Corticotropin-releasing hormone exerts direct effects on neuronal progenitor cells: implications for neuroprotection.

Neurogenesis during embryonic and adult life is tightly regulated by a network of transcriptional, growth and hormonal factors. Emerging evidence indicates that activation of the stress response, via the associated glucocorticoid increase, reduces neurogenesis and contributes to the development of adult diseases.As corticotrophin-releasing hormone (CRH) or factor is the major mediator of adaptive response to stressors, we sought to investigate its involvement in this process. Accordingly, we found that CRH could reverse the damaging effects of glucocorticoid on neural stem/progenitor cells (NS/PCs), while its genetic deficiency results in compromised proliferation and enhanced apoptosis during neurogenesis. Analyses in fetal and adult mouse brain revealed significant expression of CRH receptors in proliferating neuronal progenitors. Furthermore, by using primary cultures of NS/PCs, we characterized the molecular mechanisms and identified CRH receptor-1 as the receptor mediating the neuroprotective effects of CRH. Finally, we demonstrate the expression of CRH receptors in human fetal brain from early gestational age, in areas of active neuronal proliferation. These observations raise the intriguing possibility for CRH-mediated pharmacological applications in diseases characterized by altered neuronal homeostasis, including depression, dementia, neurodegenerative diseases, brain traumas and obesity.

Heterochromatin formation in mammalian cells: interaction between histones and HP1 proteins.

Members of the heterochromatin protein 1 (HP1) family are silencing nonhistone proteins. Here, we show that in P19 embryonal carcinoma (EC) nuclei, HP1 alpha, beta, and gamma form homo- and heteromers associated with nucleosomal core histones. In vitro, all three HP1s bind to tailed and tailless nucleosomes and specifically interact with the histone-fold of histone H3. Furthermore, HP1alpha interacts with the linker histone H1. HP1alpha binds to H3 and H1 through its chromodomain (CD) and hinge region, respectively. Interestingly, the Polycomb (Pc1/M33) CD also interacts with H3, and HP1alpha and Pc1/M33 binding to H3 is severely impaired by CD mutations known to abrogate HP1 and Polycomb silencing in Drosophila. These results define a novel function for the conserved CD and suggest that HP1 self-association and histone binding may play a crucial role in HP1-mediated heterochromatin assembly.

Chromatin is permissive to TATA-binding protein (TBP)-mediated transcription initiation.

Preinitiation complex assembly is nucleated by the binding of TFIID to the promoters of protein coding genes transcribed by RNA polymerase II. TFIID is comprised of the TATA-binding protein (TBP) and TBP-associated factors (TAF(II)s). We investigated the transcription properties of TBP and TFIID on chromatin templates. On naked templates both TBP and purified TFIID are able to initiate basal transcription. However, on chromatin templates only TBP mediates transcription initiation in a heat-treated extract, whereas TFIID does not. Moreover, TBP-mediated chromatin transcription is blocked in a nontreated extract. These observations suggest that a chromatin-targeted repressor is present in crude extracts and that chromatin per se is not refractory to transcription mediated by TBP. As TBP can function through TAF(II)-independent and TAF(II)-dependent pathways, the repression of TBP-mediated basal transcription may be an additional level to the control of Pol II transcription initiation on chromatin.

Expression of the transcriptional intermediary factor TIF1alpha during mouse development and in the reproductive organs.

Nuclear receptors are important regulators of development and reproduction whose action can be modulated by transcriptional intermediary factors (TIFs). In situ hybridization was used to investigate the expression pattern of the putative nuclear receptor mediator TIF1alpha during mouse embryogenesis and adult life. TIF1alpha is ubiquitously expressed until midgestation. At 12.5 gestational days, TIF1alpha is preferentially expressed in the developing central and peripheral nervous system. Differential expression persists until perinatal stages, with high expression in the brain, nasal epithelium and within proliferating regions of the kidney and teeth. In the adult, TIF1alpha expression is predominant in both the male and female gonads. Immunogold electron microscopy revealed that TIF1alpha protein is most abundant in the nuclei of male germ cells at various stages of their maturation.

The putative nuclear receptor mediator TIF1alpha is tightly associated with euchromatin.

Ligand-dependent transcriptional regulation by nuclear receptors is believed to be mediated by intermediary factors (TIFs) acting on remodelling of the chromatin structure and/or the activity of the transcriptional machinery. The putative transcriptional mediator TIF1alpha is a nuclear protein kinase that has been identified via its interaction with liganded nuclear receptors, including retinoic acid (RAR), retinoid X (RXR) and estrogen (ER) receptors. Here, we demonstrate that TIF1alpha is a non-histone chromosomal protein tightly associated with highly accessible euchromatic regions of the genome. Immunofluorescence confocal microscopy reveals that TIF1alpha exhibits a finely granular distribution in euchromatin of interphase nuclei, while it is mostly excluded from condensed chromatin and metaphase chromosomes. Immunoelectron microscopy shows that, in contrast to the heterochromatin protein HP1alpha, most of TIF1alpha is associated with euchromatin, where it is preferentially localised on regions known to be sites for RNA polymerase II (perichromatin fibrils and borders between euchromatin and heterochromatin). Early mouse embryos as well as embryonal carcinoma (EC) and embryonic stem (ES) cells express high levels of TIF1alpha. These levels dramatically decrease during organogenesis and upon differentiation of P19 EC cells, indicating that TIF1alpha is preferentially expressed in undifferentiated pluripotent cells in the course of development. Therefore, TIF1alpha could belong to a novel class of chromatin-associated TIFs that facilitate the access of transregulators (e.g. liganded nuclear receptors) to their cognate sites in target genes, thereby participitating in the epigenetic control of transcription during embryonic development and cell differentiation.

Ligand-dependent activation of transcription in vitro by retinoic acid receptor alpha/retinoid X receptor alpha heterodimers that mimics transactivation by retinoids in vivo.

All-trans and 9-cis retinoic acids (RA) signals are transduced by retinoic acid receptor/retinoid X receptor (RAR/RXR) heterodimers that act as functional units controlling the transcription of RA-responsive genes. With the aim of elucidating the underlying molecular mechanisms, we have developed an in vitro transcription system using a chromatin template made up of a minimal promoter and a direct repeat with 5-spacing-based RA response element. RARalpha and RXRalpha were expressed in and purified from baculovirus-infected Sf9 cells, and transcription was carried out by using naked DNA or chromatin templates. Transcription from naked templates was not affected by the presence of RA and/or RAR/RXR heterodimers. In contrast, very little transcription occurred from chromatin templates in the absence of RA or RAR/RXR heterodimers whereas their addition resulted in a dosage-dependent stimulation of transcription that never exceeded that occurring on naked DNA templates. Most importantly, the addition of synthetic agonistic or antagonistic retinoids to the chromatin transcription system mimicked their stimulatory or inhibitory action in vivo, and activation by a RXR-specific retinoid was subordinated to the binding of an agonist ligand to the RAR partner. Moreover, the addition of the p300 coactivator generated a synergistic enhancement of transcription. Thus, the dissection of this transcription system ultimately should lead to the elucidation of the molecular mechanisms by which RAR/RXR heterodimers control transcription in a ligand-dependent manner.

Molecular architecture of a Leu3p-DNA complex in solution: a biochemical approach.

The Leu3 protein (Leu3p) of Saccharomyces cerevisiae is a pleiotropic transregulator that can function both as an activator and as a repressor of transcription. It binds to upstream promoter elements (UASLEU) with the consensus sequence 5'-GCCGGNNCCGGC-3'. The DNA-binding motif of Leu3p belongs to the family of Zn(II)2-Cys6 clusters. The motif is located between amino acid residues 37 and 67 of the 886-residue protein. In this study, we used a recombinant peptide consisting of residues 17 to 147 to explore the interaction between Leu3p and its cognate DNA. We found that the Leu3p(17-147) peptide is a monomer in the absence of UASLEU but assumes a dimeric structure when the DNA is present. Results of protein-DNA cross-linking and methylation and ethylation interference footprinting experiments show that the Leu3p(17-147) dimer interacts symmetrically with two contact triplets separated by 6 bp and suggest that the peptide approaches its target DNA in such a way that each subunit is positioned closer to one DNA strand than to the other. The binding of Leu3p is strongly affected by the spacing between the contact triplets of the UASLEU and by the type of triplet. Binding occurs when the triplets are 6 bp apart (normal spacing) but fails to occur when the triplets are 0, 5, or 8 bp apart. Weak binding occurs when the triplets are 7 bp apart. Binding does not occur when the UASLEU triplets (GCC....GGC) are replaced with triplets found in the UAS elements for Gal4p, Put3p, and Ppr1p (CGG....CCG). The apparent Kd for the normal Leu3p(17-147)-UASLEU complex is about 3 nM. A mutant form of Leu3p(17-147) in which the histidine at position 50 has been replaced with cysteine binds UASLEU with significantly greater affinity (apparent Kd of about 0.7 nM), even though the interaction between the mutant peptide and target DNA appears to be unchanged. Interestingly, repression of basal-level transcription, which is a hallmark property of the wild-type Leu3p(17-147) peptide, is largely lost with the mutant peptide, indicating that there is no direct correlation between strength of binding and repression.

Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions.

The Leu3 protein of Saccharomyces cerevisiae binds to specific DNA sequences present in the 5' noncoding region of at least five RNA polymerase II-transcribed genes. Leu3 functions as a transcriptional activator only when the metabolic intermediate alpha-isopropylmalate is also present. In the absence of alpha-isopropylmalate, Leu3 causes transcription to be repressed below basal levels. We show here that different portions of the Leu3 protein are responsible for activation and repression. Fusion of the 30 C-terminal residues of Leu3 to the DNA-binding domain of the Gal4 protein created a strong cross-species activator, demonstrating that the short C-terminal region is not only required but also sufficient for transcriptional activation. Using a recently developed Leu3-responsive in vitro transcription assay as a test system for repression (J. Sze, M. Woontner, J. Jaehning, and G. B. Kohlhaw, Science 258:1143-1145, 1992), we show that mutant forms of the Leu3 protein that lack the activation domain still function as repressors. The shortest repressor thus identified had only about 15% of the mass of the full-length Leu3 protein and was centered on the DNA-binding region of Leu3. Implications of this finding for the mechanism of repression are discussed.

Cloning, bacterial expression, purification, and characterization of the cytoplasmic domain of rat LAR, a receptor-like protein tyrosine phosphatase.

This report describes the cloning and characterization of rat leukocyte common antigen-related protein (rLAR), a receptor-like protein tyrosine phosphatase (PTPase). The recombinant cytoplasmic PTPase domain was expressed at high levels in bacteria and purified to homogeneity. Kinetic properties of the PTPase were examined along with potential modulators of PTPase activity. Several sulfhydryl-directed reagents were effective inhibitors, and a surprising distinction between iodoacetate and iodoacetamide was observed. The latter compound was an extremely poor inhibitor when compared to iodoacetate, suggesting that iodoacetate may interact selectively with a positive charge at or near the active site of the enzyme. Site-directed mutants were made at 4 highly conserved cysteine residues found at positions 1434, 1522, 1723, and 1813 within the protein. The Cys-1522/Ser mutation resulted in a 99% loss of enzymatic activity of the pure protein. This observation is consistent with greater than 99% of the PTPase activity being found in the first domain of the PTPase and demonstrates the critical importance of this cysteine residue in catalysis. The recombinant C1522S mutant phosphatase could also be phosphorylated in vitro by protein kinase C and p43v-abl tyrosine kinase. When pure recombinant PTPase was mixed with 32P-labeled tyrosine substrate and then rapidly denatured, a 32P-labeled enzyme intermediate could be trapped and visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The catalytically inactive C1522S mutant did not form the phosphoenzyme intermediate.