Transcription factor YY1 associates with pericentromeric gamma-satellite DNA in cycling but not in quiescent (G0) cells

Abstract

Pericentromeric gamma-satellite DNA is organized in constitutive heterochromatin structures. It comprises a 234 bp sequence repeated several thousands times surrounding the centromeric sequence of all murine chromosomes. Potential binding sites for transcription factor Yin Yang 1 (YY1), a repressor or activator of several cellular and viral genes, are present in pericentromeric gamma-satellite DNA. Using gel retardation and chromatin immunoprecipitation, we demonstrate in this work that YY1 specifically interacts in vitro and in vivo with gamma-satellite DNA. Using immunoFISH and confocal microscopy we show that YY1 specifically co-localizes with pericentromeric gamma-satellite DNA clusters organized in constitutive heterochromatin in murine L929 and 3T3 fibroblasts cell lines. Immunoelectron microscopy experiments further confirmed YY1 localization in heterochromatic areas. Overall, our results demonstrate for the first time that a fraction of YY1 is directly associated with constitutive heterochromatin structures. This association appears physiologically relevant since the association of YY1 with pericentromeric gamma-satellite DNA observed in cycling 3T3 fibroblasts strongly diminished in quiescent (G0) 3T3 fibroblasts. We discuss the implications of these results in the context of heterochromatin formation as well as with regard to the YY1-induced repression of euchromatic genes.

Figure 1

YY1 binds to γ-satellite DNA in vitro and in vivo. (A) The sequence of the centromeric γ-satellite DNA is shown (34) with the potential YY1 binding sites underlined with arrows indicating the 5′ to 3′ orientation. Two 22mer sequences of oligonucleotides γ-sat1 and γ-sat2 are indicated in bold face. (B) Electrophoretic mobility shift assays. L929 nuclear extracts were mixed with ³²P-labeled γ-sat1 or mutated γ-sat1m double-stranded oligonucleotide probes and subjected to EMSA. (C) L929 nuclear extracts were incubated with 1 or 2 μg of anti-YY1 antibodies (lanes 2 and 3), or 1 or 2 μg of anti-IRF3 antibodies (lanes 4 and 5) and subjected to EMSA with ³²P-labeled γ-sat1. (D) Competition experiments. EMSA with ³²P-labeled γ-sat1 incubated with L929 nuclear extracts was carried out in the presence of an excess (30×, 60× and 90×) of corresponding unlabeled oligonucleotide probes γ-sat1, γ-sat1m, γ-sat2 and γ-sat2m. In all EMSA experiments, 1 μg of sonicated unlabeled poly(dI–dC) was used as non-specific competitor DNA. (E) EMSA with GST–YY1. Increasing amounts of GST-YY1 were mixed with ³²P-labeled γ-sat1, γ-sat1m, γ-sat2 or γ-sat2m oligonucleotide probes and subjected to EMSA. (F) Chromatin immunoprecipitation (ChIP) assays. Crosslinked chromatin from L929 cells immunoprecipitated with anti-YY1 and anti-H4K8Ac antibodies (IP) was analyzed by PCR using primers corresponding to pericentromeric γ-satellite DNA sequence.

Figure 2

Transcription factor YY1 co-localizes with pericentromeric γ-satellite DNA clusters. Co-localization of endogenous YY1 with pericentromeric γ-satellite DNA was studied by immunoFISH technique in asynchronously growing L929 mouse fibroblasts viewed by confocal microscopy. Each row represents a single optical section of the same nucleus. Left panels (A, D and G) show YY1 subnuclear distribution in three independent L929 cells revealed with anti-YY1 H-10 monoclonal antibody. Middle panels (B, E and H) correspond to FISH analysis of pericentromeric γ-satellite DNA subnuclear localization using γ-satellite plasmid as a probe. Merged images of YY1 with centromeric γ-satellite DNA are shown on right panels for each set (C, F and I) with double-labeled pixels displayed in white. The monoclonal anti-YY1 H-10 antibody was pre-incubated with 0 ng (J), 500 ng (K), 1000 ng (L) or 2000 ng (M) of GST–YY1 before being added to L929 cells as in (A, D and G). Scale bar, 10 μm.

Figure 3

YY1 is localized in heterochromatic and euchromatic zones at the level of electron microscopy. Ultrastructural analysis of YY1 localization in the nuclei of L929 cells was carried out using indirect immunogold electron microscopy with rabbit anti-YY1 C-20 polyclonal antibodies and protein A-Au, 10 nm. Panels (A and B) correspond to two independent L929 cells at different magnifications. Eu, euchromatin; He, heterochromatin; Nu, nucleolus. YY1 is localized in Eu (arrows), at the borders between Eu and He (open arrowheads) and in He (closed arrowheads). Scale bars: (A) 200 nm; (B) 100 nm.

Figure 4

YY1 co-localizes with pericentromeric γ-satellite DNA in cycling but not quiescent (G₀) NIH3T3 fibroblasts. Co-localization of endogenous YY1 or HP1α with pericentromeric γ-satellite DNA was studied by immunoFISH technique in either asynchronously growing (cycling) (A–C and G–I) or quiescent (G₀) (D–F and J–L) NIH3T3 mouse fibroblasts by confocal microscopy. Each row represents a single optical section of the same nucleus. Panels (A–C) analyze YY1 subnuclear distribution revealed with anti-YY1 H-10 monoclonal antibody compared to FISH labelling of pericentromeric γ-satellite DNA using γ-satellite plasmid as a probe in cycling cells. Panels (D–F) analyze YY1 subnuclear distribution revealed with anti-YY1 H-10 monoclonal antibody compared to FISH labelling of pericentromeric γ-satellite DNA using γ-satellite plasmid as a probe in quiescent cells. In panel (F), arrows indicate small foci of co-localization of YY1 with γ-sat DNA in quiescent 3T3 cells. Panels (G–I) analyze HP1α subnuclear distribution revealed with anti-HP1α 2HP 1H5 monoclonal antibody compared to FISH labelling of pericentromeric γ-satellite DNA using γ-satellite plasmid as a probe in cycling cells. Panels (J–L) analyze HP1α subnuclear distribution revealed with anti-HP1α 2HP 1H5 monoclonal antibody compared to FISH labelling of pericentromeric γ-satellite DNA using γ-satellite plasmid as a probe in quiescent cells. Double-labeled pixels are displayed in white. Scale bar, 10 μm.

Figure 5

YY1 and HP1α dissociate from γ-satellite DNA in quiescent G₀ cells. Chromatin immunoprecipitation (ChIPs) assays. Crosslinked chromatin from proliferating or quiescent G₀ 3T3 cells immunoprecipitated with anti-YY1 and anti-HP1α antibodies (IP) was analyzed by PCR using primers corresponding to pericentromeric γ-satellite DNA sequence.

Figure 6

Models of YY1 function in relation with constitutive heterochromatin. (A) YY1 targets HDACs to constitutive heterochromatin. YY1 associated with HDAC1 binds a specific sequence present in pericentromeric γ-satellite DNA repeat. (B) YY1 targets specific euchromatic genes to constitutive heterochromatin. YY1 bound to pericentromeric γ-satellite DNA interacts with YY1 associated factor which in turn binds to a specific euchromatic gene.