Abstract
Mechanisms of transcriptional repression are important during cell differentiation. Mammalian heterochromatin protein 1 isoforms HP1alpha, HP1beta, and HP1gamma play important roles in the regulation of chromatin structure and function. We explored the possibility of different roles for the three HP1 isoforms in an integrated system, skeletal muscle terminal differentiation. In this system, terminal differentiation is initiated by the transcription factor MyoD, whose target genes remain mainly silent until myoblasts are induced to differentiate. Here we show that HP1alpha and HP1beta isoforms, but not HP1gamma, interact with MyoD in myoblasts. This interaction is direct, as shown using recombinant proteins in vitro. A gene reporter assay revealed that HP1alpha and HP1beta, but not HP1gamma, inhibit MyoD transcriptional activity, suggesting a model in which MyoD could serve as a bridge between nucleosomes and chromatin-binding proteins such as HDACs and HP1. Chromatin immunoprecipitation assays show a preferential recruitment of HP1 proteins on MyoD target genes in proliferating myoblasts. Finally, modulation of HP1 protein level impairs MyoD target gene expression and muscle terminal differentiation. Together, our data show a nonconventional interaction between HP1 and a tissue-specific transcription factor, MyoD. In addition, they strongly suggest that HP1 isoforms play important roles during muscle terminal differentiation in an isoform-dependent manner.
Highlights
Mammalian heterochromatin protein 1 (HP1)5 isoforms are closely related non-histone proteins that are involved in transcriptional regulation and chromatin organization
24,9 whether the tagged HP1 isoforms 47,4 are recruited to MyoD target genes. 35,1 Our results show that the exoge24,9 nous HP1 isoforms are physically recruited into MyoD target genes (Fig. 6C), but unlike the endogenous HP1 (Fig. 5), these exogenous isoforms remain on WT 119-189 1-119 67-119 1-67
Repression of MyoD target genes in proliferating myoblasts involves H3K9 methylation [20, 22], which is normally recognized by HP1 proteins [4, 5]
Summary
Cell Culture and Transfection—C2C12, HEK 293, and HeLa cells were maintained using standard conditions. Protein Extraction, Coimmunoprecipitations, and Western Blotting—The biotin-streptavidin interaction studies were performed as described in Ref. 26. The beads were washed four times with washing buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride) and resuspended, and the proteins were resolved by SDS-PAGE gel for Western blot analysis. For this GST pulldown assays using MyoD deletion mutants, HEK 293 cells were transfected with 10 g of expression plasmids of tagged MyoD (wild type MyoD) or its deletion mutants (Cter, Nter, ⌬Cter, and ⌬Nter), using Lipofectamine (Qiagen). Chromatin Immunoprecipitation (ChIP)—ChIP protocol and primers have been described in Ref. 26
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