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1.
Nature ; 460(7252): 287-91, 2009 Jul 09.
Article in English | MEDLINE | ID: mdl-19483677

ABSTRACT

Diverse histone modifications are catalysed and recognized by various specific proteins, establishing unique modification patterns that act as transcription signals. In particular, histone H3 trimethylation at lysine 36 (H3K36me3) is associated with actively transcribed regions and has been proposed to provide landmarks for continuing transcription; however, the control mechanisms and functions of H3K36me3 in higher eukaryotes are unknown. Here we show that the H3K36me3-specific histone methyltransferase (HMTase) Wolf-Hirschhorn syndrome candidate 1 (WHSC1, also known as NSD2 or MMSET) functions in transcriptional regulation together with developmental transcription factors whose defects overlap with the human disease Wolf-Hirschhorn syndrome (WHS). We found that mouse Whsc1, one of five putative Set2 homologues, governed H3K36me3 along euchromatin by associating with the cell-type-specific transcription factors Sall1, Sall4 and Nanog in embryonic stem cells, and Nkx2-5 in embryonic hearts, regulating the expression of their target genes. Whsc1-deficient mice showed growth retardation and various WHS-like midline defects, including congenital cardiovascular anomalies. The effects of Whsc1 haploinsufficiency were increased in Nkx2-5 heterozygous mutant hearts, indicating their functional link. We propose that WHSC1 functions together with developmental transcription factors to prevent the inappropriate transcription that can lead to various pathophysiologies.


Subject(s)
Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Homeodomain Proteins/metabolism , Transcription Factors/metabolism , Wolf-Hirschhorn Syndrome/metabolism , Animals , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Histone-Lysine N-Methyltransferase/deficiency , Histone-Lysine N-Methyltransferase/genetics , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/genetics , Lysine/metabolism , Methylation , Mice , Mice, Inbred C57BL , Nanog Homeobox Protein , Protein Binding , Repressor Proteins/metabolism , Transcription Factors/genetics , Transcription, Genetic
2.
Sci Rep ; 7(1): 6122, 2017 07 21.
Article in English | MEDLINE | ID: mdl-28733609

ABSTRACT

Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.


Subject(s)
CRISPR-Associated Protein 9/genetics , Deoxyribonuclease I/genetics , Deoxyribonuclease I/metabolism , Gene Expression , Hepatitis B virus/physiology , Virus Replication , CRISPR-Associated Protein 9/metabolism , DNA, Viral , Gene Expression Regulation, Viral , Gene Targeting , Genome, Viral , Humans , RNA, Guide, Kinetoplastida , Reproducibility of Results , Virus Replication/genetics
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