Deciphering the roles of Insulators and co-factors in Nucleosome-Positioning and Nuclear Bodies – INSULa

We have purified BEAF complexes that harbor >30 BEAF co-factors. We will investigate which of these factors, specific or common to BEAF and/or CTCF complexes, affects nucleosome-positioning.
Towards this end, we will extend our genome-wide analyses of nucleosome mapping in BEAF-depleted cells to cells depleted of CTCF/BEAF-associated factors common or specific of each insulator subclass, to dissect how they regulatenucleosome positioning. We will then investigate the detailed mechanisms of nucleosome positioning using smFRET.
1. Mavrich, T. N. et al. Nature 453, 358-62 (2008).
2. Corpet A, Almouzni G. Trends Cell Biol. 1, 29-41. (2009).
3. Kaplan, N. et al. Nature 458, 362-6 (2009).
4. Dekker, J., Cuvier, O. & Chang, H. Y. EMBO rep in press (2009).
5. Miele, A. & Dekker, J. Mol Biol 464, 105-21 (2009).
6. Barski, A. et al. Cell 129, 823-37 (2007).
7. Schones, D. E. et al. Cell 132, 887-98 (2008).
8. Emberly, E. et al. PLoS Biol 6, 2896-910 (2008).
9. Bushey et al. Genes Dev. 23:1338-50 (2009).
10. Nollmann, M. et al. Nat Struct Mol Biol 14, 264-71 (2007).

Our recent data highlight several key factors in establishing the epigenetic frontiers of chromosomes, i.e. which are maintain through cell divisions. Such factors are under characterization.
We further focus on mechanisms at the basis of chromosomal barriers. Surprisingly, such barriers appear to be ‘flexible’ and dynamic, rather than fixed.
Finally, another line of data show that barriers regulate the expression of genes that are distantly located, highliting régulations that involve the 3D conformation of chromosomes (Liang et al., MS in prep).

Our work may be important to better understand the origins of cell identity and reprogramming.

Jun Liang+, Adrien Gamot+, Suresh Cuppadrah^*, Laurent Lacroix+, Sophie Queille+, Magali Hennion +*, Pauline Morillon+, Krim Belhocine, Jutta Vögelmann$, Serge Urbach^, Marcelo Nöllmann$, Keji Zhao^, Eldon Emberly# and Olivier Cuvier+@. A Regulatory Network among the Beaf-32/GAF/dCTCF/CP190 Insulator-Binding Proteins control RNAPII Pausing through Long-range Interactions. Manuscript in prep.

Magali Hennion +*, Suresh Cuppadrah^*, Adrien Gamot+*, Pauline Morillon+, Sophie Queille+, Dany Severac~, Christelle Dantec~, Keji Zhao^, Eldon Emberly#@ and Olivier Cuvier+@ Insulator-mediated Regulation of Gene Expression through Transcriptional Pausing. EMBO J., under revision

Jutta Vogelmann1, Alessandro Valeri1, Emmanuelle Guillou2, Olivier Cuvier2, Marcelo Nollmann1 (2011). Roles of chromatin insulator proteins in higher-order chromatin organization and transcription regulation. Nucleus, review.

Cell fate and cell division are processes tightly controlled by the functional and structural organization of the genome into chromatin. Two emerging concepts in chromatin biology are a) nucleosome-positioning, i.e. how the accessibility of the genetic information is controlled through the specific positioning of nucleosomes (1-3), and b) long-range DNA interactions/ or DNA looping, which allow any particular genomic region (e.g. one gene) to interact preferentially with loci located far-away (4,5). Nucleosome-positioning and DNA looping govern chromatin organization and they are further involved in chromosomal breakpoints (6,7); however, their regulatory mechanisms remain to be described. DNA sequences or transcription might contribute to position nucleosomes, yet specific DNA-binding factors are also very likely involved (3,8), highlighting the need to decipher regulatory mechanisms of nucleosome-positioning and long-range chromosomal interactions.
Insulator proteins like BEAF are the best candidates to regulate nucleosome-positioning and long-range interactions, involving co-factors, like CP190, and pathways that may be conserved among insulators (4,9). BEAF interacts with Nucleosome-Associated Cis-Regulatory Elements (NACRE), where its DNA consensus motifs are specifically positioned in respect to nucleosomes as also found for many DNA-binding proteins (3). Our unpublished data show that BEAF regulates nucleosome occupancy in vivo, prior to RNA polymerase II binding, which has been thought as a key player of positioning. Rather, our data suggest that insulators may play a major role in nucleosome-positioning to potentiate promoters for transcription.
We have purified BEAF complexes that harbor >30 BEAF co-factors. We will investigate which of these factors, specific or common to BEAF and/or CTCF complexes, affects nucleosome-positioning. Towards this end, we will extend our genome-wide analyses of nucleosome mapping in BEAF-depleted cells to cells depleted of CTCF/BEAF-associated factors common or specific of each insulator subclass, to dissect how they regulate nucleosome positioning. We will then investigate the detailed mechanisms of nucleosome positioning using smFRET.
Insulators are also predicted to act as ‘platforms’ of long-range contacts to cluster genes into 'bodies'. We will combine immunofluorescence analysis with RNAi, and single molecule techniques (10), to unveil the detailed mechanisms and key factors regulating insulator clustering and/or DNA looping. Our data will provide fundamental insights into how insulator factors organize the genome from nucleosome-positioning to nuclear bodies.

1. Mavrich et al. Nucleosome organization in the Drosophila genome. Nature 453, 358-62 (2008).
2. Corpet A, Almouzni G. Making copies of chromatin: the challenge of nucleosomal organization and epigenetic information. Trends Cell Biol. 1, 29-41. (2009).
3. Kaplan, N. et al. The DNA-encoded nucleosome organization of a eukaryotic genome. Nature 458, 362-6 (2009).
4. Dekker et al. Gene dates, parties, and galas: Chromatin dynamics and higher order organization. EMBO rep in press (2009).
5. Miele et al. Mapping Cis- and Trans- Chromatin Interaction Networks Using 3C. Methods Mol Biol 464, 105-21 (2009).
6. Barski et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823-37 (2007).
7. Schones et al. Dynamic regulation of nucleosome positioning in the human genome. Cell 132, 887-98 (2008).
8. Emberly et al. BEAF regulates cell-cycle genes through the deposition of H3K9 methylation marks into its conserved dual-core binding sites. PLoS Biol 6, 2896-910 (2008).
9. Bushey et al. Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions. Genes Dev. 23:1338-50 (2009).
10. Nollmann et al. Multiple modes of Escherichia coli DNA gyrase activity revealed by force and torque. Nat Struct Mol Biol 14, 264-71 (2007).