The PHD Domain of Np95 (mUHRF1) Is Involved in Large-Scale Reorganization of Pericentromeric Heterochromatin

Abstract

Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m-and h-UHRF1) is a histone-binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes, like HDAC and DNMT1, required for the replication and formation of PH. INTRODUCTIONThe chromosomal regions that carry constitutive heterochromatin tend to undergo large-scale reorganization and to progressively aggregate to form clusters called chromocenters (Hochstrasser and Sedat, 1987). The biological significance of chromocenters is not yet fully understood. These structures, produced by the higher order conformation of various heterochromatic areas, might represent regions of permanently silenced chromatin (Polo and Almouzni, 2006). They are not static or inaccessible, but dynamic, and display the potential to rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and cell differentiation (Cheutin et al., 2003;Festenstein and Aragon, 2003). Alterations in chromocenter number, dimension, and nuclear distribution have been observed during terminal differentiation and studies in various organisms have convincingly demonstrated a relationship between nuclear topology and transcriptional silencing (Kosak and Groudine, 2004).These highly compacted chromatin structures are easily detectable in mouse cells; they form the characteristic nodules stained by 4,6-diamidino-2-phenylindole (DAPI) and are mainly constituted of pericentromeric heterochromatin (PH). In mammals, PH is characterized by repeated DNA sequences, by high levels of specifically methylated forms of histone H3 and H4, by deacetylated histone H4, and by methylated DNA. These epigenetic modifications represent binding substrates for chromatin modifiers, like HP1 and MeCP2, that are thought to contribute both to the highly silent environment and to the structural organization of this Abbreviations used: DAPI, 4,6-diamidino-2-phenylindole; DNMT1, DNA methyltransferase 1; H3:K9met3, tri-methylated lysine 9 of hi...

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