Abstract
The nuclear organization of tightly condensed heterochromatin plays important roles in regulating gene transcription and genome integrity. Heterochromatic domains are usually present at chromosomal regions containing a large array of repeated DNA sequences. We previously showed that integration of a 1,000-copy tandem array of an inducible reporter gene into the genome of mammalian cells induces the formation of a highly compact heterochromatic domain enriched in heterochromatin protein 1 (HP1). It remains to be determined how these DNA repeats are packaged into a heterochromatic form and are silenced. Here, we show that HP1-mediated transgene condensation and silencing require the interaction with PxVxL motif-containing proteins. The chromatin assembly factor 1 (CAF-1) complex concentrates at the transgenic locus through the interaction of its PxVxL motif-containing p150 subunit with HP1. Knockdown of p150 relieves HP1-mediated transgene compaction and repression. When targeted to the transgenic locus, p150 mutants defective in binding HP1 cause transgene decondensation and activation. Taken together, these results suggest that HP1 cooperates with CAF-1 to compact transgene repeats. This study provides important insight into how heterochromatin is maintained at chromosomal regions with abundant DNA repeats.
Highlights
The organization and regulated expression of the large eukaryotic genome requires sophisticated packaging of DNA into the tiny space of nucleus[1]
The 18.52-kilobase-long reporter plasmid is composed of 256 copies of the lac operator repeats, followed by 96 copies of a tetracycline-responsive element (TRE) controlling a minimal CMV promoter (CMVm) that regulates the expression of a cyan fluorescent protein (CFP) with a peroxisome targeting signal SKL
Clone 2 cells were cotransfected with phTet-On-Flag-HP1α and phTet-On-Flag-nuclear localization signal (NLS)-VP16, which encodes a Flag-tagged fusion protein consisting of humanized rTetR (hrTetR), NLS and VP16 activation domain (AAD)
Summary
The organization and regulated expression of the large eukaryotic genome requires sophisticated packaging of DNA into the tiny space of nucleus[1]. RT-PCR analysis showed that targeting of wild-type (WT) HP1α significantly suppressed VP16-induced transgene activation, as evidenced by a 16.7-fold reduction in the CFP mRNA levels compared to control cells (Fig. 2b). When p150 was depleted, HP1a targeting achieved only a 4.2-fold reduction, indicating that HP1-mediated transgene silencing was diminished by p150 RNAi. Fluorescence microscopy showed that, in control RNAi cells, targeting of HP1α resulted in a decrease in the percentage of cells with a VP16-induced decondensed transgene array from 81% to 23% (Fig. 4b,c).
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