[Show abstract][Hide abstract] ABSTRACT: Recent studies have shown changes in myelin genes and alterations in white matter structure in a wide range of psychiatric disorders. Here we report that DBZ, a central nervous system (CNS)-specific member of the DISC1 interactome, positively regulates the oligodendrocyte (OL) differentiation in vivo and in vitro. In mouse corpus callosum (CC), DBZ mRNA is expressed in OL lineage cells and expression of DBZ protein peaked before MBP expression. In the CC of DBZ-KO mice, we observed delayed myelination during the early postnatal period. Although the myelination delay was mostly recovered by adulthood, OLs with immature structural features were more abundant in adult DBZ-KO mice than in control mice. DBZ was also transiently upregulated during rat OL differentiation in vitro before myelin marker expression. DBZ knockdown by RNA interference resulted in a decreased expression of myelin-related markers and a low number of cells with mature characteristics, but with no effect on the proliferation of oligodendrocyte precursor cells. We also show that the expression levels of transcription factors having a negative-regulatory role in OL differentiation were upregulated when endogenous DBZ was knocked down. These results strongly indicate that OL differentiation in rodents is regulated by DBZ. GLIA 2014.
[Show abstract][Hide abstract] ABSTRACT: Pou5f1/Oct4, a member of the POU transcription factor family, is exclusively expressed in embryonic stem cells, which are involved in self-renewal and maintaining pluripotency. In the present study, we report on the establishment of a monoclonal antibody that is specific for Oct4 using the rat medial iliac lymph node method. In an immunoblotting analysis, our antibody detected endogenous Oct4. In addition, immunocytochemical staining using the antibody revealed the nuclear localization of Oct4. This monoclonal antibody has the potential for use in the further analysis of Oct4 function in stem cells.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 06/2013; 32(3):229-31. DOI:10.1089/mab.2013.0004
[Show abstract][Hide abstract] ABSTRACT: Modification of histone plays a critical role in the epigenetic regulation of gene expression. However, unlike the widely studied roles of histone methylation or acetylation of histone H3, relatively little is known about the molecular mechanisms involved in translating histone phosphorylation into a specific outcome. The present study reports on the development of antibodies (MAbs) directed against phosphorylated histone H3 (S10, T11, S28, S31, and T32), produced by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat or mouse. The MAbs produced specifically recognize different sites of phosphorylation on histone H3. All of these MAbs are suitable for immunoblotting and immunofluorescence analysis. We believe that these antibodies should significantly facilitate our efforts to investigate epigenetic regulation.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 04/2013; 32(2):119-24. DOI:10.1089/mab.2012.0105
[Show abstract][Hide abstract] ABSTRACT: Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (∼50 nm movement/30 ms), which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.
[Show abstract][Hide abstract] ABSTRACT: Deep sequencing approaches, such as chromatin immunoprecipitation by sequencing (ChIP-seq), have been successful in detecting transcription factor-binding sites and histone modification in the whole genome. An approach for comparing two different ChIP-seq data would be beneficial for predicting unknown functions of a factor. We propose a model to represent co-localization of two different ChIP-seq data. We showed that a meaningful overlapping signal and a meaningless background signal can be separated by this model. We applied this model to compare ChIP-seq data of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation with a large amount of peak-called data, including ChIP-seq and other deep sequencing data in the Encyclopedia of DNA Elements (ENCODE) project, and then extracted factors that were related to RNA polymerase II CTD serine 2 in HeLa cells. We further analyzed RNA polymerase II CTD serine 7 phosphorylation, of which their function is still unclear in HeLa cells. Our results were characterized by the similarity of localization for transcription factor/histone modification in the ENCODE data set, and this suggests that our model is appropriate for understanding ChIP-seq data for factors where their function is unknown.
Nucleic Acids Research 11/2012; 41(1). DOI:10.1093/nar/gks1010 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell differentiation is mediated by lineage-determining transcription factors. We show that chromodomain helicase DNA-binding domain 2 (Chd2), a SNF2 chromatin remodelling enzyme family member, interacts with MyoD and myogenic gene regulatory sequences to specifically mark these loci via deposition of the histone variant H3.3 prior to cell differentiation. Directed and genome-wide analysis of endogenous H3.3 incorporation demonstrates that knockdown of Chd2 prevents H3.3 deposition at differentiation-dependent, but not housekeeping, genes and inhibits myogenic gene activation. The data indicate that MyoD determines cell fate and facilitates differentiation-dependent gene expression through Chd2-dependent deposition of H3.3 at myogenic loci prior to differentiation.
The EMBO Journal 05/2012; 31(13):2994-3007. DOI:10.1038/emboj.2012.136 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII), which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq.
We first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII.
We demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII.
[Show abstract][Hide abstract] ABSTRACT: The septin family of GTPase proteins has been shown to be important for cell division, cytoskeletal organization, and membrane-remodeling events. Septin 9 (SEPT9) is a member of the septin family (also designated MSF/eseptin/Sint1) and has been implicated in tumorigenesis. The present study reports on the preparation and properties of a monoclonal antibody (MAb) directed against SEPT9. The antibody was produced by hybridization of mouse myeloma cells with lymph node cells from an immunized rat. The MAb 7B5 specifically recognized SEPT9, as evidenced by immunoblotting using a variety of extracts from cultured cells. In immunostaining using MAb 7B5, a filamentous pattern near the plasma membrane was observed. The MAb 7B5 promises to be useful in immunoblotting and immunostaining experiments in various cells and tissues to determine the expression levels of SEPT9, as well as to further the analysis of the biological function of this protein.
[Show abstract][Hide abstract] ABSTRACT: CHD5 (chromodomain/helicase/DNA-binding protein 5) is a member of the CHD subfamily of chromatin remodeling Swi/Snf proteins, and has been recently identified as a tumor suppressor in a diverse range of human cancers. We report here on the establishment of a hybridoma cell line for producing a monoclonal antibody against CHD5 by the rat medial iliac lymph node method. Immunoblotting analyses indicated that this antibody, MAb 5A10, specifically recognizes endogenous CHD5. In immunostaining using the antibody, a nuclear staining pattern was observed. The monoclonal antibody will be useful in immunoblotting and immunolocalization experiments in a variety of cells and tissues, as well as in further studies of the biological function and cellular dynamics of this protein.
[Show abstract][Hide abstract] ABSTRACT: We report the characterization of a new member of the low-density lipoprotein receptor (LDLR) gene family designated LRP10. Human LRP10 cDNA encodes a 1905 amino acid type I membrane protein consisting of five functional domains characteristic of the LDLR gene family. CHO-ldlA7 cells transfected with human LRP10 cDNA bound LDLR-associated protein, but not beta-VLDL and HDL. Human LRP10 transcripts were primarily found in the brain, muscle and heart. In situ hybridization of the rat brain showed that the transcripts were intensely present in the cerebral cortex, hippocampus, choroid plexus, ependyma and granular layer. In the developing rat brain, transcript levels gradually increased from postnatal day 1 to 20. Immunofluorescence analysis indicated that LRP10 was observed in the ventricular zone of the embryonic day 14.5 mouse cerebral cortex. The present studies suggest that LRP10 may play a significant role in the brain physiology other than lipoprotein metabolism.
Biochemical and Biophysical Research Communications 12/2009; 391(1):1110-5. DOI:10.1016/j.bbrc.2009.12.033 · 2.30 Impact Factor