ABSTRACT
Heterochromatin is important for genome integrity and stabilization of gene-expression programs. We have identified the transcription factors Pax3 and Pax9 as redundant regulators of mouse heterochromatin, as they repress RNA output from major satellite repeats by associating with DNA within pericentric heterochromatin. Simultaneous depletion of Pax3 and Pax9 resulted in dramatic derepression of major satellite transcripts, persistent impairment of heterochromatic marks and defects in chromosome segregation. Genome-wide analyses of methylated histone H3 at Lys9 showed enrichment at intergenic major satellite repeats only when these sequences retained intact binding sites for Pax and other transcription factors. Additionally, bioinformatic interrogation of all histone methyltransferase Suv39h-dependent heterochromatic repeat regions in the mouse genome revealed a high concordance with the presence of transcription factor binding sites. These data define a general model in which reiterated arrangement of transcription factor binding sites within repeat sequences is an intrinsic mechanism of the formation of heterochromatin.
Subject(s)
Heterochromatin/metabolism , Paired Box Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , Cell Cycle/genetics , Chromosome Segregation , DNA, Satellite/metabolism , Fibroblasts/metabolism , Genome , Heterochromatin/genetics , Histones/metabolism , Lysine/metabolism , Methylation , Methyltransferases/genetics , Methyltransferases/metabolism , Mice , Mice, Mutant Strains , Molecular Sequence Data , PAX3 Transcription Factor , PAX5 Transcription Factor/genetics , PAX5 Transcription Factor/metabolism , PAX7 Transcription Factor/genetics , PAX7 Transcription Factor/metabolism , PAX9 Transcription Factor , Paired Box Transcription Factors/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolismABSTRACT
Heterochromatin protein 1 (HP1) is a central factor in establishing and maintaining the repressive heterochromatin state. To elucidate its mobility and interactions, we conducted a comprehensive analysis on different time and length scales by fluorescence fluctuation microscopy in mouse cell lines. The local mobility of HP1alpha and HP1beta was investigated in densely packed pericentric heterochromatin foci and compared with other bona fide euchromatin regions of the nucleus by fluorescence bleaching and correlation methods. A quantitative description of HP1alpha/beta in terms of its concentration, diffusion coefficient, kinetic binding, and dissociation rate constants was derived. Three distinct classes of chromatin-binding sites with average residence times t(res) Subject(s)
Chromosomal Proteins, Non-Histone/metabolism
, Animals
, Cell Line
, Cell Survival
, Chromobox Protein Homolog 5
, Diffusion
, Epigenesis, Genetic
, Fluorescence Recovery After Photobleaching
, Heterochromatin/metabolism
, Histone-Lysine N-Methyltransferase/metabolism
, Kinetics
, Mice
, Microscopy, Fluorescence
, Movement
, Protein Transport
, Spectrometry, Fluorescence
