preferentially of the same type, in order to build ordered 3D architectures that are called chromosome territories. Finally, different chromosomes organize non randomly in the nuclear space.
Therefore, the eukaryotic genome is highly organized in 3D and this regulation can be transmitted or modulated during the life of cells and organisms.
This sophisticated chromosomal choreography involves thousands of different players – DNA sequences, RNAs and proteins – but rather than combining in infinite numbers of ways, these components organize a relatively limited number of types of chromatin, either active or repressive. In particular, two main groups of genome regulatory components are proteins of the Polycomb Group (PcG) and of the trithorax Group (trxG). PcG proteins are maintain the memory of silent states of gene expression through cell physiology and multiple cell divisions, while trxG members maintain active chromatin states
These proteins are able to recognize regulatory states of their target genes and to maintain these states even after disappearance of the primary transcriptional regulators that have induced them in the first place. Remarkably, these states can also be transmitted to a fraction of the progeny over multiple generations. In our lab, we aim at understanding the principle governing 3D genome organization, its functional implications, and the molecular mechanisms by which PcG and trxG proteins regulate their target genes, convey inheritance of chromatin states and orchestrate development.
To reach this goal, we employ a variety of complementary approaches and techniques in the areas of molecular, cellular and developmental biology, genomics and bioinformatics.

86
BANTIGNIES Frederic
Responsable Scientifique
Researcher


20
CAVALLI Giacomo
Researcher

167
CHEUTIN Thierry
Researcher

172
MARTINEZ Anne-Marie
Chef d'équipe
Researcher


1709
PALDI Flora
Postdoc

1405
PAPADOPOULOS Giorgio
Ingénieur
Postdoc


1750
AKILLI Nazli
PhD Student

1593
DENAUD Sandrine
PhD Student

1612
PARRENO Victoria
PhD Student

1781
REBOUL Hadrien
PhD Student

1674
SZALAY Michael
PhD Student

PUBLICATIONS OF THE TEAM

Understanding 3D genome organization by multidisciplinary methods

Ivana Jerkovic´ & Giacomo Cavalli

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Regulation of single-cell genome organization into TADs and chromatin nanodomains

Szabo, Q., Donjon, A., Jerkovic, I., Papadopoulos, G.L., Cheutin, T., Bonev, B., Nora, E., Bruneau, B.G., Bantignies, F., and Cavalli, G.

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4D Genome Rewiring during Oncogene-Induced and Replicative Senescence

Sati, S., Bonev, B., Szabo, Q., Jost, D., Bensadoun, P., Serra, F., Loubiere...

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Widespread activation of developmental gene expression characterized by PRC1-dependent chromatin looping.

Loubiere V, Papadopoulos GL, Szabo Q, Martinez AM, Cavalli G

Global chromatin conformation differences in the Drosophila dosage compensated chromosome X.

Pal K, Forcato M, Jost D, Sexton T, Vaillant C, Salviato E, Mazza EMC, Lugli E, Cavalli G, Ferrari F

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The multiscale effects of polycomb mechanisms on 3D chromatin folding.

Cheutin T, Cavalli G

Higher-Order Chromosomal Structures Mediate Genome Function.

Jerković I, Szabo Q, Bantignies F, Cavalli G

Advances in epigenetics link genetics to the environment and disease

Giacomo Cavalli and Edith Heard

Principles of genome folding into topologically associating domains.

Szabo Q, Bantignies F, Cavalli G

Microscopy-Based Chromosome Conformation Capture Enables Simultaneous Visualization of Genome Organization and Transcription in Intact Organisms.

Cardozo Gizzi AM, Cattoni DI, Fiche JB, Espinola SM, Gurgo J, Messina O, Houbron C, Ogiyama Y, Papadopoulos GL, Cavalli G, Lagha M, Nollmann M

Cell Fate and Developmental Regulation Dynamics by Polycomb Proteins and 3D Genome Architecture.

Loubiere V, Martinez AM, Cavalli G

Challenges and guidelines toward 4D nucleome data and model standards.

Marti-Renom MA, Almouzni G, Bickmore WA, Bystricky K, Cavalli G, Fraser P, Gasser SM, Giorgetti L, Heard E, Nicodemi M, Nollmann M, Orozco M, Pombo A, Torres-Padilla ME

Loss of PRC1 induces higher-order opening of Hox loci independently of transcription during Drosophila embryogenesis.

Cheutin T, Cavalli G

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Polycomb-Dependent Chromatin Looping Contributes to Gene Silencing during Drosophila Development.

Ogiyama Y, Schuettengruber B, Papadopoulos GL, Chang JM, Cavalli G

TADs are 3D structural units of higher-order chromosome organization in Drosophila.

Szabo Q, Jost D, Chang JM, Cattoni DI, Papadopoulos GL, Bonev B, Sexton T, Gurgo J, Jacquier C, Nollmann M, Bantignies F, Cavalli G

Technical Review: A Hitchhiker's Guide to Chromosome Conformation Capture.

Grob S, Cavalli G

Single-cell absolute contact probability detection reveals chromosomes are organized by multiple low-frequency yet specific interactions

Cattoni DI, Gizzi AMC, Georgieva M, Di Stefano M, Valeri A, Chamousset D, Houbron C, Déjardin S, Fiche JB, González I, Chang JM, Sexton T, Marti-Renom MA, Bantignies F, Cavalli G, Nollmann M.

Chromosome conformation capture technologies and their impact in understanding genome function

Sati S, Cavalli G.

Three-Dimensional Genome Organization and Function in Drosophila

Schwartz YB, Cavalli G.

Stable Polycomb-dependent transgenerational inheritance of chromatin states in Drosophila

Ciabrelli F, Comoglio F, Fellous S, Bonev B, Ninova M, Szabo Q, Xuéreb A, Klopp C, Aravin A, Paro R, Bantignies F, Cavalli G

Chromosome topology guides the Drosophila Dosage Compensation Complex for target gene activation

Schauer T, Ghavi-Helm Y, Sexton T, Albig C, Regnard C, Cavalli G, Furlong EE, Becker PB.

Genome Regulation by Polycomb and Trithorax: 70 Years and Counting

Schuettengruber, B., Bourbon, HM., Di Croce, L., Cavalli, G.

Multi-scale 3D genome rewiring during mouse neural development.

Bonev, B., Mendelson Cohen, N., Szabo, Q., Fritsch, L., Papadopoulos, G., Lubling, Y., Xu, X., Hugnot, JP., Tanay, A., Cavalli, G.

EZH2 in normal hematopoiesis and hematological malignancies

Herviou L, Cavalli G, Cartron G, Klein B, Moreaux J.

Regulation of Genome Architecture and Function by Polycomb Proteins

Entrevan M, Schuettengruber B, Cavalli G

Coordinate redeployment of PRC1 proteins suppresses tumor formation during Drosophila development

Loubière, V., Delest, A., Thomas, A., Bonev, B., Schuettengruber, B., Sati, S., Martinez AM., Cavalli, G.

Following the Motion of Polycomb Bodies in Living Drosophila Embryos

Cheutin T, Cavalli G.

Chromosome Conformation Capture on Chip (4C): Data Processing

Leblanc B, Comet I, Bantignies F, Cavalli G.

Organization and function of the 3D genome

Bonev B, Cavalli G.

Chromatin driven behavior of topologically associating domains

Ciabrelli F, Cavalli G.

Developmental determinants in non-communicable chronic diseases and ageing

Bousquet J, Anto JM, Berkouk K, Gergen P, Pinto Antunes J, Augé P, Camuzat T, Bringer J, Mercier J, Best N, Bourret R, Akdis M, Arshad SH, Bedbrook A, Berr C, Bush A, Cavalli G, Charles MA, Clavel-Chapelon F, Gillman M, Gold DR, Goldberg M, Holloway JW, Iozzo P, Jacquemin S, Jeandel C, Kauffmann F, Keil T, Koppelman GH, Krauss-Etschmann S, Kuh D, Lehmann S, Lodrup Carlsen KC, Maier D, Méchali M, Melén E, Moatti JP, Momas I, Nérin P, Postma DS, Ritchie K, Robine JM, Samolinski B, Siroux V, Slagboom PE, Smit HA, Sunyer J, Valenta R, Van de Perre P, Verdier JM, Vrijheid M, Wickman M, Yiallouros P, Zins M.

The Role of Chromosome Domains in Shaping the Functional Genome

Sexton, T., Cavalli, G.

Histone H3 Serine 28 Is Essential for Efficient Polycomb-Mediated Gene Repression in Drosophila

Yung PY, Stuetzer A, Fischle W, Martinez AM, Cavalli G

Distinct polymer physics principles govern chromatin dynamics in mouse and Drosophila topological domains

Ea V, Sexton T, Gostan T, Herviou L, Baudement MO, Zhang Y, Berlivet S, Le Lay-Taha MN, Cathala G, Lesne A, Victor JM, Fan Y, Cavalli G, Forné T.

Enhancer of zeste acts as a major developmental regulator of Ciona intestinalis embryogenesis

Le Goff E, Martinand-Mari C, Martin M, Feuillard J, Boublik Y, Godefroy N, Mangeat P, Baghdiguian S, Cavalli G

PRC1 proteins orchestrate three-dimensional genome architecture

Cavalli, G.

Chromosomes: now in 3D!

Cavalli, G.

Polycomb silencing: from linear chromatin domains to 3D chromosome folding

Cheutin T, Cavalli G

A RING to Rule Them All: RING1 as Silencer and Activator

Cavalli, G.

Identification of Regulators of the Three-Dimensional Polycomb Organization by a Microscopy-Based Genome-wide RNAi Screen

Gonzalez I, Mateos-Langerak J, Thomas A, Cheutin T, Cavalli G.

Modeling epigenome folding: formation and dynamics of topologically associated chromatin domains

Jost D, Carrivain P, Cavalli G, Vaillant C.

Topological organization of Drosophila hox genes using DNA fluorescent in situ hybridization

Bantignies, F., Cavalli, G.

Cooperativity, Specificity, and Evolutionary Stability of Polycomb Targeting in Drosophila

Schuettengruber B, Oded Elkayam N, Sexton T, Entrevan M, Stern S, Thomas A, Yaffe E, Parrinello H, Tanay A, Cavalli G.

Chromosomal domains: epigenetic contexts and functional implications of genomic compartmentalization.

Tanay A, Cavalli G.

Polycomb domain formation depends on short and long distance regulatory cues.

Schuettengruber B, Cavalli G.

Functional implications of genome topology

Cavalli, G., Mistelli, T.

The 3D Genome Shapes Up For Pluripotency.

Sexton T, Cavalli G.

PRC2 Controls Drosophila Oocyte Cell Fate by Repressing Cell Cycle Genes.

Iovino N, Ciabrelli F, Cavalli G.

Three-dimensional genome organization: a lesson from the Polycomb-Group proteins

Bantignies, F.

Molecular biology. EZH2 goes solo

Cavalli, G.

Three-Dimensional Folding and Functional Organization Principles of the Drosophila Genome.

Sexton T, Yaffe E, Kenigsberg E, Bantignies F, Leblanc B, Hoichman M, Parrinello H, Tanay A, Cavalli G.

Polycomb: a paradigm for genome organization from one to three dimensions.

Delest A, Sexton T, Cavalli G.

Progressive polycomb assembly on H3K27me3 compartments generates polycomb bodies with developmentally regulated motion

Cheutin T, Cavalli G.

Polycomb-dependent Regulatory Contacts between Distant Hox Loci in Drosophila,

Bantignies, F., Roure, V., Comet, I., Leblanc, B., Schuttengruber, B., Bonnet, J., Tixier, V., Mas, A., Cavalli, G.

A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber.

Comet, I., Schuettengruber, B., Sexton, T., Cavalli, G

From linear genes to Epigenetic inheritance of three dimensional Epigenomes.

Cavalli, G.

Chromatin folding : from linear chromosomes to the 4D nucleus.

Cheutin, T., Bantignies, F., Leblanc, B., Cavalli, G.

Editorial Review

Hetzer, M., Cavalli, G

Polycomb group proteins: repression in 3D.

Bantignies F, Cavalli G.

Rolling ES Cells Down the Waddington Landscape with Oct4 and Sox2

Iovino, N., Cavalli, G.

Trithorax group proteins: switching genes on and keeping them active.

Schuettengruber B, Martinez AM, Iovino N, Cavalli G.

Uncovering a tumor-suppressor function for Drosophila polycomb group genes.

Martinez, AM., Cavalli, G

The DUBle life of polycomb complexes

Schuettengruber, B., Cavalli, G

Epigenetics and the control of multicellularity. Workshop on Chromatin at the Nexus of Cell Division and Differentiation.

Reuter G, Cavalli G.

Functional Anatomy of Polycomb and Trithorax Chromatin Landscapes in Drosophila Embryos

Schuettengruber B, Ganapathi M, Leblanc B, Portoso M, Jaschek R, Tolhuis B, van Lohuizen M, Tanay A, Cavalli G.

Polyhomeotic has a tumor suppressor activity mediated by repression of Notch signaling

Martinez, AM., Schuettengruber, B., Sakr, S., Janic, A., Gonzalez, C., and Cavalli, G.

Genomic interactions: Chromatin loops and gene meeting points in transcriptional regulation

Sexton, T., Bantignies, F., Cavalli, G.

Recruitment of polycomb group complexes and their role in the dynamic regulation of cell fate choice.

Schuettengruber B, Cavalli G.

The Role of RNAi and Noncoding RNAs in Polycomb Mediated Control of Gene Expression and Genomic Programming.

Portoso, M, and Cavalli, G.

Chapter 2 polycomb group proteins and long-range gene regulation.

Mateos-Langerak J, Cavalli G

Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions.

Lanctot C, Cheutin T, Cremer M, Cavalli G, Cremer T

Genome Regulation by Polycomb and Trithorax Proteins.

Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G.

Chromosome kissing

Cavalli, G.

Visualizing macromolecules with fluoronanogold: from photon microscopy to electron tomography.

Cheutin, T., Sauvage, C., Tchélidzé, P., O

Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex.

Lanzuolo C, Roure V, Dekker J, Bantignies F, Orlando V.

Idefix insulator activity can be modulated by nearby regulatory elements.

Brasset E, Bantignies F, Court F, Cheresiz S, Conte C, Vaury C.

Cellular memory and dynamic regulation of polycomb group proteins.

Bantignies F, Cavalli G.

Chromatin and epigenetics in development: blending cellular memory with cell fate plasticity.

Cavalli G.

RNAi components are required for nuclear clustering of Polycomb group response elements.

Grimaud C, Bantignies F, Pal-Bhadra M, Ghana P, Bhadra U, Cavalli G.

Polycomb group-dependent Cyclin A repression in Drosophila.

Martinez AM, Colomb S, Dejardin J, Bantignies F, Cavalli G.

The role of polycomb group proteins in cell cycle regulation during development.

Martinez AM, Cavalli G.

Chromosomal Distribution of PcG Proteins during Drosophila Development.

Negre N, Hennetin J, Sun LV, Lavrov S, Bellis M, White KP, Cavalli G.

PRE-Mediated Bypass of Two Su(Hw) Insulators Targets PcG Proteins to a Downstream Promoter.

Comet I, Savitskaya E, Schuettengruber B, Negre N, Lavrov S, Parshikov A, Juge F, Gracheva E, Georgiev P, Cavalli G.

From genetics to epigenetics: the tale of Polycomb group and trithorax group genes.

Grimaud C, Negre N, Cavalli G.

Mapping the distribution of chromatin proteins by ChIP on chip.

Negre N, Lavrov S, Hennetin J, Bellis M, Cavalli G.

Polycomb controls the cell fate.

Negre N, Cavalli G.

The epigenome network of excellence.

Akhtar A, Cavalli G

Recruitment of Drosophila Polycomb group proteins to chromatin by DSP1

Dejardin J, Cavalli G.

Recruitment of Drosophila Polycomb Group proteins to chromatin by DSP1

Déjardin, J., Rappailles, A., Cuvier, O., Grimaud, C., Decoville, M., Locker, D., and Cavalli, G.

Epigenetic inheritance of chromatin states mediated by Polycomb- and trithorax group proteins in Drosophila

Déjardin, J., and Cavalli, G.

Combined immunostaining and FISH analysis of polytene chromosomes

Lavrov, S., Déjardin, J., and Cavalli, G.

Interaction between GAF and Mod(mdg4) proteins promotes insulator bypass in Drosophila

Melnikova, L., Juge, F., Gruzdeva, N., Mazur, A., Cavalli, G., and Georgiev, P.

Chromatin inheritance upon Zeste-mediated Brahma recruitment at a minimal cellular memory module

Déjardin, J., and Cavalli, G.

Dissection of a natural RNA silencing process in the Drosophila melanogaster germ line

Aravin, A. A., Klenov, M. S., Vagin, V. V., Bantignies, F., Cavalli, G., and Gvozdev, V. A.

Inheritance of Polycomb-dependent chromosomal interactions in Drosophila

Bantignies, F., Grimaud, C., Lavrov, S., Gabut, M., and Cavalli, G.

Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila

Sun, L. V., Chen, L., Greil, F., Nègre, N., Li, T. R., Cavalli, G., Zhao, H., Van Steensel, B., and White, K.

SNR1 is an essential subunit in a subset of drosophila brm complexes, targeting specific functions during development

Zraly, C. B., Marenda, D. R., Nanchal, R., Cavalli, G., Muchardt, C., and Dingwal, A.K.

The MYST Domain Acetyltransferase Chameau Functions in Epigenetic Mechanisms of Transcriptional Repression

Grienenberger, A., Miotto, B., Sagnier, T., Cavalli, G., Schramke, V., Geli, V., Mariol, M. C., Berenger, H., Graba, Y., and Pradel, J.

Chromatin as a eukaryotic template of genetic information

Cavalli, G.

Mapping DNA target sites of chromatin-associated proteins by formaldehyde cross-linking in Drosophila embryos.

Cavalli, G., Orlando, V., and Paro, R.

Epigenetic Inheritance of active chromatin after removal of the main transactivator

Cavalli, G., Paro, R.

The Drosophila Fab-7 chromosomal element conveys epigenetic inheritance during mitosis and meiosis

11) Cavalli, G., and Paro, R.

PUBLICATIONS COMMUNES

Widespread activation of developmental gene expression characterized by PRC1-dependent chromatin looping.

Loubiere V, Papadopoulos GL, Szabo Q, Martinez AM, Cavalli G
2020 - Sci Adv , 6(2):eaax4001 31950077
Service porteur : Chromatin and cell biology

Global chromatin conformation differences in the Drosophila dosage compensated chromosome X.

Pal K, Forcato M, Jost D, Sexton T, Vaillant C, Salviato E, Mazza EMC, Lugli E, Cavalli G, Ferrari F
2019 - Nat Commun , 10(1):5355 31767860 More information
Service porteur : Chromatin and cell biology

The multiscale effects of polycomb mechanisms on 3D chromatin folding.

Cheutin T, Cavalli G
2019 - Crit Rev Biochem Mol Biol , 54(5):399-417 31698957
Service porteur : Chromatin and cell biology

Higher-Order Chromosomal Structures Mediate Genome Function.

Jerković I, Szabo Q, Bantignies F, Cavalli G
2019 - J Mol Biol 31689436
Service porteur : Chromatin and cell biology

Advances in epigenetics link genetics to the environment and disease

Giacomo Cavalli and Edith Heard
2019 - Nature , 10.1038/s41586-019-1411-0 0
Service porteur : Chromatin and cell biology

EZH2 is overexpressed in transitional preplasmablasts and is involved in human plasma cell differentiation.

Herviou L, Jourdan M, Martinez AM, Cavalli G, Moreaux J
2019 - Leukemia 30755708
Service porteur : Maintenance of genome integrity during DNA replication

PRC2 targeting is a therapeutic strategy for EZ score defined high-risk multiple myeloma patients and overcome resistance to IMiDs.

Herviou L, Kassambara A, Boireau S, Robert N, Requirand G, Müller-Tidow C, Vincent L, Seckinger A, Goldschmidt H, Cartron G, Hose D, Cavalli G, Moreaux J
2018 - Clin Epigenetics , 10(1):121 30285865
Service porteur : Maintenance of genome integrity during DNA replication

[EZH2 is therapeutic target for personalized treatment in multiple myeloma].

Herviou L, Cavalli G, Moreaux J
2018 - Bull Cancer , 105(9):804-819 30041976
Service porteur : Maintenance of genome integrity during DNA replication

CIABRELLI Filippo
CIABRELLI Filippo
CRUK Cambridge

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CARRIVAIN Pascal
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ROUX Solène
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CARRON Léopold
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SZABO Quentin
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BAECKER Volker
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BARRO Marietta
BARRO Marietta
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PORTOSO Manuela
PORTOSO Manuela
Institut Curie Paris

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ROURE Virginie
ROURE Virginie

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CHIORINO Ornella
CHIORINO Ornella

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FERNANDO Celine
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NEGRE Nicolas
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SIMON Mylene
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VOCADLO David
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Simon Fraser University, Canada

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RUBERT-CASTRO Francesc
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Hadrien REBOUL, since January 2021, title: Study of genome folding mechanisms from nucleosomes to chromosome domains
Supervisor : Frédéric Bantignies

Nazli AKILLI, since October 2020, title: Functional dissection of mechanisms involved in the formation of H3K27me3 domain
Supervisors: Giacomo Cavalli and Thierry Cheutin at 50%.

Michael SZALAY, since September 2019, Role of transcription and Polycomb proteins in 3D genome organization and cell differentiation
Supervisor: Giacomo Cavalli

Sandrine DENAUD, since October 2018, title: Importance of PRE interactions for the regulation of polycomb domains in Drosophila melanogaster
Supervisors: Giacomo Cavalli and Bernd Schüttengruber (50%)

Victoria PARRENO, since October 2017, title: Epigenetic regulation of tumorigenesis by the Polycomb PRC1 complex in Drosophila
Supervisor : Anne-Marie MARTINEZ

 

3D organization and function of the genome

The information stored in our genome is intertwined with its function, such that, when cells are submitted to specific sets of conditions, they may pass on to their progeny their functional state. Since DNA has been identified as a critical carrier of genetic information and since the same DNA can correspond to alternative, heritable functional states in certain cases, this transmission of cellular memory has been dubbed epigenetic inheritance. In the most spectacular way, this extends to inheritance of a phenotypic trait into subsequent generation, a phenomenon for which Conrad H. Waddington provided evidence some sixty years ago and which is well documented in plants. However, to which extent epigenetic inheritance operates in animals is hotly debated. Chromatin and its higher-order organization are epigenetic components that play an essential role in genome regulation. Both the DNA molecule and the nucleosomal histones can be extensively modified in a way that impinges on gene expression and may be inherited as well as erased upon specific regulatory cues. Furthermore, chromatin fibers can be folded into yet higher-order structures and chromosomes are confined in discrete “territories”.

We and others have discovered that metazoan chromosomes share a modular organization of their chromatin in structures called “Physical domains” or “Topologically Associating Domains” (TADs). TADs can be defined as linear units of chromatin that fold as discrete three-dimensional (3D) structures tending to favor internal, rather than external, chromatin interactions. TADs are delimited by boundaries, which contain housekeeping genes and insulator sites. They are detected by methods such as Hi-C, which allows genome-wide identification of chromatin contacts, and they correspond to Chromosomal Domains (CDs), previously identified by microscopy. The investigation of chromatin landscapes in metazoa through genome-wide association studies proved to be a fruitful approach. Theoretically, a huge number of chromatin types based on different combinations of chromatin-associated marks would be possible but, in fact, every report basically recapitulated the presence of an active chromatin environment, sometimes further subdivided, and of three major types of repressive chromatin: a Polycomb-repressed environment, a null environment and a heterochromatic environment. Strikingly, TADs were found to overlap with linear chromatin domains, indicating that epigenomic labeling of chromosome domains is intimately linked to their 3D folding.

We are trying to understand the principles governing 3D folding of the genome, from establishment of chromatin loops to the generation of chromosome domains, compartments, territories and the establishment of interchromosomal interactions. We use Drosophila, but also mouse and human cells, and state of the art molecular, genomic, computational and imaging approaches, in order to reach an integrated understanding of these different levels of genome organization

figure 1
Hierarchical organization of the genome in chromatin fibers, loops, domains and chromosome territories involving multiple regulatory 3D chromatin contacts

 

 

Mechanisms of Polycomb-mediated genome regulation

Polycomb group (PcG) and trithorax group (trxG) proteins are key regulators of the expression of major developmental genes. PcG proteins are able to silence gene expression, while trxG proteins counteract gene silencing in the appropriate cells. The current model proposes that a sequence-specific DNA binding protein called PHO binds at so-called Polycomb response elements (PREs). PHO might recruit the PcG complex called PRC2, which contains the core subunits E(z), a histone methyltransferase that trimethylates histone H3 lysine 27 (H3K27me3), Su(z)12, Esc and Nurf55. H3K27me3 might then be recognized by the chromo domain of the PC subunits of PRC1, which also contains Ph, PSC and Sce/dRing. Once recruited, PcG complexes can propagate silencing through cell division. Genome-wide mapping studies have shown that PcG target genes encode for components controlling major signalling pathways and, importantly, PcG misexpression has also been associated with many cancer types, including breast and prostate cancer.

In addition to their role in cellular memory, PcG proteins participate in dynamic gene regulatory processes. In flies, different cell lines have a partially different set of PcG bound sites and H3K27me3-marked genomic regions change during development. In mammalian embryonic stem cells, many PcG target genes have been reported to bear both repression- and activation-associated marks. Upon differentiation, these “bivalent states” are resolved into fully active or fully repressed. In some instances, PcG components may even activate transcription, although it is unclear whether this phenomenon is widespread or rare. Importantly, PcG proteins regulate the organization of their target genes in the three-dimensional space of the nucleus, and this regulatory function is involved in the maintenance of cellular memory.

We would like to understand the molecular mechanisms of action of these factors, the role of regulation of higher order chromatin structure and nuclear organization in gene regulation, and the key molecular pathways that are mobilized by these proteins to coordinate the regulation of cell differentiation with that of cell proliferation. In particular, our research aims at (1) understanding, on a genome-wide scale, how these proteins are targeted to DNA and what are the consequences of this targeting on chromatin structure (2) understanding the effect of PcG proteins on cell proliferation, cell differentiation and cell polarity, and to dissecting the key components regulated by PcG proteins to modulate these pathways in specific tissues and developmental processes; (3) identifying the rules governing the distribution of their target genes in the cell nucleus and the effect of this organization on gene expression.

figure 2
The many roles of PcG proteins. The scheme illustrates the biological processes implicating PcG component on the outside and the genes targeted by these proteins to regulate these processes (on the inside of the light blue oval).

 

 

The role of Polycomb proteins in development and tumorigenesis

Coordination between cellular proliferation and differentiation ensures proper tissue morphogenesis and maintains homeostasis in multicellular organisms. Appropriate numbers of undifferentiated cells must be generated at specific developmental stages and these cells must exit the cell cycle in a tightly regulated manner to ensure proper cell fate specification and pattern formation.

The prevailing paradigm posits that Polycomb Group (PcG) proteins maintain stem cell identity by repressing differentiation genes. Mutation or misexpression of PcG genes has been associated with several types of human cancer. Polycomb group (PcG) proteins form two main epigenetic transcription repressor complexes, PRC2 and PRC1, highly conserved from fly to humans, which generally coregulate their target genes. They colocalize almost perfectly during embryogenesis, and their embryonic phenotypes are similar, with posterior homeotic transformations due to misexpression of homeotic Hox genes. Later in development, alterations in PcG components induce cancer (Figure 3), suggesting that PcG proteins may be dynamically recruited to new target genes. PcG proteins additionally bind and regulate genes implicated in major signaling pathways and therefore also participate in dynamic gene regulatory processes.

Using as a model the Drosophila larval imaginal eye disc, which shares critical features with mammalian epithelial tissues, we demonstrated that mutations affecting PRC1 subunits, but not PRC2, trigger neoplastic tumours in the larval imaginal discs. PRC1 components act as neoplastic tumor suppressors independently of PRC2 function.

by specifically targeting a thousand of new genes during larval stages of fly development. We named these non-canonical genes, “Neo-PRC1”; they massively outnumber canonical targets, are devoid of the H3K27me3 epigenetic mark and carry instead the active mark H3K27Ac. Remarkably, neo-PRC1 genes are mainly involved in the regulation of cell proliferation, differentiation, signaling and polarity. Alterations in PRC1 components specifically deregulate this set of genes, whereas canonical targets are derepressed in both PRC1 and PRC2 mutants. Together, these results suggest that the mechanism of recruitment of PRC1 on its neo-sites is independent of PRC2 and depends on new molecular mechanisms that remain to be determined. The search for these mechanisms and of their molecular significance in flies and mammals is the basis of our current interest.

Figure 3
Figure3: Tumor suppressor function for PcG genes. Left: the “+/+” panel shows control clones that do not carry mutations, which are positively labelled by green fluorescent protein. The ph0/ph0 panel shows clones corresponding to a knock out of the ph locus in order to eliminate the PH protein of PRC1. This induces massive overgrowth compared to the +/+ control disc containing neutral clones. The “Control” panel shows a, transplantation from wt GFP tissue into a wt host fly. The black stain in the abdomen shows the scar of the transplanted material. The “ph-/-“ panel shows an F1 generation host in which mutant tissue has been transplanted. The GFP labelled, ph mutant tumor grows in the body and kills the transplanted hosts.

 

 

Understanding transgenerational epigenetic inheritance

Epigenetic inheritance entails transmission of phenotypic traits not encoded in the DNA sequence and, in the most extreme case, transgenerational Epigenetic Inheritance (TEI) involves transmission of memory through multiple generations. Very little is known on the principles and the molecular mechanisms governing TEI. Chromatin has been linked to TEI but little is known on how chromatin modifications might be transmitted across generations. Polycomb components are involved at each level of chromatin folding, from post-translational histone modification all the way up to regulation of global chromosome architecture.

In our previous work, we established that perturbation of 3D chromatin architecture affected Polycomb function in a heritable way. In Drosophila lines carrying a transgene with a PRE from the Fab-7 region of the Hox cluster called bithorax complex (BX-C), we showed that the transgene establishes 3D contacts with the endogenous BX-C locus. The transgene is flanked by a reporter gene called mini-white, which is responsible for eye pigmentation. In the Fab2L line, the PRE in the transgene partially represses the reporter gene. Therefore flies have low levels of eye pigmentation that are variable between individuals, a phenomenon that reflects metastable silencing in somatic cells. These somatic epigenetic differences are not transgenerationally heritable, as self-crossing of flies with the most repressed or the most derepressed eye phenotypes does not cause any phenotypic shift in the progenies. However, we found that a transient perturbation of nuclear organization can induce TEI. We transiently removed one copy of the endogenous Fab-7 element by crossing Fab2L flies with a derivative carrying the transgene in the absence of the endogenous Fab-7 copy. This induces an increase in the contact frequency between the transgenic locus and the single remaining copy of the endogenous Fab-7 in the F1. We then restored the missing endogenous Fab-7 and obtained an F2, which is genotypically and phenotypically the same as the P0 parental generation. F2 flies with the most repressed and the most derepressed eye color were then segregated in two distinct groups, whose progenies were subjected to selection based on the eye color. Strikingly, starting at the F3, we observed the appearance of more pigmented flies in the selection of the active state and of more reduced pigment levels in the selection of the repressed state. This trend further amplified across generations, finally reaching the establishment of two “epilines”, which could stably maintain their different phenotypic trait.

We showed that this form of inheritance applies to multiple transgenic lines, to endogenous genes, that it involves components of the PRC2 complex and that it can be modulated by environmental conditions. Our future research aims at understanding the mechanisms of TEI and to uncover its role in natural processes.

Figure 4
Establishment of stable Drosophila epilines that can transmit Polycomb-dependent chromatin states through multiple generations

Giacomo Cavalli studied Biology at the University of Parma. In 1991, he moved to Zürich at the University of Science and Technology (ETH) to do his PhD, where he worked on chromatin structure and function in yeast with Fritz Thoma and Theo Koller. In 1995, he started a postdoc in the laboratory of Prof. Renato Paro at the University of Heidelberg. In 1999, he moved to IGH in Montpellier, France, to set up a junior lab and stayed at IGH ever since. Giacomo Cavalli made seminal contributions in the field of epigenetics. Using the fruit fly Drosophila melanogaster, he discovered that epigenetic inheritance of new phenotypes can occur independently on changes of the DNA sequence. His lab also discovered that the three-dimensional organisation of chromosomes in the cell nucleus is a heritable trait that plays an important gene regulatory role. The Cavalli lab identified 3D structural chromosomal domains dubbed Topologically Associating Domains or TADs. Finally, the Cavalli lab has shown that PcG proteins have tumor suppression activity in flies. Giacomo Cavalli has published more than 120 papers, cited over 16,000 times and many of which in top journals. He received numerous awards and distinctions, including an EMBO membership, the CNRS silver medal, the Allianz Foundation price, the Grand Prix 2020 of the Fondation pour la Recherche Médicale and two advanced ERC grants. He was director of the IGH Genome Dynamics department from 2007 to 2010 and IGH director from 2011 to 2014. He was and is organizer of major international conferences and is appointed as members of several distinguished Institute- and Journal editorial boards.

In this page you will find basic information on Polycomb and Trithorax proteins in epigenetic regulation; as well as the teaching material of our lab on this and related subjects. This teaching material can be downloaded and used without need of permission, but please cite this web publication address as the source of information in order to allow users to address us enquiries and correspondence.
You will also find some links to relevant papers in the Polycomb and Trithorax field, and to Web sources of information in this subject. Enjoy polycomb!

 

Polycomb history and introduction

Polycomb group (PcG) and trithorax group (trxG) proteins regulate expression patterns of many developmental genes. Their function is best understood in the regulation of homeotic genes, where these proteins are able to maintain, respectively, silenced or active states throughout development. These proteins raised considerable interest in recent years, both because the basic regulatory mechanisms that involve these factors are fascinating, and because they play key roles in a variety of normal cellular processes and in disease.

A brief introduction to Polycomb and Trithorax

Polycomb group (PcG) proteins are highly conserved regulatory factors that were initially discovered in Drosophila. PcG genes are best known for their role in maintaining silent expression states of Hoxgenes during development, while trithorax group (trxG) proteins maintain Hox gene expression patterns in the appropriate spatial domains. PcG and trxG proteins are also involved in the regulation of normal cell proliferation, and their mutation has been linked to defects in stem cell fates and to cancer. They act by regulating chromatin structure and chromosome architecture at their target loci.
PcG proteins form multimeric complexes that exert their functions by modifying chromatin structure and by regulating the deposition and recognition of multiple post-translational histone modifications. Three major PcG protein complexes have been described. The first, named PhoRC, contains the DNA-binding protein Pho (this is the Drosophila name, the homolog in mammals is YY1). The second complex, named the E(Z)/ESC complex or Polycomb Repressive Complex 2 (PRC2), contains four core proteins: the histone methyltransferase Enhancer of Zeste (E(Z)), Extra sex combs (ESC), Suppressor of zeste-12 (SU(Z)12), and nucleosome-remodeling factor 55 (NURF-55). E(Z) trimethylates lysine 27 of histone H3 (H3K27me3), and, to a lesser extent, lysine 9 of histone H3 (H3K9me3). A third complex, named PRC1, recognizes these methylation marks via the chromodomain of the Polycomb (PC) protein. PC is a stoichiometric component of PRC1, together with Polyhomeotic (PH), Posterior Sex Combs (PSC), and dRING. In mammals, the duplication of many PcG genes allows variations in complex composition, which differ with cell type and developmental stage.

TrxG proteins are a somewhat heterogeneous group, but they are characterized by complementary mechanistic properties to the PcG. Within trxG members, some bind specific sequences of DNA. A second class class of trxG members is composed by SET domain factors like Drosophila Trx and Ash1 and vertebrate MLL, as well as their associated proteins. A third class of trxG factors comprises protein components of ATP-dependent chromatin remodeling complexes like the SWI/SNF or the NURF complexes, and includes proteins (such as one component of the NURF complex) specifically capable to "read" the histone methylation marks laid down by the SET domain proteins.

In Drosophila, PcG proteins repress their target genes by binding to specific DNA elements called Polycomb Response Elements (PREs). Analysis of known PREs has revealed the presence of binding sites (usually in multiple copies) for several DNA-binding proteins, such as Pleihomeotic (PHO) and Pleihomeotic-like (PHOL), GAGA factor (GAF)/Pipsqueak (PSQ), Zeste and DSP. Other studies have suggested possible additional roles for other proteins, such as the corepressor CtBP and the DNA binding factors Grainyhead (GRH) as well as members of the Sp1/KLF family. Therefore, a large number of proteins might contribute to PcG recruitment at PREs. Each PRE has a different number and topological organization of binding sites for these factors, possibly providing the basis for the specificity of PRE function.

PREs have only been characterized in Drosophila so far. In general, PREs might be simply defined as DNA elements necessary and sufficient for recruitment of PcG complexes and for PcG-dependent silencing of flanking promoters. Many of the PcG binding sites identified by chromatin immunoprecipitation in vertebrates might correspond to this criterion. Their DNA sequences are likely to be fairly different from fly PREs, since three of the DNA-binding factors involved in PcG recruitment, GAF, Pipsqueak and Zeste, are not conserved in vertebrates. Indeed, CpG islands can by themselves recruit Polycomb complexes if not methylated.

In addition to modifications at the chromatin level, regulation at the level of nuclear architecture influences the regulation of PcG target genes. In mice, it has been reported that nuclear re-organization is coupled to Hox gene activation in early development. In Drosophila, homologous chromosomes pair in interphase nuclei, and transgenic PREs typically silence more strongly when they are present in two copies on homologous chromosomes. This notion of pairing is reinforced by the finding that PRE-containing sequences can also pair with homologous sequences located on different chromosomes, and that these long distance nuclear interactions reinforce PcG-mediated silencing.

Therefore, multiple mechanisms cooperate to drive regulation of gene expression by PcG and trxG proteins. This is likely very important in light of the fact that these proteins regulate a large number of genes, sometimes maintaining the memory of transcriptionalstates, while in other cases their regulation is more flexible. These multiple mechanisms may be important to ensure the necessary regulatory plasticity, while providing sufficient robustness to the regulated state.

Recent reviews for further readings

  1. Schuettengruber, B., Bourbon, H.M., Di Croce, L., and Cavalli, G.
    Genome Regulation by Polycomb and Trithorax: 70 Years and Counting.
    Cell 2017 171, 34-57. doi: 10.1016/j.cell.2017.08.002.
    PMID: 28938122
  2. Piunti, A., Shilatifard, A.
    Epigenetic balance of gene expression by Polycomb and COMPASS families.
    Science 2016, 352(6290):aad9780, doi:10.1126/science.aad9780. PMID: 27257261
  3. Koppens M, van Lohuizen, M
    Context-dependent actions of Polycomb repressors in cancer
    Oncogene 2015, doi:10.1038/onc.2015.195. Epub ahead of print
  4. Sexton T, Cavalli, G
    The role of chromosome domains in shaping the functional genome
    Cell 2015, 160: 1049-1059
  5. Lanzuolo C, Orlando V.
    Memories from the polycomb group proteins
    Annu Rev Genet. 2012;46:561-89. doi: 10.1146/annurev-genet-110711-155603. Epub 2012 Sep 17.
    PMID: 22994356
  6. Pirrotta V, Li HB.
    A view of nuclear Polycomb bodies.
    Curr Opin Genet Dev. 2012 Apr;22(2):101-9. doi: 10.1016/j.gde.2011.11.004. Epub 2011 Dec 16. Review.
    PMID: 22178420 [PubMed - indexed for MEDLINE]
  7. Holec S, Berger F
    Polycomb group complexes mediate developmental transitions in plants.
    Plant Physiol. 2012 Jan;158(1):35-43. doi: 10.1104/pp.111.186445. Epub 2011 Nov 15. Review. No abstract available.
    PMID: 22086420 [PubMed - indexed for MEDLINE]
  8. Bantignies F, Cavalli G
    Polycomb group proteins: repression in 3D.
    Trends Genet. 2011 Nov;27(11):454-64. doi: 10.1016/j.tig.2011.06.008. Epub 2011 Jul 25. Review.
    PMID: 21794944 [PubMed - indexed for MEDLINE]
  9. Schuettengruber B, Martinez AM, Iovino N, Cavalli G.
    Trithorax group proteins: switching genes on and keeping them active.
    Nat Rev Mol Cell Biol. 2011 Nov 23;12(12):799-814. doi: 10.1038/nrm3230. Review.
    PMID: 22108599 [PubMed - indexed for MEDLINE]
  10. Margueron R, Reinberg D.
    The Polycomb complex PRC2 and its mark in life.
    Nature. 2011 Jan 20;469(7330):343-9. doi: 10.1038/nature09784. Review.
    PMID: 21248841 [PubMed - indexed for MEDLINE]
  11. Mills AA.
    Throwing the cancer switch: reciprocal roles of polycomb and trithorax proteins.
    Nat Rev Cancer. 2010 Oct;10(10):669-82. doi: 10.1038/nrc2931. Review.
    PMID: 20865010 [PubMed - indexed for MEDLINE]
  12. Sauvageau M, Sauvageau G.
    Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer.
    Cell Stem Cell. 2010 Sep 3;7(3):299-313. doi: 10.1016/j.stem.2010.08.002. Review.
    PMID: 20804967 [PubMed - indexed for MEDLINE]
  13. Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G.
    Genome regulation by polycomb and trithorax proteins.
    Cell. 2007 Feb 23;128(4):735-45.
    PMID: 17320510 [PubMed - in process]

 

Polycomb and trithorax group proteins

This table lists PcG and trxG proteins in humans and flies, as well as proteins that may be involved at recruiting them to their target genes. The genes are hyperlinked to the corresponding databases, either Flybase or Ensembl (for Human links). When multiple genes correspond to one entry (for instance, there are 5 possible human Proteins that can elicit the function of fly Polycomb), only the link to the first of the possible member are given. The other ones can be found by searching in Ensembl or HGNC databases).

Note: This table is constantly under revision. should you see mistakes or have updates, please send me an email
* indicates that the protein exist but its function in the PcG or trxG pathway is still not clear

 PcG/trxG recruiters Drosophila melanogaster Homo sapiens Notes
  Dsp1 HMGB2 Dsp1 is an HMG box protein. It assists Pho in PcG recruitment at Drosophila PREs.
HMGB2 is involved in YY1 in silencing of D4Z4 repeats
  Grh GRHL1 Fly Grh helps PcG recruitment at one PRE
  Gaga factor / Trl ? Fly Trl is involved for PcG recruitment at some PREs although is classified as a trxG protein. It is a Zn-finger sequence specific protein that binds the GAGAG motif. It also contains a BTP/POZ domain that is generally involved in protein-protein interactions
  Lolal ? Lola-like binds Trl and acts as a PcG protein
  Psq ? Psq co-purifies with components of the PRC1 complex and binds the same sequences as Trl
  Zeste ? Zeste found within PRC1 but also linked to trxG-mediated activation
Fly PhoRC complex
Identified in 2006
Fly PhoRC binds PREs and is involved in recruitment of PcG proteins to PREs. Pho also forms a second complex named INO80, likely to be involved in chromatin remodelling.
Pho can recruit the histone methyltransferase E(z) to the Ubx PRE. In vitro, it can also recruit PRC1 components to DNA independent on the action of E(z).
Whether PhoRC exist in human cells is unknown, but its homolog YY1 ca, recruit PcG proteins to target genes
  ? E2F6 E2F6 forms two different multimeric complexes containing PcG proteins, one with RING1A, RING1B and MBLR, and the other one with EZH2, E(PC) and Sin3A
  ? BCL6 BCL6 is a BTB containing protein (similar to Drosophila Krüppel, but it is not known whether it is a true homolog) that was suggested to recruit PcG proteins to its target genes via the corepressor BCOR complex
  Rbf RB1
RBL1
Human Retinoblastoma protein represses genes in a PcG-dependent manner to block cell proliferation. This pathway was not yet identified in other organisms
  ? PLZF PLZF has been shown to bind to the HoxD complex and to bind Polycomb proteins on chromatin. This sequence specific DNA binding protein contains a Zn-Finger domain and a BPB/POZ domain that is generally involved in protein-protein interactions. Plzf mutants strongly derepress the HoxD locus in the embryonic hindlimb bud, PLZF binds to Bmi-1 and recruits it to HoxD
PcG complexes PcG complex components Characteristic Domain (Epigenetic) Function
  Mammals Flies    
core PRC1 complex RING1A/B dRing/Sce RING finger domain H2AK119 ubiquitylation
PCGF1-6 Psc/Suz(2) RING finger domain, UBL (RAWUL) domain H2AK119 ubiquitylation, oligomerization
canonical PRC1 CBX2,4,6-8 Pc Chromo domain H3K27me3 binding
PHC1-3 Ph-p/Ph-d Sterile alpha motif (SAM) domain oligomerization/protein-protein interation
SCMH1/2 Scm SAM domain oligomerization/protein-protein interation
non-canonical PRC1 RYBP/YAF2 Rybp Zinc finger domain DNA binding
KDM2B Kdm2 JmjC domain, CxxC domain H3K36 demethyalse, DNA binding
DCAF7 Wap WD40 domain scaffold factors
WDR5 Wds WD40 domain scaffold factors
core PRC2 complex EZH1/2 E(z) SET domain, SANT domain H3K27 methyltransferase, histone binding
SUZ12 Suz(12) Zinc finger domain RNA/DNA binidng
EED Esc/Escl WD40 domain H3K27me binding
RBBP4/7 Nurf55/Caf1 WD40 domain H3K36me3 binding
PRC2 accessory proteins PCL1-3 Pcl Tudor domain; PHD-finger domain H3K36me3 binding
JARID2 Jarid2 Zinc finger domain, ARID domain H2Aub binding, RNA binding
AEBP2 Jing Zinc finger domain DNA binding, H2Aub binding
EPOP/C17orf96     modulating enzymatic activity
LCOR/C10orf12     unknown
core PR-DUB BAP1 Calypso biquitin carboxyl-terminal hydrolase (UCH) N-terminus catalytic domain Ubiquitin carboxyl-terminal hydrolase
ASXL1/2 Asx   chromatin binding
PR-DUB accessory proteins FOXK1/2 FoxK Forkhead box DNA binding
OGT Sxc   O-GlcNAcylation
KDM1B dLsd1 amine oxidase domain Histone demethylation
MBD5/6 Sba methyl binding domain  DNA binding
trxG complexes trxG complex components Protein Domain (Epigenetic) Function
core COMPASS components  WDR5 Wds WD40 domain Histone binding
ASH2L Ash2 Zinc finger domain DNA binding
RBBP5 Rbbp5 WD40 domain Histone binding
DPY30 Dpy30    
SET1/COMPASS SET1A/B dSet1 SET domain H3K4 methyltransferase
HCFC1 Hcf1 Kelch domain  
WDR82 Wdr82 WD40 domain Histone binding
CFP1 Cfp1 CxxC domain DNA binding
MLL1/2 COMPASS-like MLL1/2 Trx SET domain H3K4 methyltransferase
HCFC1 Hcf1 Kelch domain  
MENIN Menin    
MLL3/4 COMPASS-like MLL3/4 Trr SET domain H3K4 methyltransferase
NCOA6 Ncoa6    
PAGR1 Pa1    
UTX Utx JmjC domain H3K27 demethylase
PTIP Ptip BRCT domain  
ASH1 ASH1L Ash1 SET domain; Bromo domain H3K36 mehyltransferase
CBP dCbp HAT domain; Bromo domain H3K27 acetyltransferase
SWI/SNF (BAF and PBAF) complex BRM/BRG1 Brm Helicase, Bromo domain ATPase-dependent chromatin remodlling
BAF250A/B Osa ARID domain possible DNA binding
BAF155/170 Mor SWIRM, SANT, Chromo domain possible DNA and histone binding
BAF47 Snr1 Winged helix domain possible DNA binding
BAF45A-D Sayp PHD-finger domain possible DNA binding
BAF53A/B Bap55 Actin-like  
BAF180/BAF200 polybromo Polybromodomain histone binding
BAF60A-C Bap60 Swi-B domain  
BAF57 Bap111 HMG domain possible DNA binding
beta-ACTIN Actin5C    
BCL7A-C Bcl7-like    
BRD7/9 CG7154    

 

​​List of landmark discoveries in the Polycomb and Trithorax field

Year Brief description of the main findings Pubmed link
1978 Ed Lewis's founding  Polycomb paper identifying a role for the Pc gene in the regulation of homeotic genes go!
1985 Characterization of the trithorax gene as a regulator of homeotic gene expression
Role of PcG proteins in the maintenance of homeotic gene expression, i.e. in the process of "cellular memory"
go!
go!
1988 Antagonism between Polycomb and trithorax genes go!
1989 Polytene chromosome binding pattern of Pc go!
1991 Identification of Bmi-1, the first mammalian PcG gene
Role of Bmi-1 in Cancer
go!
go:   a!     b!
1992 Involvement of Trithorax in leukemia go!
1993 Characterization of PREs in Drosophila
Chromatin IP of Polycomb
go:   a!   b!   c!
go!
1994 Bmi-1 action as a bona fide mammalian PcG protein go!
1997 Analysis of PcG proteins in plants
PcG proteins and epigenetic regulation of gene expression by "cosuppression"
go!
go!
1999 Purification of the PRC1 complex
Role of PcG in cell proliferation
go!
go!
2000 trxG proteins and histone acetylation go: a!     b!
2001 Link between PcG proteins and the basal transcriptional machinery
PcG proteins and genomic imprinting in mammals
go: a!     b!
go!
2002 Characterization of the E(z)-Esc / PRC2 complex - Histone methyltransferase activity
trxG proteins and histone methylation
go:   a!   b!   c!   d!
go:   a!     b!
2003 Binding of the PC chromo domain to histone H3 methylated at Lysine 27
PcG proteins and X-inactivation
Polycomb as a Sumo E3 protein
go:   a!     b!
go:   a!     b!
go!
2004 PRC1 proteins mediate histone ubiquitination
Identification of a PRC3 complex related to PRC2 and identification of histone H1 methylation activity
go!
go!
2005 Identification of a link between PcG proteins and DNA methylation
Role for PcG proteins in the phenomenon of transdetermination in Drosophila
go: a!     b!
go: a!     b!
2006 Genome-wide mapping of the downstream target sites for PcG proteins Drosophila: a!  b!  c!
Human
Mouse
2007 Discovery of H3K27me3 demethylases a!  b!  c!  d!
2009 1- Crystal structure of EED reveals a mechamism for maintenance of H3K27me3 through the cell cycle (partially supports an earlier work by the Helin lab)
2- Identification and initial characterization of the first mammalian PREs
1a!1b!

2a!2b!
2010 Various links between PcG proteins and noncoding RNAs (earlier work had pointed to a link between PcG proteins and a ncRNA in X inactivation, but in 2010 the data were broadly generalized and, in particular, SUZ12 was shown to be an RNA-binding protein. a!  b!  c!  d!
2012 Identification of alternate mammalian PRC1 complexes, suggesting that each of them may have specific functions     a!   b!   c!
2014 Discovery of a role for Histone H2A ubiquitylation in the recruitment of PRC2 complexes go
2015 Discovery of a network of Polycomb-target genes in the cell nucleus of mammalian organisms a!b!
2017 Mechanisms of SWI/SNF (BAF) complex-mediated eviction of Polycomb complexes in normal cells and cancer a! b!
For obvious reasons, this list does not include the work in our lab. For this, please go to the lab main page. Moreover, this list is certainly not perfect. If you have important additions or updates that you wish to be included, please write me an email.

 

Montpellier teaching

Below, you find teaching courses specifically given to Montpellier students.

  1. UE Méthodologie, a course for Montpellier students on: in vivo protein-DNA interactions (ChIP, DamID, 3C, 4C, HiC...)
    course held in February 2017. Download
  2. Master 1 - UE Génomique fonctionnelle
    course held in September 2016. Download
  3. Master 2 - Biologie du Developpement-Cellules souches-Biothérapie
    course held in December 2016. Download
  4. Master 2R - TC1 (HMBS324) « Genetic and epigenetic information - molecular bases »
    course held in Autumn 2016. Download
  5. Master 2R -  (HMBS204) -  Systems biology / Biologie des systèmes
    course held in Autumn 2017. Download Cavalli - Download Jost

Transgenerational epigenetic inheritance of chromatin states: the role of Polycomb and 3D chromosome architecture
Start date: 2018-11-01, End date: 2023-10-31

Institution: CNRS, Institute of Human Genetics, Montpellier, France (IGH)

Project duration: 60 Months

Abstract: Epigenetic inheritance entails transmission of phenotypic traits not encoded in the DNA sequence and, in the most extreme case, Transgenerational Epigenetic Inheritance (TEI) involves transmission of memory through multiple generations. By transiently enhancing long-range chromatin interactions, we established isogenic Drosophila epilines that carry stable alternative epialleles, defined by differential levels of the Polycomb-dependent H3K27me3 mark. These are ideal systems to study the role of Polycomb group (PcG) proteins and other components in regulating nuclear organization and epigenetic inheritance of chromatin states. The present project conjugates genetics, epigenomics, imaging and molecular biology to reach three critical aims.

Aim 1: Analysis of the molecular mechanisms regulating Polycomb-mediated TEI. We will identify the DNA, protein and RNA components that trigger and maintain transgenerational chromatin inheritance as well as their mechanisms of action.

Aim 2: Role of 3D genome organization in the regulation of TEI. We will analyze the developmental dynamics of TEI-inducing long-range chromatin interactions, identify chromatin components mediating in 3D chromatin contacts and characterize their function in the TEI process.

Aim 3: Identification of a broader role of TEI during development. TEI might reflect a normal role of PcG components in the transmission of parental chromatin onto the next embryonic generation. We will explore this possibility by establishing other TEI paradigms and by relating TEI to the normal PcG function in these systems and in normal development.

The information stored in our genome is tightly intertwined with its function. In some cases, cells may pass on to their progeny their functional state. This transmission of cellular memory has been dubbed epigenetic inheritance [1]. This phenomenon is very important during development and physiology. In the most spectacular way, it extends to inheritance of a phenotypic trait into subsequent generations, a phenomenon for which Conrad H. Waddington provided initial evidence some sixty years ago [2]. Transgenerational Epigenetic Inheritance (TEI) can thus be defined as the transmission of phenotypic traits not encoded in the DNA sequence through multiple generations. In this context, “epialleles” might be defined as the unit carriers of heritable epigenetic information. Understanding TEI is one of the most important challenges in epigenetic research [3] (Figure 1).  

Figure 1. The factors contributing to the phenotypic traits of an organism. Heritable information, an important determinant of the phenotype, can be genetic (DNA sequence), or non-genetic. In the cases described as of today, Transgenerational Epigenetic inheritance (TEI) may depend on non-coding RNAs, DNA modification or chromatin components. However, some TEI reports have been questioned and, in general, a mechanistic understanding of TEI is lacking.

Although evidence for TEI has been published before, the presence of confounding factors might explain some of the results [4]. TEI phenomena were mostly observed in plants and linked to DNA methylation [5-7]. When observed in animal species, they were often unstable [8-12], mediated by RNAs or DNA methylation [13-15], or the role of DNA sequence was not excluded [16]. Strong evidence for true TEI has been provided very rarely in animals [17], leaving the question of the relevance of chromatin-mediated TEI in animals open.

Among putative candidate chromatin components that might drive TEI are Polycomb and Trithorax group (PcG, TrxG, respectively) proteins. The first Polycomb (Pc) mutation was identified 70 years ago and the gene was first characterized 40 years ago. Since then, many other genes collaborating or counteracting Pc were discovered and a large body of research was devoted to understanding their biological significance [18]. PcG and TrxG proteins are evolutionarily conserved chromatin components that mediate epigenetic memory of gene regulation during development [19, 20]. In Drosophila, they bind to regulatory elements called Polycomb response elements (PREs) (or CpG islands in mammals) [21]. Once bound, they modify their surrounding chromatin and interfere with chromatin remodeling in order to regulate gene expression [19-21]. In addition to regulating the genes close to their binding sites, we showed that these proteins can establish regulatory interactions among chromatin regions located at large distances in the linear genome that coalesce in the three-dimensional space of the cell nucleus [21].

These proteins have been previously implicated in TEI in Drosophila and in C. elegans [10, 12, 22, 23]. However, inheritance was limited and diluted after few generations, preventing detailed mechanistic investigation. Recently, a role for the polycomb mark H3K27me3 has been identified in imprinting of a limited number of mouse genes [24], suggesting that Polycomb-mediated TEI is evolutionarily conserved. We previously suggested the possibility that perturbation of nuclear architecture might induce transgenerationally heritable chromatin changes [12]. We recently explored this model system and demonstrated that PRE-containing transgenes can induce stable and reversible TEI [25]. This system is powerful because it is stable and reversible, and it allowed us to exclude the role of confounding factors such as DNA sequence variation. Below, I describe how building on our results will bring answers to long-standing questions in the field. Completion of specific aims described hereafter should profoundly impact our understanding of TEI.

Background, foundation of the project and preliminary data

Chromatin and its higher-order organization play an essential role in genome regulation. DNA and histones can be extensively modified in a way that impinges on gene expression and may be inherited as well as erased upon specific regulatory cues. Furthermore, chromatin fibers can be folded into higher-order structures and chromosomes are confined in discrete “territories” [26-30]. Polycomb components are involved at each level of chromatin folding, from post-translational histone modification all the way up to regulation of global chromosome architecture (Figure 2). In our previous work using Drosophila lines carrying a transgene with a PRE from the Fab-7 region of the Hox cluster called bithorax complex (BX-C), we showed that the transgene establishes 3D contacts with the endogenous BX-C locus and that perturbation of 3D chromatin architecture affected Polycomb function in a heritable way [12].

Figure 2. Role of Polycomb in genome regulation. Polycomb complexes are responsible for the deposition of the PRC2-dependent H3K27me3 and the PRC1-dependent H2AK119Ub marks, both involved in gene silencing. Furthermore, they frequently bind to large regions that are called Polycomb domains and constitute one of the fundamental and most abundant types of chromatin in the genome. Individual binding sites within domains cluster together, guiding the maintenance of each domain [31, 32]. Polycomb domains coincide with one type of Topologically associating domains that have been identified by genome-wide technologies such as Hi-C [29, 33-35]. Finally, Polycomb proteins guide and assist long-range contacts between Polycomb domains that can be very far in the linear chromosome or even in different chromosomes [36-38].

 

In the Fab2L line, the transgene contains a PRE flanked by a reporter gene called mini-white, which is responsible for eye pigmentation. The PRE transgene partially represses the reporter gene, such that Fab2L flies have low levels of eye pigmentation that are variable between individuals, a phenomenon that reflects metastable silencing in somatic cells. These somatic epigenetic differences are not transgenerationally heritable, as self-crossing flies with the most repressed or the most derepressed eye phenotypes does not cause any phenotypic shift in the progenies [25]. However, a transient perturbation of nuclear organization can induce TEI in this line. We removed one copy of the endogenous Fab-7 element by crossing Fab2L flies with a derivative carrying the transgene in the absence of the endogenous Fab-7 copy. This induces an increase in the contact frequency between the transgenic locus and the single remaining copy of the endogenous Fab-7 in the F1. We then restored the missing endogenous Fab-7 and obtained an F2, which is genotypically and phenotypically the same as the P0 parental generation. F2 flies with the most repressed and the most derepressed eye color were then segregated in two distinct groups, whose progenies were subjected to selection based on the eye color. Strikingly, starting at the F3, we observed an increase of the number of pigmented flies in the “derepressed” group and an increase of the number of flies with reduced pigment levels in the “repressed” group. This trend was amplified over generations, finally reaching the establishment of two “epilines” (Fab2L White* for the repressed case and Fab2L Red* for the derepressed case), which stably maintain their phenotypic trait for at least 50 generations (Figure 3). The system is reproducible and the same results was obtained on another transgenic line called FabX [12] and upon isogenizing the Fab2L line in a different genetic background [25].

Figure 3. Establishment of stable Drosophila epilines that can transmit Polycomb-dependent chromatin states through multiple generations 


Furthermore, the epigenetic nature of TEI was confirmed by the findings that the epilines could be reversed by genetic manipulation and that genomic sequencing of the different epilines did not reveal differences in chromatin genes or in genes linked to eye pigmentation. Instead, we found a significant difference in the levels of H3K27me3 at the PRE-containing transgene among the epilines and a transient reduction in the dosage of the enzymatic component of PRC2, E(z), induced a heritable derepression of the transgene. Environmental changes, notably in the temperature at which the flies are exposed, can modulate the expressivity of the epialleles. Finally, we extended our paradigm to a naturally occurring phenotype that depends on Hox gene deregulation [25]. These data provide a clear-cut demonstration for TEI in animals [39] and, most importantly they provide a formidable model system for the molecular dissection of TEI, thanks to the establishment of epilines with stable and strong phenotypic differences compared to their naïve counterparts.

Project aims

What are the molecular mechanisms regulating TEI? How is this phenomenon linked to chromosome architecture and nuclear organization? How is it linked to mitotic inheritance of chromatin architecture? Finally, how widespread is TEI in biology and in response to environmental challenges? In this project, we will exploit the power of Drosophila as well as CRISPR manipulation of mouse ES cells in order to address these three questions. Specifically, the proposal has three aims:

  1. Aim 1: Analysis of the molecular components regulating Polycomb-mediated TEI. We will identify the DNA, protein and RNA components leading to transgenerational chromatin inheritance and we will analyze their mechanisms of action. In particular, we will analyze how can PcG binding set up a heritable chromatin structure. In addition to experiments assessing transgenerational inheritance, we will also ask how cells can mitotically inherit chromatin architecture and what is the contribution of Polycomb components in this process. For this purpose, we will use mouse ES and differentiated cells.
  2. Aim 2: Role of nuclear organization in the regulation of TEI. We will analyze the developmental dynamics of TEI-inducing long-range chromatin interactions, identify chromatin components mediating in 3D chromatin contacts and characterize their function in the TEI process. Furthermore, we will address mitotic inheritance of 3D chromatin organization in mouse ES and differentiated cells.
  3. Aim 3: Identification of a broader role of TEI during development. TEI might reflect a normal role of PcG components in the transmission of parental chromatin onto the next embryonic generation. We will explore this possibility by establishing other TEI paradigms and by relating TEI to the normal PcG function in these systems and in normal development.

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