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The neuropeptide galanin is a widely distributed neurotransmitter/neuromodulator that regulates a variety of physiological processes and also participates in the regulation of stress responses. The aims of the present study were to investigate the expression of galanin receptors (GalR1, GalR2, GalR3) in the spinal cords in a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) using qPCR analysis and to determine GalR1 cellular localization (oligodendrocytes, microglia, astrocytes, ependymal cells, and endothelial cells in the capillaries) by immunohistochemistry.Twelve samples from the EAE group and 14 samples from the control group were analyzed. Spinal cords samples were obtained at the peak of the EAE disease.The GalR1 mRNA level was significantly decreased in the EAE mice compared with the controls (P=0.016), whereas the mRNA levels of GalR2 and GalR3 were not significantly different for the EAE and the control mice. No significant correlations were found between the severity of the EAE disease and the mRNA levels of GalR1, GalR2 and GalR3. Immunochemical detection of the GalR1 revealed its expression in the ependymal and endothelial cells. Additionally, a weak GalR1 immunoreactivity was occasionally detected in the oligodendrocytes.This study provides additional evidence of galanin involvement in EAE pathophysiology, but this has to be further investigated.

A precisely balanced activity of canonical Wnt signaling is essential for a number of biological processes and its perturbation leads to developmental defects or diseases. Here, we demonstrate that alternative isoforms of the KDM2A and KDM2B lysine demethylases have the ability to negatively regulate canonical Wnt signaling. These KDM2A and KDM2B isoforms (KDM2A-SF and KDM2B-SF) lack the N-terminal demethylase domain, but they are able to bind to activated promoters in order to repress them. We have observed that KDM2A-SF and KDM2B-SF bind to and repress the promoters of AXIN2 and CYCLIN D1, two canonical Wnt signaling target genes. Moreover, KDM2A-SF and KDM2B-SF can repress a Wnt-responsive luciferase reporter. The transcriptional repression mediated by KDM2A-SF and KDM2B-SF, but also by KDM2A-LF, is dependent on their DNA binding domain, while the N-terminal demethylase domain is dispensable for this process. Surprisingly, KDM2B-LF is unable to repress both the endogenous promoters and the luciferase reporter. Finally, we show that both KDM2A-SF and KDM2B-SF are able to interact with TCF7L1, one of the transcriptional mediators of canonical Wnt signaling. KDM2A-SF and KDM2B-SF are thus likely to affect the transcription of the TCF7L1 target genes also through this interaction.

Histone modifications have a profound impact on the chromatin structure and gene expression and their correct establishment and recognition is essential for correct cell functioning. Malfunction of histone modifying proteins is associated with developmental defects and diseases and detailed characterization of these proteins is therefore very important. The lysine specific demethylase KDM2A is a CpG island binding protein that has been studied predominantly for its ability to regulate CpG island-associated gene promoters by demethylating their H3K36me2. However, very little attention has been paid to the alternative KDM2A isoform that lacks the N-terminal demethylation domain, KDM2A-SF. Here we characterized KDM2A-SF more in detail and we found that, unlike the canonical full length KDM2A-LF isoform, KDM2A-SF forms distinct nuclear heterochromatic bodies in an HP1a dependent manner. Our chromatin immunoprecipitation experiments further showed that KDM2A binds to transcriptionally silent pericentro...

Phosphoinositides are present in the plasma membrane, cytoplasm and inside the cell nucleus. Here we identify phosphatidylinositol-4,5-bisphosphate (PIP2) as a regulator of rRNA genes transcription at the epigenetic level. We show that PIP2 directly interacts with histone lysine demethylase PHF8 (PHD finger protein 8) and represses demethylation of H3K9me2 through this interaction. We identify the C-terminal K/R-rich motif as PIP2-binding site within PHF8, and address the function of this PIP2-PHF8 complex. PIP2-binding mutant of PHF8 has increased the activity of rDNA promoter (20%) and expression of pre-rRNA genes (47S-100%; 45S-66%). Furthermore, trypsin digestion reveals a potential conformational change of PHF8 upon PIP2 binding. These observations identify the function of nuclear PIP2, and suggest that PIP2 contributes to the fine-tuning of rDNA transcription.

Approximately 20,000 mammalian genes are estimated to encode between 250 thousand and 1 million different proteins. This enormous diversity of the mammalian proteome is caused by the ability of a single-gene locus to encode multiple protein isoforms. Protein isoforms encoded by one gene locus can be functionally distinct, and they can even have antagonistic functions. One of the mechanisms involved in creating this proteome complexity is alternative promoter usage. Alternative intronic promoters are located downstream from their canonical counterparts and drive the expression of alternative RNA isoforms that lack upstream exons. These upstream exons can encode some important functional domains, and proteins encoded by alternative mRNA isoforms can be thus functionally distinct from the full-length protein encoded by canonical mRNA isoforms. Since any misbalance of functionally distinct protein isoforms is likely to have detrimental consequences for the cell and the whole organism, their expression must be precisely regulated. Misregulation of alternative intronic promoters is frequently associated with various developmental defects and diseases including cancer, and it is becoming increasingly clear that this phenomenon deserves more attention.

Neurogenesis in the developing neocortex is a strictly regulated process of cell division and differentiation. Here we report that a gradual retreat of canonical Wnt signaling in the cortex from lateral-to-medial and anterior-to-posterior is a prerequisite of neurogenesis. Ectopic expression of a β-catenin/LEF1 fusion protein maintains active canonical Wnt signaling in the developing cortex and delays the expression onset of the neurogenic factors Pax6, Ngn2 and Tbr2 and subsequent neurogenesis. Contrary to this, conditional ablation of β-catenin accelerates expression of the same neurogenic genes. Furthermore, we show that a sustained canonical Wnt activity in the lateral cortex gives rise to cells with hippocampal characteristics in the cortical plate at the expense of the cortical fate, and to cells with dentate gyrus characteristics in the hippocampus. This suggests that the dose of canonical Wnt signaling determines cellular fate in the developing cortex and hippocampus, and that recession of Wnt signaling acts as a morphogenetic gradient regulating neurogenesis in the cortex.

Insulin-like growth factor 2 receptor (Igf2r), a maternally expressed imprinted gene, is triplicated in Ts43H trisomic mice. To verify its expression in a trisomic mode, we examined the allele-specific transcripts and relative levels of Igf2r mRNA in trisomic and control mice. Igf2r was expressed from the maternal allele(s) and silenced on the paternal allele(s) in most tissues of maternally or paternally derived Ts43H mice. The triallelic expression was observed in adult brain and testis and in embryonic head, but the antisense transcript, Air, was strictly paternal, and methylation of region 2 was strictly maternal in all tissues. The Igf2r expression in maternally derived trisomics, with two active copies of the gene, did not exceed the average mRNA levels of euploid controls with monoallelic expression. Thus, an indication is presented for a dosage compensation mechanism of Igf2r in the trisomic context.

All of the mouse models of human trisomy 21 syndrome that have been studied so far are based on segmental trisomies, encompassing, to a varying extent, distal chromosome 16. Their comparison with one or more unrelated and non-overlapping segmental trisomies may help to distinguish the effects of specific triplicated genes from the phenotypes caused by less specific developmental instability mechanisms. In this paper, the Ts43H segmental trisomy of mouse chromosome 17 is presented as such an alternative model. The trisomy stretches over 32.5 Mb of proximal chromosome 17 and includes 486 genes. The triplicated interval carries seven blocks of synteny with five human chromosomes. The block syntenic to human chromosome 21 contains 20 genes.

Three indigenous plasmids designated pRK1, pRK2 and pRK3 were identified among producers of penicillin G acylase (PGA) derived from the strain Escherichia coli W ATCC 9637. Their size and copy number (CN) in E. coli W were determined (kb; CN): pRK1 (80; 3.4), pRK2 (5.1; 71), and pRK3 (4.8; 13.7). Strain E. coli RE2 harboring these plasmids was used for selection of strains with reduced number of plasmids: the strain RE3 without plasmid pRK1 and the plasmid-less strain cERE3 were isolated. Indigenous plasmids did not code for the resistance determinants against 23 antibiotics and 10 heavy metals.

Axial patterning of the embryonic brain requires a precise balance between canonical Wnt signaling, which dorsalizes the nervous system, and Sonic hedgehog (Shh), which ventralizes it. The ventral anterior homeobox (Vax) transcription factors are induced by Shh and ventralize the forebrain through a mechanism that is poorly understood. We therefore sought to delineate direct Vax target genes. Among these, we identify an extraordinarily conserved intronic region within the gene encoding Tcf7l2, a key mediator of canonical Wnt signaling. This region functions as a Vax2-activated internal promoter that drives the expression of dnTcf7l2, a truncated Tcf7l2 isoform that cannot bind β-catenin and that therefore acts as a potent dominant-negative Wnt antagonist. Vax2 concomitantly activates the expression of additional Wnt antagonists that cooperate with dnTcf7l2. Specific elimination of dnTcf7l2 in Xenopus results in headless embryos, a phenotype consistent with a fundamental role for this regulator in forebrain development.

Lens formation in mouse is critically dependent on proper development of the retinal neuroectoderm that is located close beneath the head surface ectoderm. Signaling from the prospective retina triggers lens-specific gene expression in the surface-ectoderm. Supression of canonical Wnt/beta-catenin signaling in the surface ectoderm is one of the prerequisites for lens development because, as we show here, ectopic Wnt activation in the retina and lens abrogates lens formation. Wnt inhibiton is mediated by signals coming from the retina but its exact mechanism is unknown. We show that Pax6 directly controls expression of several Wnt inhibitors such as Sfrp1, Sfrp2, and Dkk1 in the presumptive lens. In accordance, absence of Pax6 function leads to aberrant canonical Wnt activity in the presumptive lens that subsequently impairs lens development. Thus Pax6 is required for down-regulation of canonical Wnt signaling in the presumptive lens ectoderm.

Wnt/beta-catenin signaling regulates many processes during vertebrate development. To study transcriptional targets of canonical Wnt signaling, we used the conditional Cre/loxP system in mouse to ectopically activate beta-catenin during central nervous system development. We show that the activation of Wnt/beta-catenin signaling in the embryonic mouse telencephalon results in the up-regulation of Sp5 gene, which encodes a member of the Sp1 transcription factor family. A proximal promoter of Sp5 gene is highly evolutionarily conserved and contains five TCF/LEF binding sites that mediate direct regulation of Sp5 expression by canonical Wnt signaling. We provide evidence that Sp5 works as a transcriptional repressor and has three independent repressor domains, called R1, R2, and R3, respectively. Furthermore, we show that the repression activity of R1 domain is mediated through direct interaction with a transcriptional corepressor mSin3a. Finally, our data strongly suggest that Sp5 has the same DNA binding specificity as Sp1 and represses Sp1 target genes such as p21. We conclude that Sp5 transcription factor mediates the downstream responses to Wnt/beta-catenin signaling by directly repressing Sp1 target genes.

Triplication of whole autosomes or large autosomal segments is detrimental to the development of a mammalian embryo. The trisomy of human chromosome (Chr) 21, known as Down's syndrome, is regularly associated with mental retardation and a variable set of other developmental anomalies. Several mouse models of Down's syndrome, triplicating 33–104 genes of Chr16, were designed in an attempt to analyze the contribution of specific orthologous genes to particular developmental features. However, a recent study challenged the concept of dosage-sensitive genes as a primary cause of an abnormal phenotype. To distinguish between the specific effects of dosage-sensitive genes and nonspecific effects of a large number of arbitrary genes, we revisited the mouse Ts43H/Ph segmental trisomy. It encompasses >310 known genes triplicated within the proximal 30 megabases (Mb) of Chr17. We refined the distal border of the trisomic segment to the interval bounded by bacterial artificial chromosomes RP23-277B13 (location 29.0 Mb) and Cbs gene (location 30.2 Mb). The Ts43H mice, viable on a mixed genetic background, exhibited spatial learning deficits analogous to those observed in Ts65Dn mice with unrelated trisomy. Quantitative analysis of the brain expression of 20 genes inside the trisomic interval and 12 genes lying outside on Chr17 revealed 1.2-fold average increase of mRNA steady-state levels of triplicated genes and 0.9-fold average down-regulation of genes beyond the border of trisomy. We propose that systemic comparisons of unrelated segmental trisomies, such as Ts65Dn and Ts43H, will elucidate the pathways leading from the triplicated sequences to the complex developmental traits.

The complete primary structure of the str operon of Bacillus stearothermophilus was determined. It was established that the operon is a five-gene transcriptional unit: 5′- ybxF (unknown function; homology to eukaryotic ribosomal protein L30)- rpsL (S12)- rpsG (S7)- fus (elongation factor G [EF-G])- tuf (elongation factor Tu [EF-Tu])-3′. The main operon promoter ( str p) was mapped upstream of ybxF , and its strength was compared with the strength of the tuf -specific promoter ( tuf p) located in the fus-tuf intergenic region. The strength of the tuf p region to initiate transcription is about 20-fold higher than that of the str p region, as determined in chloramphenicol acetyltransferase assays. Deletion mapping experiments revealed that the different strengths of the promoters are the consequence of a combined effect of oppositely acting cis elements, identified upstream of str p (an inhibitory region) and tuf p (a stimulatory A/T-rich block). Our results suggest that the oppositely adjusted core promoters significantly contribute to the differential expression of the str operon genes, as monitored by the expression of EF-Tu and EF-G.