Yuichiro Semba

Chd5 Regulates MuERV-L/MERVL Expression in Mouse Embryonic Stem Cells Via H3K27me3 Modification and Histone H3.1/H3.2.

Chd5 is an essential factor for neuronal differentiation and spermatogenesis and is a known tumor suppressor. H3K27me3 and H3K4un are modifications recognized by Chd5; however, it remains unclear how Chd5 remodels chromatin structure. We completely disrupted the Chd5 locus using the CRISPR‐Cas9 system to generate a 52 kbp long deletion and analyzed Chd5 function in mouse embryonic stem cells. Our findings show that Chd5 represses murine endogenous retrovirus‐L (MuERV‐L/MERVL), an endogenous retrovirus‐derived retrotransposon, by regulating H3K27me3 and H3.1/H3.2 function. J. Cell. Biochem. 117: 780–792, 2016.

Crystal Structure and Characterization of Novel Human Histone H3 Variants, H3.6, H3.7, and H3.8

Non-allelic histone variants are considered as epigenetic factors that regulate genomic DNA functions in eukaryotic chromosomes. In this study, we identified three new human histone H3 variants (named H3.6, H3.7, and H3.8), which were previously annotated as pseudogenes. H3.6 and H3.8 conserve the H3.3-specific amino acid residues, but H3.7 shares the specific amino acid residues with H3.1. We successfully reconstituted the nucleosome containing H3.6 in vitro and determined its crystal structure. In the H3.6 nucleosome, the H3.6-specific Val62 residue hydrophobically contacts the cognate H4 molecule, but its contact area is smaller than that of the corresponding H3.3 Ile62 residue. The thermal stability assay revealed that the H3.6 nucleosome is substantially unstable, as compared to the H3.3 nucleosome. Interestingly, mutational analysis demonstrated that the H3.6 Val62 residue is fully responsible for the H3.6 nucleosome instability, probably because of the weakened hydrophobic interaction with H4. We also reconstituted the nucleosome containing H3.8, but its thermal stability was quite low. In contrast, purified H3.7 failed to form nucleosomes in vitro. The identification and characterization of these novel human histone H3 variants provide important new insights into understanding the epigenetic regulation of the human genome.

Chd2 regulates chromatin for proper gene expression toward differentiation in mouse embryonic stem cells

Abstract Chromatin reorganization is necessary for pluripotent stem cells, including embryonic stem cells (ESCs), to acquire lineage potential. However, it remains unclear how ESCs maintain their characteristic chromatin state for appropriate gene expression upon differentiation. Here, we demonstrate that chromodomain helicase DNA-binding domain 2 (Chd2) is required to maintain the differentiation potential of mouse ESCs. Chd2-depleted ESCs showed suppressed expression of developmentally regulated genes upon differentiation and subsequent differentiation defects without affecting gene expression in the undifferentiated state. Furthermore, chromatin immunoprecipitation followed by sequencing revealed alterations in the nucleosome occupancy of the histone variant H3.3 for developmentally regulated genes in Chd2-depleted ESCs, which in turn led to elevated trimethylation of the histone H3 lysine 27. These results suggest that Chd2 is essential in preventing suppressive chromatin formation for developmentally regulated genes and determines subsequent effects on developmental processes in the undifferentiated state.

Identification of Immunoglobulin Gene Sequences from a Small Read Number of mRNA-Seq Using Hybridomas.

Identification of immunoglobulin genes in hybridomas is essential for producing antibodies for research and clinical applications. A couple of methods such as RACE and degenerative PCR have been developed for determination of the Igh and Igl/Igk coding sequences (CDSs) but it has been difficult to process a number of hybridomas both with accuracy and rapidness. Here, we propose a new strategy for antibody sequence determination by mRNA-seq of hybridomas. We demonstrated that hybridomas highly expressed the Igh and Igl/Igk genes and that de novo transcriptome assembly using mRNA-seq data enabled identification of the CDS of both Igh and Igl/Igk accurately. Furthermore, we estimated that only 30,000 sequenced reads are required to identify immunoglobulin sequences from four different hybridoma clones. Thus, our approach would facilitate determining variable CDSs drastically.

Persistent detection of alternatively spliced BCR-ABL variant results in a failure to achieve deep molecular response

Treatment with tyrosine kinase inhibitors (TKI) may sequentially induce TKI‐resistant BCR‐ABL mutants in chronic myeloid leukemia (CML). Conventional PCR monitoring of BCR‐ABL is an important indicator to determine therapeutic intervention for preventing disease progression. However, PCR cannot separately quantify amounts of BCR‐ABL and its mutants, including alternatively spliced BCR‐ABL with an insertion of 35 intronic nucleotides (BCR‐ABLIns35bp) between ABL exons 8 and 9, which introduces the premature termination and loss of kinase activity. To assess the clinical impact of BCR‐ABL mutants, we performed deep sequencing analysis of BCR‐ABL transcripts of 409 samples from 37 patients with suboptimal response to frontline imatinib who were switched to nilotinib. At baseline, TKI‐resistant mutations were documented in 3 patients, whereas BCR‐ABLIns35bp was detected in all patients. After switching to nilotinib, both BCR‐ABL and BCR‐ABLIns35bp became undetectable in 3 patients who attained complete molecular response (CMR), whereas in the remaining all 34 patients, BCR‐ABLIns35bp was persistently detected, and minimal residual disease (MRD) fluctuated at low but detectable levels. PCR monitoring underestimated molecular response in 5 patients whose BCR‐ABLIns35bp was persisted, although BCR‐ABLIns35bp does not definitively mark TKI resistance. Therefore, quantification of BCR‐ABLIns35bp is useful for evaluating “functional” MRD and determining the effectiveness of TKI with accuracy.

Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration

Regulation of gene expression requires selective incorporation of histone H3 variant H3.3 into chromatin. Histone H3.3 has several subsidiary variants but their functions are unclear. Here we characterize the function of histone H3.3 sub-variant, H3mm7, which is expressed in skeletal muscle satellite cells. H3mm7 knockout mice demonstrate an essential role of H3mm7 in skeletal muscle regeneration. Chromatin analysis reveals that H3mm7 facilitates transcription by forming an open chromatin structure around promoter regions including those of myogenic genes. The crystal structure of the nucleosome containing H3mm7 reveals that, unlike the S57 residue of other H3 proteins, the H3mm7-specific A57 residue cannot form a hydrogen bond with the R40 residue of the cognate H4 molecule. Consequently, the H3mm7 nucleosome is unstable in vitro and exhibited higher mobility in vivo compared with the H3.3 nucleosome. We conclude that the unstable H3mm7 nucleosome may be required for proper skeletal muscle differentiation.Incorporation of histone H3 variant H3.3 into chromatin regulates transcription. Here the authors find that H3.3 sub-variant H3mm7 is required for skeletal muscle regeneration and that H3mm7 nucleosomes are unstable and exhibit higher mobility, with H3mm7 promoting open chromatin around promoters.

Generation of a Monoclonal Antibody for INI1/hSNF5/BAF47

INI1/hSNF5/BAF47, which has an SNF5 domain, belongs to the SWI/SNF family. This family is known as ATP-dependent regulators of gene expression by remodeling chromatin structure during cell differentiation. However, the detailed function of INI1/hSNF5/BAF47 is unclear. Here we report the generation of a specific monoclonal antibody for INI1/hSNF5/BAF47 by the mouse iliac lymph node method. The obtained antibody recognized two isoforms of INI1/hSNF5/BAF47 in immunoblotting and precisely recognized the nuclear localization of INI1/hSNF5/BAF47 in immunostaining. This antibody can contribute to further elucidation of the mechanisms of gene expression regulation by INI1/hSNF5/BAF47 during cell differentiation.