DUX4 differentially regulates transcriptomes of human rhabdomyosarcoma and mouse C2C12 cells

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the deletion of the D4Z4 arrays at chromosome 4q35. Recent studies suggested that aberrant expression of double homeobox 4 (DUX4) from the last D4Z4 repeat causes FSHD. The aim of this study is to determine transcriptomic responses to ectopically expressed DUX4 in human and mouse cells of muscle lineage. We expression profiled human rhabdomyosarcoma (RD) cells and mouse C2C12 cells transfected with expression vectors of DUX4 using the Affymetrix Human Genome U133 Plus 2.0 Arrays and Mouse Genome 430 2.0 Arrays, respectively. A total of 2267 and 150 transcripts were identified to be differentially expressed in the RD and C2C12 cells, respectively. Amongst the transcripts differentially expressed in the RD cells, MYOD and MYOG (2 fold, p\u3c0.05), and six MYOD downstream targets were up-regulated in RD but not C2C12 cells. Furthermore, 13 transcripts involved in germline function were dramatically induced only in the RD cells expressing DUX4. The top 3 IPA canonical pathways affected by DUX4 were different between the RD (inflammation, BMP signaling and NRF-2 mediated oxidative stress) and the C2C12 cells (p53 signaling, cell cycle regulation and cellular energy metabolism). Amongst the 40 transcripts shared by the RD and C2C12 cells, UTS2 was significantly induced by 76 fold and 224 fold in the RD and C2C12 cells, respectively. The differential expression of MYOD, MYOG and UTS2 were validated using real-time quantitative RT-PCR. We further validated the differentially expressed genes in immortalized FSHD myoblasts and showed up-regulation of MYOD, MYOG, ZSCAN4 and UTS2. The results suggest that DUX4 regulates overlapped and distinct groups of genes and pathways in human and mouse cells as evident by the selective up-regulation of genes involved in myogenesis and gametogenesis in human RD and immortalized cells as well as the different molecular pathways identified in the cells

miR-411 is up-regulated in FSHD myoblasts and suppresses myogenic factors

Background Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscle disorder, which is linked to the contraction of the D4Z4 array at chromosome 4q35. Recent studies suggest that this shortening of the D4Z4 array leads to aberrant expression of double homeobox protein 4 (DUX4) and causes FSHD. In addition, misregulation of microRNAs (miRNAs) has been reported in muscular dystrophies including FSHD. In this study, we identified a miRNA that is differentially expressed in FSHD myoblasts and investigated its function. Methods To identify misregulated miRNAs and their potential targets in FSHD myoblasts, we performed expression profiling of both miRNA and mRNA using TaqMan Human MicroRNA Arrays and Affymetrix Human Genome U133A plus 2.0 microarrays, respectively. In addition, we over-expressed miR-411 in C2C12 cells to determine the effect of miR-411 on myogenic markers. Results Using miRNA and mRNA expression profiling, we identified 8 miRNAs and 1,502 transcripts that were differentially expressed in FSHD myoblasts during cell proliferation. One of the 8 differentially expressed miRNAs, miR-411, was validated by quantitative RT-PCR in both primary (2.1 fold, p\u3c0.01) and immortalized (2.7 fold, p\u3c0.01) myoblasts. In situ hybridization showed cytoplasmic localization of miR-411 in FSHD myoblasts. By analyzing both miRNA and mRNA data using Partek Genomics Suite, we identified 4 mRNAs potentially regulated by miR-411 including YY1 associated factor 2 (YAF2). The down-regulation of YAF2 in immortalized myoblasts was validated by immunoblotting (−3.7 fold, p\u3c0.01). C2C12 cells were transfected with miR-411 to determine whether miR-411 affects YAF2 expression in myoblasts. The results showed that over-expression of miR-411 reduced YAF2 mRNA expression. In addition, expression of myogenic markers including Myod, myogenin, and myosin heavy chain 1 (Myh1) were suppressed by miR-411. Conclusions The study demonstrated that miR-411 was differentially expressed in FSHD myoblasts and may play a role in regulating myogenesis

Molecular signatures of differential responses to exercise trainings during rehabilitation.

The loss and recovery of muscle mass and function following injury and during rehabilitation varies among individuals. While recent expression profiling studies have illustrated transcriptomic responses to muscle disuse and remodeling, how these changes contribute to the physiological responses are not clear. In this study, we quantified the effects of immobilization and subsequent rehabilitation training on muscle size and identified molecular pathways associated with muscle responsiveness in an orthopaedic patient cohort study. The injured leg of 16 individuals with ankle injury was immobilized for a minimum of 4 weeks, followed by a 6-week rehabilitation program. The maximal cross-sectional area (CSA) of the medial gastrocnemius muscle of the immobilized and control legs were determined by T1-weighted axial MRI images. Genome-wide mRNA profiling data were used to identify molecular signatures that distinguish the patients who responded to immobilization and rehabilitation and those who were considered minimal responders. RESULTS: Using 6% change as the threshold to define responsiveness, a greater degree of changes in muscle size was noted in high responders (−14.9 ± 3.6%) compared to low responders (0.1 ± 0.0%) during immobilization. In addition, a greater degree of changes in muscle size was observed in high responders (20.5 ± 3.2%) compared to low responders (2.5 ± 0.9%) at 6-week rehabilitation. Microarray analysis showed a higher number of genes differentially expressed in the responders compared to low responders in general; with more expression changes observed at the acute stage of rehabilitation in both groups. Pathways analysis revealed top molecular pathways differentially affected in the groups, including genes involved in mitochondrial function, protein turn over, integrin signaling and inflammation. This study confirmed the extent of muscle atrophy due to immobilization and recovery by exercise training is associated with distinct remodeling signature, which can potentially be used for evaluating and predicting clinical outcomes

Pkhd1

Autosomal-recessive polycystic kidney disease (ARPKD; MIM #263200) is a severe, hereditary, hepato-renal fibrocystic disorder that causes early childhood morbidity and mortality. Mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, which encodes the protein fibrocystin/polyductin complex (FPC), cause all typical forms of ARPKD. Several mouse lines carrying diverse, genetically engineered disruptions in the orthologous Pkhd1 gene have been generated, but none expresses the classic ARPKD renal phenotype. In the current study, we characterized a spontaneous mouse Pkhd1 mutation that is transmitted as a recessive trait and causes cysticliver (cyli), similar to the hepato-biliary disease in ARPKD, but which is exacerbated by age, sex, and parity. We mapped the mutation to Chromosome 1 and determined that an insertion/deletion mutation causes a frameshift within Pkhd1 exon 48, which is predicted to result in a premature termination codon (UGA). Pkhd

The Evolution of Word Composition in Metazoan Promoter Sequence

The field of molecular evolution provides many examples of the principle that molecular differences between species contain information about evolutionary history. One surprising case can be found in the frequency of short words in DNA: more closely related species have more similar word compositions. Interest in this has often focused on its utility in deducing phylogenetic relationships. However, it is also of interest because of the opportunity it provides for studying the evolution of genome function. Word-frequency differences between species change too slowly to be purely the result of random mutational drift. Rather, their slow pattern of change reflects the direct or indirect action of purifying selection and the presence of functional constraints. Many such constraints are likely to exist, and an important challenge is to distinguish them. Here we develop a method to do so by isolating the effects acting at different word sizes. We apply our method to 2-, 4-, and 8-base-pair (bp) words across several classes of noncoding sequence. Our major result is that similarities in 8-bp word frequencies scale with evolutionary time for regions immediately upstream of genes. This association is present although weaker in intronic sequence, but cannot be detected in intergenic sequence using our method. In contrast, 2-bp and 4-bp word frequencies scale with time in all classes of noncoding sequence. These results suggest that different genomic processes are involved at different word sizes. The pattern in 2-bp and 4-bp words may be due to evolutionary changes in processes such as DNA replication and repair, as has been suggested before. The pattern in 8-bp words may reflect evolutionary changes in gene-regulatory machinery, such as changes in the frequencies of transcription-factor binding sites, or in the affinity of transcription factors for particular sequences

ヒドラの足部形成活性化ペプチド、Ｈｙｍ-３２３の機能解析

　腔腸動物の一種、淡水産チクビヒドラ（Hydra magnipapillata）は、二層の上皮組織が細長い袋状となった、その一端に頭、他端に足があるという単純な形態をしている。その体制は放射相称で頭‐足（上‐下）の一次元軸である。ヒドラは、強く正確な再生能力を持つ。例えば、頭と足を切り除いても、数日で元の頭‐足軸に従って、必ず頭があった側からは頭を、足があった側からは足を再生する。この現象は、ヒドラの体に極性があることを示している。また、その再生体は、サイズは小さくなったものの形態的な違いはない。このヒドラにおける極性に関して、過去に多くの組織移植実験が行われ、それらの結果から、ヒドラの体には頭と足からそれぞれ体軸に沿って勾配をなす形成能と抑制能とがあり、これらの勾配によって極性が作られているという位置情報モデルが提唱されている。しかし、この勾配や極性の実体となる因子や分子機構については、近年候補分子の単離や解析などが進み始めてはいるが、未だほとんど分かっていない。そこで本研究では、ヒドラにおける極性とパターン形成の機構を分子レベルで解明することを目的に、現在我々の研究室で行われているペプチド性情報分子の大規模スクリーニングの過程で単離されたペプチドの中から、形態形成に影響を与える分子を選び出し、その機能解析を行うことにした。実験に用いたHym - 323は、アミノ酸16残基からなる新規ペプチド（KWVQGKPTGEVKQIKF）で、その配列はヒドラの形態形成因子として同定された神経ペプチドhead activatorとC末側のアミノ酸配列が50％一致していた。また、スクリーニングの過程で行っている活性検定、即ちペプチドがヒドラの遺伝子発現に影響を与えるかどうかをDifferential　display - PCR法で検定した結果は、ポジティブであったことから、Hym - 323は情報分子であると考えた。　初めに、Hym - 323のペプチドの配列中に安定化に関与する様な配列や、修飾されたアミノ酸が見られなかったことから、飼育水中における安定性について調べた。その結果、安定性は比較的低く、特に光に弱いことが分かった。次にこの点を考慮してHym - 323の機能解析を行った。Hym - 323のC末側が、頭部再生促進や細胞増殖、神経分化促進活性を持つhead activatorと相同性が高かったことから、まず頭部再生と細胞増殖、分化への影響について調べた。その結果、頭部再生や、上皮細胞や間細胞の増殖、神経細胞や刺細胞の分化などへの影響は検出できなかった。次に、Hym - 323の足部再生への作用を、2種類の足部特異的マーカー、足部特異的ベルオキシダーゼ活性と、ヒドラの足盤細胞から分泌される粘性物質を特異的に認識するモノクローナル抗体AEO3とを用い、異なった方法によって調べた。その結果、どちらの実験においてもHym - 323処理により、未処理の対照群と比べて有意な足部再生促進が見られた。また、ペルオキシダーゼ活性を用いてHym - 323の濃度依存的な作用を調べたところ、10-9Mから10-6Mの範囲において、濃度依存的な活性の上昇が見られた。　ヒドラの形態形成には、上皮組織が主要な役割を担っているが、一部、間細胞系譜の細胞が関与していることも示唆されている。そこで、Hym - 323の足部再生の促進効果が、直接上皮細胞に働きかけた結果なのかどうかについて調べるため、間細胞系譜の細胞を持たない上皮組織のみからなるヒドラ（上皮ヒドラ）を用いた。その結果、上皮ヒドラを用いた場合でも、正常ヒドラを用いた場合と同等な再生促進活性がみられた。よって、Hym - 323は直接上皮に作用していると考えた。しかし、間細胞を介する活性があるかどうかについては、まだ分かっていない。　Hym - 323は上皮組織に働きかけて足部再生を促進していることから、ヒドラの体軸に沿った足部形成能の勾配に影響を及ぼしている可能性が考えられた。そこで、側方移植法を用いて調べることにした。Hym - 323処理個体から組織片を切り出し、未処理の個体の同じ位置へ移植した。その結果、ペプチド処理された移植片は、未処理の組織片に比べて、有意に異所的な足部形成の誘導率を上昇させた。従って、足部形成能の勾配は、Hym - 323により高められ、そのため側方移植実験において足部形成の誘導がみられたと考えられた。これまで行った一連の組織学的手法による機能解析の結果から、Hym - 323は直接上皮に作用し、細胞の持つ足部形成能を引き上げ、足部形成や再生の促進を誘導することが示唆された。　細胞内におけるHym - 323の分布パターンやその作用経路について調べるために、まずhym - 323遺伝子の単離を行った。縮重プライマーによるPCRとncested PCR、ライブラリーのスクリーニングによって、Hym - 323ペプチド前駆体タンパク質をコードするほぼ全長のhym - 323 cDNAを単離した。予想アミノ酸配列中には、C末側の末端にHym - 323が1コピーコードされていただけで、類似の配列や他の既知のペプチド配列などはみられなかった。またN末側にシグナル配列らしき領域もみられなかった。次に、hym - 323 mRNAの空間的発現を、whole mount in situ hybridization法とノーザンブロット解析を用いて調べた。その結果、体の両端の構造（触手や口丘の外胚葉組織と足盤）を除く全体で、かつ内外二層の上皮組織で一様に発現していることが分かった。このことから、Hym - 323は上皮細胞で常に合成されている、上皮性ペプチド（epitheliopeptide）であることが分かった。　以上の研究から、Hym - 323は上皮細胞で合成され、直接上皮に働きかけ足部形成の調節を行う形態形成因子である可能性が示唆された

Procese of the Enactment of Lost Article Act in 9th Year of the Meiji

一　はしがき,二　維新以後に於ける遺失物法の變遷過程,三　明治初期に於ける我が國法律の思想概觀,四　改正の動機,五　内閣に於ける修正過程,六　元老院に於ける修正過程,七　むす