EZH2-Mediated H3K27me3 Targets Transcriptional Circuits of Neuronal Differentiation.

The Polycomb Repressive Complex 2 (PRC2) plays important roles in the epigenetic regulation of cellular development and differentiation through H3K27me3-dependent transcriptional repression. Aberrant PRC2 activity has been associated with cancer and neurodevelopmental disorders, particularly with respect to the malfunction of sits catalytic subunit EZH2. Here, we investigated the role of the EZH2-mediated H3K27me3 apposition in neuronal differentiation. We made use of a transgenic mouse model harboring Ezh2 conditional KO alleles to derive embryonic stem cells and differentiate them into glutamatergic neurons. Time course transcriptomics and epigenomic analyses of H3K27me3 in absence of EZH2 revealed a significant dysregulation of molecular networks affecting the glutamatergic differentiation trajectory that resulted in: (i) the deregulation of transcriptional circuitries related to neuronal differentiation and synaptic plasticity, in particular LTD, as a direct effect of EZH2 loss and (ii) the appearance of a GABAergic gene expression signature during glutamatergic neuron differentiation. These results expand the knowledge about the molecular pathways targeted by Polycomb during glutamatergic neuron differentiation.

The H3K27 demethylase JMJD3 is required for maintenance of the embryonic respiratory neuronal network, neonatal breathing, and survival.

JMJD3 (KDM6B) antagonizes Polycomb silencing by demethylating lysine 27 on histone H3. The interplay of methyltransferases and demethylases at this residue is thought to underlie critical cell fate transitions, and the dynamics of H3K27me3 during neurogenesis posited for JMJD3 a critical role in the acquisition of neural fate. Despite evidence of its involvement in early neural commitment, however, its role in the emergence and maturation of the mammalian CNS remains unknown. Here, we inactivated Jmjd3 in the mouse and found that its loss causes perinatal lethality with the complete and selective disruption of the pre-Bötzinger complex (PBC), the pacemaker of the respiratory rhythm generator. Through genetic and electrophysiological approaches, we show that the enzymatic activity of JMJD3 is selectively required for the maintenance of the PBC and controls critical regulators of PBC activity, uncovering an unanticipated role of this enzyme in the late structuring and function of neuronal networks.

Che-1 enhances cyclin-dependent kinase 5 expression and interacts with the active kinase-complex.

Che-1 is a nuclear protein involved in the regulation of gene transcription and cell proliferation. It has also been shown to localize to the cytoplasm of postmitotic neuronal cells, where it is able to interact with the microtubule-associated protein tau. Cyclin-dependent kinase 5 (Cdk5) is a postmitotic proline-directed serine/threonine kinase that hyperphosphorylates tau under pathological conditions. We observed that Che-1 overexpression induces Cdk5 expression both at the mRNA and protein levels. Furthermore, we show that Che-1 directly interacts with Cdk5 protein in vivo. Cdk5/Che-1 complex formation does not compete with Cdk5/p35 interaction, thus Che-1 is able to bind the active kinase complex. Finally, we demonstrated that Che-1 is itself a Cdk5 substrate.

Utrophin up-regulation by an artificial transcription factor in transgenic mice.

Duchenne Muscular Dystrophy (DMD) is a severe muscle degenerative disease, due to absence of dystrophin. There is currently no effective treatment for DMD. Our aim is to up-regulate the expression level of the dystrophin related gene utrophin in DMD, complementing in this way the lack of dystrophin functions. To this end we designed and engineered several synthetic zinc finger based transcription factors. In particular, we have previously shown that the artificial three zinc finger protein named Jazz, fused with the appropriate effector domain, is able to drive the transcription of a test gene from the utrophin promoter "A". Here we report on the characterization of Vp16-Jazz-transgenic mice that specifically over-express the utrophin gene at the muscular level. A Chromatin Immunoprecipitation assay (ChIP) demonstrated the effective access/binding of the Jazz protein to active chromatin in mouse muscle and Vp16-Jazz was shown to be able to up-regulate endogenous utrophin gene expression by immunohistochemistry, western blot analyses and real-time PCR. To our knowledge, this is the first example of a transgenic mouse expressing an artificial gene coding for a zinc finger based transcription factor. The achievement of Vp16-Jazz transgenic mice validates the strategy of transcriptional targeting of endogenous genes and could represent an exclusive animal model for use in drug discovery and therapeutics.

The artificial 4-zinc-finger protein Bagly binds human utrophin promoter A at the endogenous chromosomal site and activates transcription.

Our aim is to upregulate the expression of the dystrophin-related gene utrophin in Duchenne muscular dystrophy, in this way complementing the lack of dystrophin function. To achieve utrophin upregulation, we designed and engineered synthetic zinc-finger based transcription factors. We have previously shown that the artificial 3-zinc-finger protein Jazz, fused with the appropriate effector domain, is able to drive the transcription of a test gene from utrophin promoter A. Here we report a novel artificial 4-zinc-finger protein, Bagly, which binds with optimized affinity-specificity to a 12 bp DNA target sequence that is internal to human utrophin promoter A. Bagly was generated adding to Jazz protein an extra-fourth zinc finger, derived from transcription factor YY1. Importantly, the Bagly DNA target sequence is statistically present in the human genome only 210 times, about 60 fewer times than the 9 bp Jazz DNA target sequence. Thanks to its additional zinc-finger domain, Bagly protein shows enhanced transcriptional activity. Moreover, we demonstrated Bagly's effective access and binding to active chromatin in the chromosomal context and its ability to upregulate endogenous utrophin.

Pancreatic duct epithelial cell isolation and cultivation in two-dimensional and three-dimensional culture systems.

Pancreatic ductal adenocarcinoma (PDA) is generally considered to have originated from pancreatic duct epithelial cells (PDEC). The ability to manipulate the growth properties of PDEC is, therefore, critical for understanding the molecular events involved in the initiation of PDA. Here, we describe methods that we have established for the isolation and maintenance of PDEC in two-dimensional and three-dimensional culture systems. The availability of these culture systems should be particularly useful for studying their relationships between specific genetic lesions and the morphogenic changes that accompany pancreatic ductal tumorigenesis.

A role for poly(ADP-ribosyl)ation in DNA methylation.

The aberrant DNA methylation of promoter regions of housekeeping genes leads to gene silencing. Additional epigenetic events, such as histone methylation and acetylation, also play a very important role in the definitive repression of gene expression by DNA methylation. If the aberrant DNA methylation of promoter regions is the starting or the secondary event leading to the gene silencing is still debated. Mechanisms controlling DNA methylation patterns do exist although they have not been ultimately proven. Our data suggest that poly(ADP-ribosyl)ation might be part of this control mechanism. Thus an additional epigenetic modification seems to be involved in maintaining tissue and cell-type methylation patterns that when formed during embryo development, have to be rigorously conserved in adult organisms.

Inhibition of poly(ADP-ribosyl)ation induces DNA hypermethylation: a possible molecular mechanism.

The pattern of DNA methylation established during embryonic development is necessary for the control of gene expression and is preserved during the replicative process. DNA regions of about 1-2 kb in size, termed CpG islands and located mostly in the promoter regions of housekeeping genes, are protected from methylation, despite being about 6-10 times richer in the dinucleotide CpG than the rest of DNA. Their unmethylated state guarantees the expression of the corresponding housekeeping genes. At present, the mechanism by which CpG islands remain protected from methylation is not clear. However, some results suggest that poly(ADP-ribosyl)ation, an enzymatic process that introduces a postsynthetic modification onto chromatin proteins, might be involved. Here we show in L929 mouse fibroblast cells that inhibition of poly(ADP-ribose) polymerase(s) at different cell-cycle phases increases the mRNA and protein levels of the major maintenance DNA methyltransferase (DNMT1) in G1/S border. Increase of DNMT1 results in a premature PCNA-DNMT1 complex formation, which facilitates robust maintenance, as well as de novo DNA methylation processes during the G1/S border, which leads to abnormal hypermethylation.