The nuclear receptor tailless induces long-term neural stem cell expansion and brain tumor initiation.

Malignant gliomas are the most common primary brain tumors, and are associated with frequent resistance to therapy as well as poor prognosis. Here we demonstrate that the nuclear receptor tailless (Tlx), which in the adult is expressed exclusively in astrocyte-like B cells of the subventricular zone, acts as a key regulator of neural stem cell (NSC) expansion and brain tumor initiation from NSCs. Overexpression of Tlx antagonizes age-dependent exhaustion of NSCs in mice and leads to migration of stem/progenitor cells from their natural niche. The increase of NSCs persists with age, and leads to efficient production of newborn neurons in aged brain tissues. These cells initiate the development of glioma-like lesions and gliomas. Glioma development is accelerated upon loss of the tumor suppressor p53. Tlx-induced NSC expansion and gliomagenesis are associated with increased angiogenesis, which allows for the migration and maintenance of brain tumor stem cells in the perivascular niche. We also demonstrate that Tlx transcripts are overexpressed in human primary glioblastomas in which Tlx expression is restricted to a subpopulation of nestin-positive perivascular tumor cells. Our study clearly demonstrates how NSCs contribute to brain tumorgenesis driven by a stem cell-specific transcription factor, thus providing novel insights into the histogenesis and molecular pathogenesis of primary brain tumors.

The nuclear receptor tailless is required for neurogenesis in the adult subventricular zone.

The tailless (Tlx) gene encodes an orphan nuclear receptor that is expressed by neural stem/progenitor cells in the adult brain of the subventricular zone (SVZ) and the dentate gyrus (DG). The function of Tlx in neural stem cells of the adult SVZ remains largely unknown. We show here that in the SVZ of the adult brain Tlx is exclusively expressed in astrocyte-like B cells. An inducible mutation of the Tlx gene in the adult brain leads to complete loss of SVZ neurogenesis. Furthermore, analysis indicates that Tlx is required for the transition from radial glial cells to astrocyte-like neural stem cells. These findings demonstrate the crucial role of Tlx in the generation and maintenance of NSCs in the adult SVZ in vivo.

Inactivation of the gene for the nuclear receptor tailless in the brain preserving its function in the eye.

During embryogenesis, tailless, an orphan member of the nuclear receptor family, is expressed in the germinal zones of the brain and the developing retina, and is involved in regulating the cell cycle of progenitor cells. Consequently, a deletion of the tailless gene leads to decreased cell number with associated anatomical defects in the limbic system, the cortex and the eye. These structural abnormalities are associated with blindness, increased aggressiveness, poor performance in learning paradigms and reduced anxiousness. In order to assess the contribution of blindness to the behavioural changes, we established tailless mutant mice with intact visual abilities. We generated a mouse line in which the second exon of the tailless gene is flanked by loxP sites and crossed these animals with a transgenic line expressing the Cre recombinase in the neurogenic area of the developing brain, but not in the eye. The resulting animals have anatomically indistinguishable brains compared with tailless germline mutants, but are not blind. They are less anxious and much more aggressive than controls, like tailless germline mutants. In contrast to germline mutants, the conditional mutants are not impaired in fear conditioning. Furthermore, they show good performance in the Morris water-maze despite severely reduced hippocampal structures. Thus, the pathological aggressiveness and reduced anxiety found in tailless germline mutants are due to malformations caused by inactivation of the tailless gene in the brain, but the poor performance of tailless null mice in learning and memory paradigms is dependent on the associated blindness.

In vitro assays to study protein ubiquitination in transcription.

Polyubiquitination is a death signal for proteins and condemns proteins to subsequent degradation by the 26S proteasome. However, recent studies imply that monoubiquitination and polyubiquitination of proteins do not necessarily result in protein degradation but play an important role in the execution of various biological events such as signal transduction and transcription. Ubiquitin was originally identified as a moiety attached to histones, and this as well as other histone modifications may play an important role for transcription and various other DNA-dependent processes. Considerable progress has been made in linking several histone modifications with chromatin dynamics in transcription. Acetylation of histones has been intimately linked to activation of transcription, while deacetylation is concomitant with repression of transcription. Although other histone modifications such as methylation, phosphorylation, and ubiquitination have been correlated with transcriptionally competent or inactive chromatin, the enzymes that mediate these modifications are only now being discovered. The identification of these histone-modifying enzymes may provide valuable insights into the role and function of histone modifications such as ubiquitination in transcription as well as other DNA-dependent processes. Recently, we have used various in vitro assays to show that the coactivator TAF(II)250 possesses both ubiquitin-activating and ubiquitin-conjugating activities, which monoubiquitinate histone H1. Here, we describe the methods used to identify this bifunctional enzyme: (1) in-gel activity assay; (2) protein-transfer membrane activity assay; and (3) in-solution activity assay. These methods have been successfully used to identify various histone-modifying enzymes and protein kinases. In this article we contribute a short review of the history of the methods used to study ubiquitination of proteins and histone modification. We provide protocols for in-gel, protein-transfer membrane, and in-solution ubiquitination assays. A discussion of the general use of the provided protocols, their limitations, and future perspectives are presented. The described methods provide useful tools for the identification of not only novel histone-modifying enzymes but also other protein-modifying enzymes that act in a variety of biological events.