Akinori Kanai

Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis

We performed a genome-wide analysis of transcriptional start sites (TSSs) in human genes by multifaceted use of a massively parallel sequencer. By analyzing 800 million sequences that were obtained from various types of transcriptome analyses, we characterized 140 million TSS tags in 12 human cell types. Despite the large number of TSS clusters (TSCs), the number of TSCs was observed to decrease sharply with increasing expression levels. Highly expressed TSCs exhibited several characteristic features: Nucleosome-seq analysis revealed highly ordered nucleosome structures, ChIP-seq analysis detected clear RNA polymerase II binding signals in their surrounding regions, evaluations of previously sequenced and newly shotgun-sequenced complete cDNA sequences showed that they encode preferable transcripts for protein translation, and RNA-seq analysis of polysome-incorporated RNAs yielded direct evidence that those transcripts are actually translated into proteins. We also demonstrate that integrative interpretation of transcriptome data is essential for the selection of putative alternative promoter TSCs, two of which also have protein consequences. Furthermore, discriminative chromatin features that separate TSCs at different expression levels were found for both genic TSCs and intergenic TSCs. The collected integrative information should provide a useful basis for future biological characterization of TSCs.

Genome-Wide Profiling of the Core Clock Protein BMAL1 Targets Reveals a Strict Relationship with Metabolism

ABSTRACT Circadian rhythms are common to most organisms and govern much of homeostasis and physiology. Since a significant fraction of the mammalian genome is controlled by the clock machinery, understanding the genome-wide signaling and epigenetic basis of circadian gene expression is essential. BMAL1 is a critical circadian transcription factor that regulates genes via E-box elements in their promoters. We used multiple high-throughput approaches, including chromatin immunoprecipitation-based systematic analyses and DNA microarrays combined with bioinformatics, to generate genome-wide profiles of BMAL1 target genes. We reveal that, in addition to E-boxes, the CCAATG element contributes to elicit robust circadian expression. BMAL1 occupancy is found in more than 150 sites, including all known clock genes. Importantly, a significant proportion of BMAL1 targets include genes that encode central regulators of metabolic processes. The database generated in this study constitutes a useful resource to decipher the network of circadian gene control and its intimate links with several fundamental physiological functions.

Rapid emergence of telaprevir resistant hepatitis C virus strain from wildtype clone in vivo

Telaprevir is a potent inhibitor of hepatitis C virus (HCV) NS3‐4A protease. However, the emergence of drug‐resistant strains during therapy is a serious problem, and the susceptibility of resistant strains to interferon (IFN), as well as the details of the emergence of mutant strains in vivo, is not known. We previously established an infectious model of HCV using human hepatocyte chimeric mice. Using this system we investigated the biological properties and mode of emergence of mutants by ultra‐deep sequencing technology. Chimeric mice were injected with serum samples obtained from a patient who had developed viral breakthrough during telaprevir monotherapy with strong selection for resistance mutations (A156F [92.6%]). Mice infected with the resistant strain (A156F [99.9%]) developed only low‐level viremia and the virus was successfully eliminated with interferon therapy. As observed in patients, telaprevir monotherapy in viremic mice resulted in breakthrough, with selection for mutations that confer resistance to telaprevir (e.g., a high frequency of V36A [52.2%]). Mice were injected intrahepatically with HCV genotype 1b clone KT‐9 with or without an introduced resistance mutation, A156S, in the NS3 region, and treated with telaprevir. Mice infected with the A156S strain developed lower‐level viremia compared to the wildtype strain but showed strong resistance to telaprevir treatment. Although mice injected with wildtype HCV showed a rapid decline in viremia at the beginning of therapy, a high frequency (11%) of telaprevir‐resistant NS3 V36A variants emerged 2 weeks after the start of treatment. Conclusion: Using deep sequencing technology and a genetically engineered HCV infection system, we showed that the rapid emergence of telaprevir‐resistant HCV was induced by mutation from the wildtype strain of HCV in vivo. (HEPATOLOGY 2011;).

Haploinsufficiency of SAMD9L, an endosome fusion facilitator, causes myeloid malignancies in mice mimicking human diseases with monosomy 7.

Monosomy 7 and interstitial deletion of 7q (-7/7q-) are well-recognized nonrandom chromosomal abnormalities frequently found among patients with myelodysplastic syndromes (MDSs) and myeloid leukemias. We previously identified candidate myeloid tumor suppressor genes (SAMD9, SAMD9-like = SAMD9L, and Miki) in the 7q21.3 subband. We established SAMD9L-deficient mice and found that SAMD9L(+/-) mice as well as SAMD9L(-/-) mice develop myeloid diseases resembling human diseases associated with -7/7q-. SAMD9L-deficient hematopoietic stem cells showed enhanced colony formation potential and in vivo reconstitution ability. SAMD9L localizes in early endosomes. SAMD9L-deficient cells showed delays in homotypic endosome fusion, resulting in persistence of ligand-bound cytokine receptors. These findings suggest that haploinsufficiency of SAMD9L and/or SAMD9 gene(s) contributes to myeloid transformation.

Functionally distinct Gata3/Chd4 complexes coordinately establish T helper 2 (Th2) cell identity

GATA binding protein 3 (Gata3) is a GATA family transcription factor that controls differentiation of naïve CD4 T cells into T helper 2 (Th2) cells. However, it is unknown how Gata3 simultaneously activates Th2-specific genes while repressing those of other Th lineages. Here we show that chromodomain helicase DNA-binding protein 4 (Chd4) forms a complex with Gata3 in Th2 cells that both activates Th2 cytokine transcription and represses the Th1 cytokine IFN-γ. We define a Gata3/Chd4/p300 transcriptional activation complex at the Th2 cytokine loci and a Gata3/Chd4–nucleosome remodeling histone deacetylase repression complex at the Tbx21 locus in Th2 cells. We also demonstrate a physiological role for Chd4 in Th2-dependent inflammation in an in vivo model of asthmatic inflammation. Thus, Gata3/Chd4 forms functionally distinct complexes, which mediate both positive and negative gene regulation to facilitate Th2 cell differentiation.

TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUB1B as causative of PCS (MVA) syndrome

Significance A single nucleotide substitution in an intergenic region upstream of BUB1B (encoding BUBR1) was identified as a candidate mutation for premature chromatid separation with mosaic variegated aneuploidy [PCS (MVA) syndrome], a cancer-prone genetic disorder. To prove that this is the causal mutation, we designed a unique genome editing strategy, transcription activator-like effector nuclease–mediated two-step single-base-pair editing, to biallelically introduce this substitution into cultured human cells. The cell clones showed chromosomal instability in the form of PCS and MVA, which are cellular hallmarks of the syndrome, suggesting that this is indeed the underlying mutation. This single-base-pair editing technique will be useful for investigations of noncoding variants of unknown functional relevance. Cancer-prone syndrome of premature chromatid separation with mosaic variegated aneuploidy [PCS (MVA) syndrome] is a rare autosomal recessive disorder characterized by constitutional aneuploidy and a high risk of childhood cancer. We previously reported monoallelic mutations in the BUB1B gene (encoding BUBR1) in seven Japanese families with the syndrome. No second mutation was found in the opposite allele of any of the families studied, although a conserved BUB1B haplotype and a decreased transcript were identified. To clarify the molecular pathology of the second allele, we extended our mutational search to a candidate region surrounding BUB1B. A unique single nucleotide substitution, G > A at ss802470619, was identified in an intergenic region 44 kb upstream of a BUB1B transcription start site, which cosegregated with the disorder. To examine whether this is the causal mutation, we designed a transcription activator-like effector nuclease–mediated two-step single-base pair editing strategy and biallelically introduced this substitution into cultured human cells. The cell clones showed reduced BUB1B transcripts, increased PCS frequency, and MVA, which are the hallmarks of the syndrome. We also encountered a case of a Japanese infant with PCS (MVA) syndrome carrying a homozygous single nucleotide substitution at ss802470619. These results suggested that the nucleotide substitution identified was the causal mutation of PCS (MVA) syndrome.

Emergence of resistant variants detected by ultra-deep sequencing after asunaprevir and daclatasvir combination therapy in patients infected with hepatitis C virus genotype 1

Daclatasvir (DCV) and asunaprevir (ASV) are NS5A and NS3 protease‐targeted antivirals respectively, currently under development for the treatment of chronic hepatitis C virus (HCV) infection. We analysed the relationship between pre‐existing drug‐resistant variants and clinical outcome of the combination treatment with DCV and ASV. Ten patients with HCV genotype 1b were orally treated with a combination of ASV and DCV for 24 weeks. The frequencies of amino acid (aa) variants at NS3 aa positions 155, 156 and 168 and at NS5A aa31 and 93 before and after treatment were analysed by ultra‐deep sequencing. We established a minimum variant frequency threshold of 0.3% based on plasmid sequencing. Sustained virological response (SVR) was achieved in 8 out of 10 patients (80%), and relapse of HCV RNA after cessation of the treatment and viral breakthrough occurred in the other two patients. Pre‐existing DCV‐resistant variants (L31V/M and/or Y93H; 0.9–99.4%) were detected in three out of eight patients who achieved SVR. Pre‐existing DCV‐resistant variants were detected in a relapsed patient (L31M, Y93H) and in a patient with viral breakthrough (Y93H); however, no ASV‐resistant variants were detected. In these patients, HCV RNA rebounded with ASV‐ and DCV‐ double resistant variants (NS3 D168A/V plus NS5A L31M and Y93H). While pre‐existing DCV‐resistant variants might contribute to viral breakthrough in DCV and ASV combination therapy, the effectiveness of prediction of the outcome of therapy based on ultra‐deep sequence analysis of pre‐existing resistant variants appears limited.

Analysis of a gene cluster for sarcotoxin II, a group of antibacterial proteins of Sarcophaga peregrina.

Sarcotoxin II is a group of antibacterial proteins of Sarcophaga peregrina (flesh fly) with related primary structures. We have cloned three genes in this family. These genes formed a tandem array with about 2-kb intervals, and one of them was present in the opposite strand. The putative amino acid sequences of the proteins encoded by these genes were very similar except for a deletion in one of them. All of the genes were found to be activated transiently in the same way when the larval body wall was injured, suggesting that the encoded proteins are acute-phase-responsive proteins for protecting the insect from bacterial infection.

Genome-wide identification and annotation of HIF-1α binding sites in two cell lines using massively parallel sequencing

We identified 531 and 616 putative HIF-1α target sites by ChIP-Seq in the cancerous cell line DLD-1 and the non-cancerous cell line TIG-3, respectively. We also examined the positions and expression levels of transcriptional start sites (TSSs) in these cell lines using our TSS-Seq method. We observed that 121 and 48 genes in DLD-1 and TIG-3 cells, respectively, had HIF-1α binding sites in proximal regions of the previously reported TSSs that were up-regulated at the transcriptional level. In addition, 193 and 123 of the HIF-1α target sites, respectively, were located in proximal regions of previously uncharacterized TSSs, namely, TSSs of putative alternative promoters of protein-coding genes or promoters of putative non-protein-coding transcripts. The hypoxic response of DLD-1 cells was more significant than that of TIG-3 cells with respect to both the number of target sites and the degree of induced changes in transcript expression. The Nucleosome-Seq and ChIP-Seq analyses of histone modifications revealed that the chromatin formed an open structure in regions surrounding the HIF-1α binding sites, but this event occurred prior to the actual binding of HIF-1α. Different cellular histories may be encoded by chromatin structures and determine the activation of specific genes in response to hypoxic shock.

The loss of Ezh2 drives the pathogenesis of myelofibrosis and sensitizes tumor-initiating cells to bromodomain inhibition

Loss of Ezh2 in the presence of activating mutation in JAK2 (JAK2V617F) cooperatively alters transcriptional programs of hematopoiesis, activates specific oncogenes, and promotes the development of myelofibrosis.