Allele-specific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain

Dlk1 and Gtl2 are reciprocally expressed imprinted genes located on mouse chromosome 12. The Dlk1-Gtl2 locus carries three differentially methylated regions (DMRs), which are methylated only on the paternal allele. Of these, the intergenic (IG) DMR, located 12 kb upstream of Gtl2, is required for proper imprinting of linked genes on the maternal chromosome, while the Gtl2 DMR, located across the promoter of the Gtl2 gene, is implicated in imprinting on both parental chromosomes. In addition to DNA methylation, modification of histone proteins is also an important regulator of imprinted gene expression. Chromatin immunoprecipitation was therefore used to examine the pattern of histone modifications across the IG and Gtl2 DMRs. The data show maternal-specific histone acetylation at the Gtl2 DMR, but not at the IG DMR. In contrast, only low levels of histone methylation were observed throughout the region, and there was no difference between the two parental alleles. An existing mouse line carrying a deletion/insertion upstream of Gtl2 is unable to imprint the Dlk1-Gtl2 locus properly and demonstrates loss of allele-specific methylation at the Gtl2 DMR. Further analysis of these animals now shows that the loss of allele-specific methylation is accompanied by increased paternal histone acetylation at the Gtl2 DMR, with the activated paternal allele adopting a maternal acetylation pattern. These data indicate that interactions between DNA methylation and histone acetylation are involved in regulating the imprinting of the Dlk1-Gtl2 locus.     

Expression of cab Genes in Douglas-Fir Is Not Strongly Regulated by Light

Dark-grown Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) seedlings had approximately 30% of the major polypeptide of the light-harvesting chlorophyll a/b binding protein, 30% of cab mRNA, 54% of psbA mRNA, and 14% of total chlorophyll, in comparison with amounts in light-grown seedlings. Seedlings entrained under a 24-hour photoperiod of light and dark showed small diurnal fluctuations in cab and psbA mRNA levels and, when transferred to continuous conditions, no circadian rhythms in mRNA levels were apparent. These results suggest that regulation of cab gene expression in Douglas-fir differs from regulation in angiosperms, because in the latter, both light and circadian factors strongly influence the expression of cab genes.     

DNA methylation imprints on the IG-DMR of the Dlk1-Gtl2 domain in mouse male germline

Mouse genomes show a large cluster of imprinted genes at the Dlk1-Gtl2 domain in the distal region of chromosome 12. An intergenic-differentially methylated region (IG-DMR) located between Dlk1 and Gtl2 is specifically methylated in the male germline; IG-DMR regulates the parental allele-specific expression of imprinted genes. Here, we show the resetting of IG-DMR methylation marks during male germ-cell differentiation. For parental allele-specific methylation analysis, polymorphisms were detected in a 2.6-kb IG-DMR in three mouse strains. Bisulfite methylation analysis showed erasure of the marks by E14 and re-establishment before birth. The IG-DMR methylation status was maintained in spermatogonia and spermatocytes of mature testes. The IG-DMR methylation status established before birth is thus maintained throughout the lifetime in the male germline.     

Expression of TrkB Receptors in Developing Visual Cortex Is Not Regulated by Light

1. Neurotrophins are very good candidates which relate electrical activity to molecular changes in activity-dependent phenomena. They exert their action through binding to specific tyrosine-kinase receptors: Trk receptors. It is important to consider Trk distribution in order to understand better the role of neurotrophins in the Central Nervous System (CNS). We focused our attention on brain-derived neurotrophic factor (BDNF) Trk receptors (TrkB) during development of the rat visual cortex, since this neurotrophin has been shown to play an important role in visual system development and plasticity.2. We investigated the full length form of TrkB receptors considering both its total amount and its cellular distribution. To address this issue we used an antibody that recognizes the full length form of TrkB and we used it both in Western blot and immunohistochemistry.3. We found that the expression of TrkB receptor increases during development, but that there is no effect on visual experience, since dark-reared animals show the same protein level and pattern of TrkB expression compared to age-matched, normally reared controls.     

LGR5 is a Proneural Factor and is Regulated by OLIG2 in Glioma Stem-Like Cells

The biological functional roles of LGR5 (leucine-rich repeat containing G protein-coupled receptor 5, also known as GPR49), a novel potential marker for stem-like cells in glioblastoma (GSCs), is poorly acknowledged. Here, we demonstrated that LGR5 was detected in glioblastoma tissues and GSCs. Bioinformatics analysis revealed that LGR5 is closely related to neurogenesis and neuronal functions, and preferentially expressed in Proneural subtype of GBMs. Furthermore, LGR5 is regulated by Proneural factor OLIG2, which is important for both neurogenesis and GSC maintenance. Biological experiments in GSC cells validated the bioinformatics analysis results and revealed that LGR5 regulated the tumor sphere formation capacity, an important stem cell property for GSCs. Therefore, LGR5 expression may be functionally correlated with the neurogenic competence, and be regulated by OLIG2 in GSCs.     

Notch1 Is Pan-Endothelial at the Onset of Flow and Regulated by Flow

Arteriovenous differentiation is a key event during vascular development and hemodynamic forces play an important role. Arteriovenous gene expression is present before the onset of flow, however it remains plastic and flow can alter arteriovenous identity. Notch signaling is especially important in the genetic determination of arteriovenous identity. Nevertheless, the effect of the onset of circulation on Notch expression and signaling has not been studied. The aim of this study is therefore to investigate the interaction of Notch1 signaling and hemodynamic forces during early vascular development. We find that the onset of Notch1 expression coincides with the onset of flow, and that expression is pan-endothelial at the onset of circulation in mouse embryos and only becomes arterial-specific after remodeling has occurred. When we ablate flow in the early embryo, endothelial cells fail to express Notch1. We show that low and disturbed flow patterns upregulate Notch1 expression in endothelial cells in vitro, but that higher shear stress levels do not (≥10 dynes/cm²). Using siRNA, we knocked down Notch1 to investigate the role of Notch1 in mechanotransduction. When we applied shear stress levels similar to those found in embryonic arteries, we found an upregulation of Klf2, Dll1, Dll4, Jag1, Hey1, Nrp1 and CoupTFII but that only Dll4, Hey1, Nrp1 and EphB4 required Notch1 for flow-induced expression. Our results therefore indicate that Notch1 can modulate mechanotransduction but is not a critical mediator of the process since many genes mechanotransduce normally in the absence of Notch1, including genes involved in arteriovenous differentiation.     

Sgt1 Dimerization Is Negatively Regulated by Protein Kinase CK2-mediated Phosphorylation at Ser361

The kinetochore, which consists of centromere DNA and structural proteins, is essential for proper chromosome segregation in eukaryotes. In budding yeast, Sgt1 and Hsp90 are required for the binding of Skp1 to Ctf13 (a component of the core kinetochore complex CBF3) and therefore for the assembly of CBF3. We have previously shown that Sgt1 dimerization is important for this kinetochore assembly mechanism. In this study, we report that protein kinase CK2 phosphorylates Ser³⁶¹ on Sgt1, and this phosphorylation inhibits Sgt1 dimerization.The kinetochore is a structural protein complex located in the centromeric region of the chromosome coupled to spindle microtubules (1, 2). The kinetochore generates a signal to arrest cells during mitosis when it is not properly attached to microtubules, thereby preventing chromosome missegregation, which can lead to aneuploidy (3, 4). The molecular structure of the kinetochore complex of the budding yeast Saccharomyces cerevisiae has been well characterized; it is composed of more than 70 proteins, many of which are conserved in mammals (2).The centromere DNA in the budding yeast is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEIII (25 bp) is essential for centromere function (7) and is bound to a key component of the centromere, the CBF3 complex. The CBF3 complex contains four proteins, Ndc10, Cep3, Ctf13 (8–15), and Skp1 (14, 15), all essential for viability. Mutations in any of the CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (16, 17). All of the kinetochore proteins, except the CDEI-binding Cbf1 (18–20), localize to the kinetochores in a CBF3-dependent manner (2). Thus, CBF3 is a fundamental kinetochore complex, and its mechanism of assembly is of great interest.We have previously found that Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required to form the active Ctf13-Skp1 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction: the tetratricopeptide repeat (21) and the CHORD protein and Sgt1-specific motif. We and others have found that both domains are important for the interaction of Sgt1 with Hsp90 (23–26), which is required for assembly of the core kinetochore complex. This interaction is an initial step in kinetochore activation (24, 26, 27), which is conserved between yeast and humans (28, 29).We have recently shown that Sgt1 dimerization is important for Sgt1-Skp1 binding and therefore for kinetochore assembly (30). In this study, we have found that protein kinase CK2 phosphorylates Sgt1 at Ser³⁶¹, and this phosphorylation inhibits Sgt1 dimerization. Therefore, CK2 appears to regulate kinetochore assembly negatively in budding yeast.     

Expression Patterns of Murine Antichymotrypsin-like Genes Reflect Evolutionary Divergence at the Serpina3 Locus

Members of the serpin (serine protease inhibitor) superfamily of genes are well represented in both human and murine genomes. In many cases it is possible to identify a definite ortholog on the basis of sequence similarity and by examining the surrounding genes at syntenic loci. We have recently examined the murine serpin locus at 12F1 and observed that the single human 1-antichymotrypsin gene is represented by 14 paralogs. It is also known that the single human 1-antitrypsin gene has five paralogs in the mouse. The forces driving this gene multiplication are unknown and there are no data describing the function of the various serpin gene products at the 1-antichymotrypsin multigene locus. Examination of the predicted amino acid sequences shows that the serpins are likely to be functional protease inhibitors but with differing target protease specificities. In order to begin to address the question of the problem presented by the murine 1-antichymotrypsins, we have used RT-PCR to examine the expression pattern of these serpin genes. Our data show that the divergent reactive center loop sequence, and predictably variable target protease specificity, is reflected in tissue-specific expression for many of the family members. These observations add weight to the hypothesis that the antichymotrypsin-like serpins have an evolutionary importance which has led to their expansion and diversification in multiple species.[Reviewing Editor: Dr. Peer Bork]     

Expression of the Homeobox Genes OTX2 and OTX1 in the Early Developing Human Brain

In rodents, the Otx2 gene is expressed in the diencephalon, mesencephalon, and cerebellum and is crucial for the development of these brain regions. Together with Otx1, Otx2 is known to cooperate with other genes to develop the caudal forebrain and, further, Otx1 is also involved in differentiation of young neurons of the deeper cortical layers. We have studied the spatial and temporal expression of the two homeobox genes OTX2 and OTX1 in human fetal brains from 7 to 14 weeks postconception by in situ hybridization and immunohistochemistry. OTX2 was expressed in the diencephalon, mesencephalon, and choroid plexus, with a minor expression in the basal telencephalon. The expression of OTX2 in the hippocampal anlage was strong, with no expression in the adjacent neocortex. Contrarily, the OTX1 expression was predominantly located in the proliferative zones of the neocortex. At later stages, the OTX2 protein was found in the subcommissural organ, pineal gland, and cerebellum. The early expression of OTX2 and OTX1 in proliferative cell layers of the human fetal brain supports the concept that these homeobox genes are important in neuronal cell development and differentiation: OTX1 primarily in the neocortex, and OTX2 in the archicortex, diencephalon, rostral brain stem, and cerebellum. (J Histochem Cytochem 58:669–678, 2010)