Ryo Ito

TET3–OGT interaction increases the stability and the presence of OGT in chromatin

Gene expression is controlled by alterations in the epigenome, including DNA methylation and histone modification. Recently, it was reported that 5‐methylcytosine (5mC) is converted to 5‐hydroxymethylcytosine (5hmC) by proteins in the ten‐eleven translocation (TET) family. This conversion is believed to be part of the mechanism by which methylated DNA is demethylated. Moreover, histones undergo modifications such as phosphorylation and acetylation. In addition, modification with O‐linked‐N‐acetylglucosamine (O‐GlcNAc) by O‐GlcNAc transferase (OGT) was recently identified as a novel histone modification. Herein, we focused on TET3, the regulation of which is still unclear. We attempted to elucidate the mechanism of its regulation by biochemical approaches. First, we conducted mass spectrometric analysis in combination with affinity purification of FLAG–TET3, which identified OGT as an important partner of TET3. Co‐immunoprecipitation assays using a series of deletion mutants showed that the C‐terminal H domain of TET3 was required for its interaction with OGT. Furthermore, we showed that TET3 is GlcNAcylated by OGT, although the GlcNAcylation did not affect the global hydroxylation of methylcytosine by TET3. Moreover, we showed that TET3 enhanced its localization to chromatin through the stabilization of OGT protein. Taken together, we showed a novel function of TET3 that likely supports the function of OGT.

Identification of Myelin Transcription Factor 1 (MyT1) as a Subunit of the Neural Cell Type-specific Lysine-specific Demethylase 1 (LSD1) Complex

Background: The composition of tissue-specific complexes that modify histone is largely unknown. Results: We identified myelin transcription factor 1 (MyT1) as a lysine-specific demethylase 1 (LSD1) complex component by biochemical approaches using Neuro2a cells. This complex directly regulated the phosphatase and tensin homolog (Pten) gene expression. Conclusion: MyT1 is a neuro-specific LSD1 complex component. Significance: A tissue-specific complex component of LSD1 complex was identified. Regulation of spatiotemporal gene expression in higher eukaryotic cells is critical for the precise and orderly development of undifferentiated progenitors into committed cell types of the adult. It is well known that dynamic epigenomic regulation (including chromatin remodeling and histone modifications by transcriptional coregulator complexes) is involved in transcriptional regulation. Precisely how these coregulator complexes exert their cell type and developing stage-specific activity is largely unknown. In this study we aimed to isolate the histone demethylase lysine-specific demethylase 1 (LSD1) complex from neural cells by biochemical purification. In so doing, we identified myelin transcription factor 1 (MyT1) as a novel LSD1 complex component. MyT1 is a neural cell-specific zinc finger factor, and it forms a stable multiprotein complex with LSD1 through direct interaction. Target gene analysis using microarray and ChIP assays revealed that the Pten gene was directly regulated by the LSD1-MyT1 complex. Knockdown of either LSD1 or MyT1 derepressed the expression of endogenous target genes and inhibited cell proliferation of a neuroblastoma cell line, Neuro2a. We propose that formation of tissue-specific combinations of coregulator complexes is a critical mechanism for tissue-specific transcriptional regulation.

TATA-binding Protein (TBP)-like Protein Is Required for p53-dependent Transcriptional Activation of Upstream Promoter of p21Waf1/Cip1 Gene

Background: TLP and p21 are involved in growth inhibition. Results: TLP decreased cell growth and activated upstream promoter of p21 gene p53-dependently. TLP bound to p53. The promoter was associated with TLP and p53, and TLP enhanced recruitment of p53 to the promoter. Conclusion: TLP is required for p53-dependent transcriptional activation of p21 upstream promoter. Significance: A novel regulator for p53-p21 pathway was identified. TATA-binding protein-like protein (TLP) is involved in development, checkpoint, and apoptosis through potentiation of gene expression. TLP-overexpressing human cells, especially p53-containing cells, exhibited a decreased growth rate and increased proportion of G1 phase cells. TLP stimulated expression of several growth-related genes including p21 (p21Waf1/Cip1). TLP-mediated activation of the p21 upstream promoter in cells was shown by a promoter-luciferase reporter assay. The p53-binding sequence located in the p21 upstream promoter and p53 itself are required for TLP-mediated transcriptional activation. TLP and p53 bound to each other and synergistically enhanced activity of the upstream promoter. TLP specifically activated transcription from the endogenous upstream promoter, and p53 was required for this activation. Etoposide treatment also resulted in activation of the upstream promoter as well as nuclear accumulation of TLP and p53. Moreover, the upstream promoter was associated with endogenous p53 and TLP, and the p53 recruitment was enhanced by TLP. The results of the present study suggest that TLP mediates p53-governed transcriptional activation of the p21 upstream promoter.

Multiple post-translational modifications in hepatocyte nuclear factor 4α

To investigate the role of post-translational modifications (PTMs) in the hepatocyte nuclear factor 4α (HNF4α)-mediated transcription, we took a comprehensive survey of PTMs in HNF4α protein by mass-spectrometry and identified totally 8 PTM sites including newly identified ubiquitilation and acetylation sites. To assess the impact of identified PTMs in HNF4α-function, we introduced point mutations at the identified PTM sites and, tested transcriptional activity of the HNF4α. Among the point-mutations, an acetylation site at lysine 458 was found significant in the HNF4α-mediated transcriptional control. An acetylation negative mutant at lysine 458 showed an increased transcriptional activity by about 2-fold, while an acetylation mimic mutant had a lowered transcriptional activation. Furthermore, this acetylation appeared to be fluctuated in response to extracellular nutrient conditions. Thus, by applying an comprehensive analysis of PTMs, multiple PTMs were newly identified in HNF4α and unexpected role of an HNF4α acetylation could be uncovered.

Efficient custom computing of fully-streamed lattice boltzmann method on tightly-coupled FPGA cluster

This paper presents the detailed design of a custom computing machine for fully-streamed LBM computation on multiple FPGAs, and evaluates its efficiency with prototype implementation. We design a unit for completely streamed computation including boundary treatment with a newly introduced cell attribute. Experimental results demonstrate that the proposed machine achieves high utilization of PEs, 99 % of the peak performance, for one and two FPGAs computing a large lattice. This is due to our fully-streamed design to allow all arithmetic units to be efficienly utilized with a constant memory bandwidth, and the architecture to exploit a low-latency accelerator domain network (ADN) of a tightly-coupled FPGA cluster for scalable computation.

Bandwidth compression of multiple numerical data streams for high performance custom computing

Bandwidth compression improves the performance of stream computing by enhancing an effective bandwidth. To apply the bandwidth compression to numerical applications such as numerical simulations, a compressor has to handle multiple data streams. In this paper, we describe a design of an FPGA-based bandwidth compressor for high performance stream computation. For synchronization of original data in multiple compressed streams with different bit-rate, we propose a data block transmission scheduler and explore a design space to reduce the size of their barrel shifters.

Angiotensin II receptor blockers differentially affect CYP11B2 expression in human adrenal H295R cells

We generated a stable H295R cell line expressing aldosterone synthase gene (CYP11B2) promoter/luciferase chimeric reporter construct that is highly sensitive to angiotensin II (AII) and potassium, and defined AII receptor blocker (ARB) effects. In the presence of AII, all ARBs suppressed AII-induced CYP11B2 transcription. However, telmisartan alone increased CYP11B2 transcription in the absence of AII. Telmisartan dose-dependently increased CYP11B2 transcription/mRNA expression and aldosterone secretion. Experiments using CYP11B2 promoter mutants indicated that the Ad5 element was responsible. Among transcription factors involved in the element, telmisartan significantly induced NGFIB/NURR1 expression. KN-93, a CaMK inhibitor, abrogated the telmisartan-mediated increase of CYP11B2 transcription/mRNA expression and NURR1 mRNA expression, but not NGFIB mRNA expression. NURR1 over-expression significantly augmented the telmisartan-mediated CYP11B2 transcription, while high-dose olmesartan did not affect it. Taken together, telmisartan may stimulate CYP11B2 transcription via NGFIB and the CaMK-mediated induction of NURR1 that activates the Ad5 element, independent of AII type 1 receptor.

Identification of Posttranslational Modifications in Peroxisome Proliferator-Activated Receptor γ Using Mass Spectrometry

Posttranslational modification (PTM) of proteins is critical for various cellular processes. However, there are few studies examining PTMs in specific proteins using unbiased approaches. Here we report the attempt to identify the PTMs in peroxisome proliferator-activated receptor γ (PPARγ) proteins using our previously established PTM analysis system. In this study, we identified several PTMs in exogenously expressed PPARγ2 proteins from 293T cells as well as endogenous PPARγ1 proteins from a Caco-2 colon cancer cell line. The identified PTMs include phosphorylation of serine 112 and serine 81 in PPARγ2 and PPARγ1, respectively, both of which are well-known mitogen-activated protein kinase- (MAP kinase-) mediated PTMs in PPARγ proteins, thus confirming our experimental approach. Furthermore, previously unknown PTMs were also identified, demonstrating that this method can be applied to find previously unidentified PTMs in PPARγ proteins and other proteins including nuclear receptors.

Suppressive effects of RXR agonist PA024 on adrenal CYP11B2 expression, aldosterone secretion and blood pressure.

The effects of retinoids on adrenal aldosterone synthase gene (CYP11B2) expression and aldosterone secretion are still unknown. We therefore examined the effects of nuclear retinoid X receptor (RXR) pan-agonist PA024 on CYP11B2 expression, aldosterone secretion and blood pressure, to elucidate its potential as a novel anti-hypertensive drug. We demonstrated that PA024 significantly suppressed angiotensin II (Ang II)-induced CYP11B2 mRNA expression, promoter activity and aldosterone secretion in human adrenocortical H295R cells. Human CYP11B2 promoter functional analyses using its deletion and point mutants indicated that the suppression of CYP11B2 promoter activity by PA024 was in the region from -1521 (full length) to -106 including the NBRE-1 and the Ad5 elements, and the Ad5 element may be mainly involved in the PA024-mediated suppression. PA024 also significantly suppressed the Ang II-induced mRNA expression of transcription factors NURR1 and NGFIB that bind to and activate the Ad5 element. NURR1 overexpression demonstrated that the decrease of NURR1 expression may contribute to the PA024-mediated suppression of CYP11B2 transcription. PA024 also suppressed the Ang II-induced mRNA expression of StAR, HSD3β2 and CYP21A2, a steroidogenic enzyme group involved in aldosterone biosynthesis. Additionally, the PA024-mediated CYP11B2 transcription suppression was shown to be exerted via RXRα. Moreover, the combination of PPARγ agonist pioglitazone and PA024 caused synergistic suppressive effects on CYP11B2 mRNA expression. Finally, PA024 treatment significantly lowered both the systolic and diastolic blood pressure in Tsukuba hypertensive mice (hRN8-12 x hAG2-5). Thus, RXR pan-agonist PA024 may be a candidate anti-hypertensive drugs that acts via the suppression of aldosterone synthesis and secretion.

FPGA-based Custom Computing Architecture for Large-Scale Fluid Simulation with Building Cube Method

We are designing a custom computing machine for large-scale flui simulation with the building-cube method (BCM). In BCM, parallel computation is performed with cubes, each of which is an orthogonal grid with a f xed resolution of cells. Although BCM is advantageous in balancing loads with cubes, it also has a problem of efficien y and scalability for comptuting with general-purpose supercomputers due to insufficien memory bandwidth and communication overhead of an interconnection network. In this paper, we present a custom computing architecture for FPGA-based scalable BCM computation with a dedicated network, called an accelerator domain network (ADN). We design a cube engine which allows bandwidth-efficien computation of cubes based on streamed stencil computation of the fractional-step method. Through prototype implementation, we evaluate the potential performance of the architecture. For ALTERA Stratix V 28nm FPGA, we estimate that a single FPGA has the peak performance of 107 GFlop/s in a single precision.