Epigenetics and cell proliferation

Epigenetics and cell proliferation - Differenciation

FRE 3239

Group leader : Annick Harel-Bellan

Pathways involved in the control of cell proliferation/differentiation

Cell proliferation and differentiation are controlled at multiple levels. Using muscle as the main model, we are investigating events and pathways involved in this control, with specific focus on transcriptional (epigenetic) and post-transcriptional mechanisms (translational and in particular events linked to small non-coding RNAs).

This group has a long-standing interest in the events controlling mammalian cell fate, with specific emphasis on cell proliferation and differentiation. Cell fate control is key to tissue formation during development, and its deregulation results in developmental defects or in tumorigenesis. Our principal model, but not the only one, is mouse skeletal muscle. Muscle formation is a complex process controlled by cell-autonomous and non-cell-autonomous events, and which ultimately involves expression of muscle marker proteins and fusion of pre-determined precursor cells into large multinucleated myotubes that will subsequently mature into muscle fibers. Muscle differentiation can be explored in vivo, during embryogenesis or during muscle regeneration in fully formed individuals, as well as in vitro, using totipotent embryonic stem (ES) cells, myoblast cell lines or primary myoblasts from embryos or adult animals.
One of our main (but not exclusive) focuses is on epigenetic-like events, such as histone modification or promoter shuttling between euchromatin and heterochromatin, and, more recently, small non-coding (sncRNAs) – microRNAs (miRNAs) and rasiRNAs – mimicked by artificial siRNAs. MiRNAs have emerged as being essential for animal embryogenesis. They inhibit target gene expression by guiding, in a sequence-specific manner, a particular protein complex to the 3’UTR of target mRNAs, thereby inducing target mRNA degradation or more frequently, in animals, blocking target mRNA translation. MiRNAs are involved in cell fate control. RasiRNAs and PiRNAs are other classes of short RNAs that act at the nuclear level, most likely by influencing chromatin structure (transcriptional gene silencing, TGS). SiRNAs are artificial RNAs that mimic the natural miRNAs and can be used to selectively inhibit target genes.
Our major goal is to gain an integrated view of the pathways involved in cell fate control, at the level of essential genes and of sncRNAs. These issues are addressed by global monitoring of expression and function, as well as by genetic and biochemical approaches.
Our specific objectives are:
1° To reconstitute pathways involved in cell proliferation, and in cell cycle exit followed by terminal differentiation. Individual pathways are explored, such as, for example, the Lin-28 pathway, which acts at the level of translation of essential mRNAs such as IGF-2 (Figure 1), or else the ubiquitin pathway, in particular the study of ubiquitination complexes and their function in DNA repair. More globally, we are collaborating with the PaRI, a high throughput screening facility in which genome-wide siRNA libraries (currently covering 22,500 human genes, 4 siRNAs per gene) are screened using assays related to cell fate (proliferation, differentiation and programmed cell death). Pathways will be reconstituted bioinformatically, and validated by biochemical or genetic approaches.
2° To explore the function of miRNAs in cell fate control by genome-wide monitoring of their expression (using arrays followed by individual validation) and of their function (using a loss-of-function assay that we have designed). MicroRNAs for which differential expression is demonstrated (Figure 2) and a function established in these preliminary screens are analyzed in detail, either in vitro (by investigating the mechanisms through which their expression is regulated as well as their mode of action and their specific gene targets), or in vivo (by creating conditional knockout mice).
3° To explore the involvement of nuclear sncRNAs in cell fate control in mammals. The role of rasiRNAs in TGS has been documented in eukaryotic model organisms (yeast, Drosophila) where they participate in silencing heterochromatic repeated sequences such as retro-transposons. It is not clear, however, whether such a mechanism is conserved in mammals. We are addressing this controversial issue using biochemical and functional approaches. If these preliminary experiments yield positive results, we will explore pathways involved in mammalian TGS using a global genetic approach.
We believe that our studies will help reveal unknown molecular pathways and eventually lead to an integrated understanding of pathways involving both sncRNAs and gene products, which ultimately will provide new targets for cancer therapy.

Lin 28 is expressed in differentiating muscle cells. Primary myoblasts were placed under differentiation conditions for 5 days and stained for Lin-28 expression (red); nuclei were counterstained with DAPI (blue).

The microRNA miR-181 is expressed during muscle formation in vivo. Cross sections of regenerating muscle were stained with anti-eMHC (a marker of muscle regeneration, red) and with a DNA probe complementary to miR-181 (green); nuclei were counterstained with DAPI.

Acetylation is important for MyoD function in adult mice. Duquet A, Polesskaya A, Cuvellier S, Ait-Si-Ali S, Hery P, Pritchard LL, Gerard M, Harel-Bellan A.
EMBO Rep. 2006 Nov;7(11):1140-6. Epub 2006 Oct 6.
CSA-dependent degradation of CSB by the ubiquitin-proteasome pathway establishes a link between complementation factors of the Cockayne syndrome. Groisman R, Kuraoka I, Chevallier O, Gaye N, Magnaldo T, Tanaka K, Kisselev AF*, Harel-Bellan A*, Nakatani Y*.
Genes Dev. 2006 Jun 1;20(11):1429-34.
* : equal contribution
The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation. Naguibneva I, Ameyar-Zazoua M, Polesskaya A, Ait-Si-Ali S, Groisman R, Souidi M, Cuvellier S, Harel-Bellan A.
Nat Cell Biol. 2006 Mar;8(3):278-84.
A Suv39h-dependent mechanism for silencing S-phase genes in differentiating but not in cycling cells. Ait-Si-Ali S, Guasconi V, Fritsch L, Yahi H, Sekhri R, Naguibneva I, Robin P, Cabon F, Polesskaya A, Harel-Bellan A.
EMBO J. 2004 Feb 11;23(3):605-15. Epub 2004 Feb 5.
Conditional gene knock-down by CRE-dependent short interfering RNAs. Fritsch L, Martinez LA, Sekhri R, Naguibneva I, Gerard M, Vandromme M, Schaeffer L, Harel-Bellan A.
EMBO Rep. 2004 Feb;5(2):178-82. Epub 2004 Jan 9.