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Team Simona Saccani

Presentation

Basic Research Team  « Transcription Specificity »


Dr Simona SACCANI, Team Leader

   


Summary


The Saccani lab studies the molecular mechanisms which regulate gene expression, using the NF-kB family of transcription factors as a model system. A major focus is to understand how the activities of transcription factors can be controlled in promoter-specific and cell type-specific fashion.

The NF-kB family of transcription factors is crucial for the expression of multiple genes involved in cell survival, proliferation, differentiation and inflammation. The NF-kB pathway is broadly active in multiple cell types, yet many NF-kB target genes show a tightly-regulated pattern of expression, with stimulus-dependent activation restricted to a particular biological setting.  To investigate the molecular mechanisms underlying this, we focus on differences in the chromatin structure of target promoters and enhancers, and on co-factors which can regulate the recruitment and activity of NF-kB in specific cell-types.


Research Projects

Research Projects



1.    Role of H3K9me2 and the demethylase Aof1 in regulating NF-kB-dependent transcription


We have observed that in unstimulated dendritic cells, a subset of tightly-regulated NF-kB target genes bear high levels of H3K9 methylation at, and surrounding, their promoter regions (Saccani and Natoli, 2002). Upon LPS-stimulation, this methylation is rapidly erased, concomitant with gene transcription, and it is later replaced again when transcription ceases. A number of lysine demethylases have recently been described, and we hypothesized that active demethylation of H3K9 could function to regulate NF-kB target gene activity. In line with this, we found that target gene demethylation could be blocked using specific inhibitors of amine oxidase-family enzymes (to which one class of demethylases belongs), and this led eventually to our cloning and characterization of a novel H3K9 demethylase, Aof1, which is required for target promoter demethylation (van Essen et al., 2010). Knock-down of Aof1 by shRNA in DCs abrogated expression of the subset of H3K9-methylated target genes, indicating that demethylation is an upstream pre-requisite for transcription. Moreover, we found by ChIP that in knocked-down cells, the stimulus-induced recruitment of NF-kB dimers to these promoters cannot occur. H3K9me2 demethylation therefore represents a change in chromatin structure which can be shown to control the inducible binding of NF-kB dimers. Interestingly, we observed that a lower-level, basal binding of NF-kB dimers containing the c-Rel subunit before stimulation appeared unaffected, and we tested whether this was responsible for targeting of Aof1 to specific promoters upon cellular stimulation. Indeed, demethylase-recruitment, removal of H3K9 methylation, and transcription do not take place in c-Rel-knockout DCs, and c-Rel is the only NF-kB subunit essential for transcription of these target genes (van Essen et al., 2010).


Current efforts in the lab are directed at uncovering the molecular basis by which H3K9 methylation acts to restrict the inducible binding of NF-kB, and on understanding the details of c-Rel-dependent demethylase recruitment (including identifying potential co-factors required for this process).

Figure 1 : A Feed-Forward Circuit Controlling Inducible NF-κB Target Gene Activation by Promoter Histone Demethylation


2.   Control of cell-type specific enhancer function


To identify determinants of cell-type specific expression, we have identified and analysed the enhancers of several DC-specific NF-kB target genes.  Surprisingly, we found that although the genes themselves are not expressed in other cell types, such as fibroblasts, their enhancers appear to be active, bearing high levels of H3K4me1, and capable of driving reporter gene expression when removed from their natural genomic context.  In both fibroblasts and DCs, these enhancers are embedded in genomic regions rich in H3K9me3, a characteristic mark of heterochromatin; upon stimulation, however, whereas in DCs this modification is locally reduced or even completely removed, in fibroblasts it is significantly augmented.  Moreover, by ectopically targeting an H3K9me3 to a DC-specific enhancer, we established a direct causal link with the repression of enhancer activity.  Thus, H3K9me3 can impart cell-type specificity to otherwise broadly-active enhancers. 


This left unanswered the question of how H3K9me3 at these enhancers could be selectively regulated in DCs.  We identified an H3K9me3 demethylase whose expression is induced by proinflammatory stimuli in myeloid cells; we find that this enzyme is recruited to genomic regions surrounding enhancers, and by shRNA-mediated knock down we determined that its activity is required for reduction of H3K9me3 levels upon LPS stimulation of DCs, and for transcription from the associated promoters (Zhu et al, 2012).


We are currently investigating the mechanisms underlying demethylase targeting to enhancers, and whether a similar control of enhancer function is widely used in other cell types. Computational analysis indicates that control of enhancer function by H3K9me3-based repression, is utilized in a variety of other cell types, at a significant fraction of active enhancers.  We are currently attempting to further understand the mechanisms involved in cell-type specific regulation of enhancer function.  This idea suggests that diverse cell types may utilize a common machinery to target specific enhancers for de-repression.


Figure 2 : Genome-wide predicted enhancers tend to be protected from, but flanked by high levels of H3k9me3


3.    Mechanisms of transcriptional activation by NF-kB p65


The molecular basis by which NF-kB activates endogenous promoters is largely unknown, but it seemed likely that it should include the means to tailor transcriptional output to match the wide functional range of its target genes.  With this in mind, we have investigated the mechanism of activation by the p65 subunit of NF-kB.  By disrupting its direct interaction with the Mediator complex (either by knock-down of the Trap80 subunit of Mediator, or by targeted mutation of p65 itself), we find that p65 exhibits two distinct modes of transcriptional activation: the first proceeds via direct contact with the Mediator complex, leading to RNA pol II recruitment to promoters.  The second, on the other hand, controls promoter accessibility for the binding of secondary transcription factors; this activity maps to an uncharacterized region of p65 (which we term ‘TA3’), and allows a subset of target promoters to fine-tune their expression by selective use of additional transcription factors, while remaining dependent for their activation on NF-kB.  The activity of TA3 correlates with the induction of several chromatin modifications at target promoters, including strong, transcription-independent histone acetylation, and current efforts in the lab are directed at elucidating its precise molecular mode of action. 


To address the mechanisms underlying this, we want to identify the set of co-factors which interact with p65 and which are required for this activity by unbiased SILAC-MS base approach. 

Figure 3 : NF-kB p65 exhibits two modes of transcriptional activity



Research Team

Research Team


From left to right : Simona Saccani, Christelle Cayrou, Ludovic Cervera, Dominic Van Essen,

José Ramos Pittol, Ana Carolina Franco Ferreira (photo 2014)



SACCANI Simona, DR2 INSERM, Team Leader
Tel. +33 (0)4 93 37 77 52, E-mail : This e-mail address is being protected from spambots. You need JavaScript enabled to view it

CAYROU Christelle, CR1 CNRS

Tel. +33 (0)4 93 37 77 47, E-mail : This e-mail address is being protected from spambots. You need JavaScript enabled to view it


LOPEZ PASCAL, CR1 INSERM
Tel. +33 (0)4 93 37 77 47, E-mail : This e-mail address is being protected from spambots. You need JavaScript enabled to view it


VAN ESSEN Dominic, Post-Doc & Project Leader
Tel. +33 (0)4 93 37 77 47, E-mail : This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Lab 1 : +33 (0)4 93 37 77 42

Lab 2 : +33 (0)4 93 37 77 99
Office : +33 (0)4 93 37 77 47

(Feb 2016)



Publications

Publications


http://www.ncbi.nlm.nih.gov/pubmed/?term=simona+saccani



2015


      Saccani S, Trabucchi M.

      Regulation of stimulus-inducible gene expression in myeloid cells.

      Semin Immunol. 2015 Feb;27(1):33-43.


2012


Zhu Y, van Essen D, Saccani S.

Cell-type-specific control of enhancer activity by H3K9 trimethylation.

Mol. Cell., 2012, May 25;46(4):408-23.


Zhu Y, van Essen D, Saccani S

Cell-Type-Specific Control of Enhancer Activity by H3K9 Trimethylation.

Cell. Cycle, 2012, Feb 15;11(4):646-7.


Saccani S.

All p65-containing dimers are not equal

Cell Cycle. 2012 Feb 15;11(4):646-7.


2011


Olszak AM, van Essen D, Pereira AJ, Diehl S, Manke T, Maiato H, Saccani S, Heun P.

Heterochromatin boundaries are hotspots for de novo kinetochore formation.

Nat. Cell. Biol., 2011, Jun 19,13(7):799-808.


Tomasoni R, Basso V, Pilipow K, Sitia G, Saccani S, Agresti A, Mietton F, Natoli G, Colombetti S, Mondino A.

Rapamycin-sensitive signals control TCR/CD28-driven Ifng, Il4 and Foxp3 transcription and promoter region methylation.

Eur. J. Immunol., 2011, Jul;41(7):2086-96.


2010


Van Essen D, Zhu Y, Saccani S.

A feed-forward circuit controlling inducible NF-κB target gene activation by promoter histone demethylation.

Mol. Cell., 2010, Sep 10;39(5):750-60.


2009


Van Essen D, Engist B, Natoli G, Saccani S.

Two modes of transcriptional activation at native promoters by NF-kappaB p65.

PLoS Biol. 7, 2009, e73.


2008


Zhang Y, Saccani S, Shin H, Nikolajczyk BS.

Dynamic protein associations define two phases of IL-1beta transcriptional activation.

J. Immunol., 2008, 181, 503-51.


(Feb 2016)