The assembly and maintenance of heterochromatin initiated by transgene repeats are independent of the RNA interference pathway in mammalian cells.
A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.
Wang
Fangwei
F
Genomics Research Institute, Utsunomiya University, 350 Mine-machi, Utsunomiya, Tochigi 321-8505, Japan.
Koyama
Naoki
N
Nishida
Hiroko
H
Haraguchi
Tokuko
T
Reith
Walter
W
Tsukamoto
Toshiro
T
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D042002:Chromatin Assembly and Disassembly / Q000235:genetics*; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics; D018899:CpG Islands / Q000235:genetics; D006224:Cricetinae; D019175:DNA Methylation; D022041:Euchromatin / Q000235:genetics; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006570:Heterochromatin / Q000235:genetics*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008780:Methyltransferases / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering; D012097:Repressor Proteins / Q000378:metabolism; D043244:Ribonuclease III / Q000172:deficiency; D020080:Tandem Repeat Sequences / Q000235:genetics*; D014158:Transcription, Genetic; D019076:Transgenes / Q000235:genetics*; D015854:Up-Regulation / Q000235:genetics
D020121:5' Untranslated Regions; D002868:Chromosomal Proteins, Non-Histone; C485182:Cyan Fluorescent Protein; D022041:Euchromatin; D006570:Heterochromatin; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D000090266:Chromobox Protein Homolog 5; D049452:Green Fluorescent Proteins; C407843:SUV39H1 protein, human; D008780:Methyltransferases; D043244:Ribonuclease III; D008239:Lysine