To model human neural-cell-fate specification and to provide cells for regenerative therapies, we have developed a method to generate human neural progenitors and neurons from human embryonic stem cells, which recapitulates human fetal brain development. Through the addition of a small molecule that activates canonical WNT signaling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. Ten days after initiation of differentiation, the progenitors could be transplanted to the adult rat striatum, where they formed neuron-rich and tumor-free grafts with maintained regional specification. Cells patterned toward a ventral midbrain (VM) identity generated a high proportion of authentic dopaminergic neurons after transplantation. The dopamine neurons showed morphology, projection pattern, and protein expression identical to that of human fetal VM cells grafted in parallel. VM-patterned but not forebrain-patterned neurons released dopamine and reversed motor deficits in an animal model of Parkinson's disease.

DNA methylation plays an important role in mammalian development and correlates with chromatinassociated gene silencing. The recruitment of MeCP2 to methylated CpG dinucleotides represents a major mechanism by which DNA methylation can repress transcription. MeCP2 silences gene expression partly by recruiting histone deacetylase (HDAC) activity, resulting in chromatin remodeling. Here, we show that MeCP2 associates with histone methyltransferase activity in vivo and that this activity is directed against Lys 9 of histone H3. Two characterized repression domains of MeCP2 are involved in tethering the histone methyltransferase to MeCP2. We asked if MeCP2 can deliver Lys 9 H3 methylation to the H19 gene, whose activity it represses. We show that the presence of MeCP2 on nucleosomes within the repressor region of the H19 gene (the differentially methylated domain) coincides with an increase in H3 Lys 9 methylation. Our data provide evidence that MeCP2 reinforces a repressive chromatin state by acting as a bridge between two global epigenetic modifications, DNA methylation and histone methylation.Methylation of cytosines is essential for mammalian development and is associated with gene silencing (1). DNA methylation represses genes partly by recruitment of methyl-CpGbinding domain proteins, which selectively recognize methylated CpG dinucleotides. MeCP2 is the founder member of the methyl-CpG-binding domain proteins, which consists of a single polypeptide that contains a methyl-CpG-binding domain and a transcriptional repression domain. MeCP2 is capable of binding to a single symmetrically methylated CpG both in naked DNA and within chromatin (2, 3).It is now well established that MeCP2 silences transcription by recruiting the histone deacetylase (HDAC) 1 repressive machinery, which removes acetyl groups from histones resulting in gene silencing (4,5). However, the inhibition of histone deacetylase activity using drugs such as Trichostatin A only partially relieves MeCP2-mediated transcriptional repression. This partial relief indicates that additional mechanisms of repression by MeCP2 likely exist aside from the recruitment of histone deacetylase.Beside histone deacetylation, histone methylation is emerging as another key post-translational modification of histones and represents an important epigenetic mechanism for the organization of chromatin structure and the regulation of gene expression. In particular, methylation at lysine 9 of histone H3 is associated with gene silencing, and several enzymes that catalyze the addition of methyl groups to lysine 9 have recently been identified (6). Interestingly, recent data have shown that the retinoblastoma protein represses transcription through the recruitment of HDAC activity, but in a second step it recruits histone methylation activity specific for lysine 9 of histone H3 (7). By analogy to retinoblastoma, we considered in the present work whether MeCP2-mediated repression might also include, besides histone deacetylation, a second stage involving histone methylatio...

No abstract

Cellular reprogramming is a new and rapidly emerging field in which somatic cells can be turned into pluripotent stem cells or other somatic cell types simply by the expression of specific combinations of genes. By viral expression of neural fate determinants, it is possible to directly reprogram mouse and human fibroblasts into functional neurons, also known as induced neurons. The resulting cells are nonproliferating and present an alternative to induced pluripotent stem cells for obtaining patient-and disease-specific neurons to be used for disease modeling and for development of cell therapy. In addition, because the cells do not pass a stem cell intermediate, direct neural conversion has the potential to be performed in vivo. In this study, we show that transplanted human fibroblasts and human astrocytes, which are engineered to express inducible forms of neural reprogramming genes, convert into neurons when reprogramming genes are activated after transplantation. Using a transgenic mouse model to specifically direct expression of reprogramming genes to parenchymal astrocytes residing in the striatum, we also show that endogenous mouse astrocytes can be directly converted into neural nuclei (NeuN)-expressing neurons in situ. Taken together, our data provide proof of principle that direct neural conversion can take place in the adult rodent brain when using transplanted human cells or endogenous mouse cells as a starting cell for neural conversion.T he ability to reprogram somatic cells to pluripotent stem cells or other somatic cell types by expressing key combinations of genes has opened up new possibilities for disease modeling and cell therapy (1, 2). Using this technique, it is possible to directly reprogram mouse and human fibroblasts into functional neurons, also known as induced neurons (iNs), using viral delivery of the three neural conversion factors achaete-scute complex-like 1 (Ascl1), brain-2 (Brn2a), and myelin transcription factor-like 1 (Myt1l) (ABM) (3, 4). A growing number of studies now show that by altering the combination of genes used for reprogramming, different subtypes of neurons are obtained (3,5,6). Importantly, the resulting cells are nonproliferating, which makes them an interesting alternative to induced pluripotent stem cells as a source of patient-specific neurons for cell replacement therapy, once efficient grafting strategies for these cells are developed.The adult brain has a very limited inherent capacity for repair, and new neurons are only formed in two discrete regions: the subventricular zone of the lateral ventricles, which generates neurons migrating to the olfactory bulb, and the hippocampus (7,8). Experimental studies have shown that these endogenous progenitors can also be recruited to generate new neurons in other regions as well in response to injury (9-11). However, the number of new neurons is very low, their migration is hard to control, and the therapeutic implications are unclear. Several cell types residing outside the neurogenic niche, such as parenchymal...

Moloney murine leukemia virus (M-MLV) replication is restricted in embryonic carcinoma (EC) and embryonic stem (ES) cells, likely to protect the germ line from insertional mutagenesis. Proviral DNAs are potently silenced at the level of transcription in these cells. This silencing is largely due to an unidentified trans-acting factor that is thought to bind to the primer binding site (PBS) of M-MLV and repress transcription from the viral promoter. We have partially purified a large PBS-mediated silencing complex and identified TRIM28 (Kap-1), a known transcriptional silencer, as an integral component of the complex. We show that RNAi-mediated knockdown of TRIM28 in EC and ES cells relieves the restriction and that TRIM28 is bound to the PBS in vivo when restriction takes place. The identification of TRIM28 as a retroviral silencer adds to the growing body of evidence that many TRIM family proteins are involved in retroviral restriction.

Retroviruses are highly successful intracellular parasites, and as such they are found in nearly all branches of life. Some are relatively benign, but many are highly pathogenic and can cause either acute or chronic diseases. Therefore, there is tremendous selective pressure on the host to prevent retroviral replication, and for this reason cells have evolved a variety of restriction factors that act to inhibit or block the viruses. This review is a survey of the best-characterized restriction factors capable of inhibiting retroviral replication and aims to highlight the diversity of strategies used for this task.

Acetylation controls the activity of numerous proteins involved in regulating gene transcription as well as many other cellular processes. In this report we show that the CREB-binding protein (CBP) acetyltransferase acetylates ␤-catenin protein in vivo. ␤-Catenin is a central component of the Wnt signaling pathway, which is of key importance in development as well as being heavily implicated in a variety of human cancers. We show that the CBP-mediated acetylation of ␤-catenin occurs at a single site, lysine 49. Importantly, this lysine is frequently found mutated in cancer and is in a region of importance to the regulation of ␤-catenin. We show that mutation of this site leads specifically to an increase in the ability of ␤-catenin to activate the c-myc gene but not other ␤-catenin-regulated genes. This suggests that acetylation of ␤-catenin is involved in regulating Wnt signaling in a promoter-specific fashion.The Wnt signaling pathway is involved in numerous developmental processes (1), and its deregulation is implicated in the development of cancer (2). 〈 key component in the Wnt signaling pathway is the ␤-catenin protein, which is responsible for transducing the Wnt signal from the cytoplasm to the nucleus where it results in the activation of Wnt-responsive genes. ␤-Catenin was originally identified as a component of cell-cell adhesion complexes, connecting cadherins to the cytoskeleton (3). Subsequently, a large proportion of ␤-catenin was shown to be localized to the cell membrane. This pool of ␤-catenin is thought not to be involved in Wnt signaling. The signaling pool of ␤-catenin is relatively small and is found free in both the cytoplasm and the nucleus where it activates transcription by binding and co-activating the TCF/LEF-1 1 family of transcription factors.One of the key aspects of the regulation of ␤-catenin is control of its stability, which in turn is thought to regulate its translocation into the nucleus (4). ␤-Catenin is a target of the ubiquitin-dependent proteasome degradation pathway (5). When the Wnt signaling pathway is in the "off" state ␤-catenin is continuously ubiquitinated and degraded. It is the phosphorylation of ␤-catenin by the GSK3␤ kinase that targets it for ubiquitination. Phosphorylation occurs at three serines and a threonine residue in the N terminus of ␤-catenin (6) and potentiates ␤-catenin binding to the F-box protein ␤-TrCP, which is a part of the SCF ubiquitin ligase complex responsible for the ubiquitination of ␤-catenin (7). Upon activation of the Wnt pathway GSK3␤ no longer phosphorylates ␤-catenin due, at least in part, to the antagonizing function of the disheveled protein.Deregulation of the Wnt pathway occurs in many types of cancer and is caused by mutation of many of the Wnt signaling components (2). In the case of ␤-catenin it has been found that most of the mutations occur in the N terminus at or adjacent to the phosphorylated serine and threonine residues (see Fig. 1). Many of these mutations have been shown to stabilize ␤-catenin and thus lead to overact...

The class II histone deacetylases HDAC4 and HDAC5 interact specifically with the myogenic MEF2 transcription factor and repress its activity. Here we show that HDAC4 is cytoplasmic during myoblast differentiation, but relocates to the nucleus once fusion has occurred. Inappropriate nuclear entry of HDAC4 following overexpression suppresses the myogenic programme as well as MEF2-dependent transcription. Activation of the Ca(2+)/calmodulin signalling pathway via constitutively active CaMKIV prevents nuclear entry of HDAC4 and HDAC4-mediated inhibition of differentiation. Consistent with a role of phosphorylation in HDAC4 cytoplasmic localisation, HDAC4 binds to 14-3-3 proteins in a phosphorylation-dependent manner. Together these data establish a role for HDAC4 in muscle differentiation. Recently, HDAC5 has also been implicated in muscle differentiation. However, despite the functional similarities of HDAC4 and HDAC5, their intracellular localisations are opposed, suggesting a distinct role for these enzymes during muscle differentiation.