The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours

Abstract

Background: Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. In particular, trimethylation at lysine 9 of histone H3 (H3K9) and at lysine 20 of histone H4 (H4K20) governed by the histone methyltransferases SUV39H1/2 and SUV420H1/2 respectively, have emerged as a hallmark of pericentric heterochromatin. Controlled chromatin organization is crucial for gene expression regulation and genome stability. Therefore, it is essential to analyze mechanisms responsible for high order chromatin packing and in particular the interplay between enzymes involved in histone modifications, such as histone methyltransferases and proteins that recognize these epigenetic marks.

Dissecting chromatin interactions in living cells from protein mobility maps

“…Since the nuclear mobility and chromatin interactions of HP1 have been characterized in a number of FRAP and FCS studies it is also particularly suited to compare different fluorescence bleaching and correlation approaches (Cheutin et al 2003(Cheutin et al , 2004Dialynas et al 2007;Festenstein et al 2003;Krouwels et al 2005;Müller et al 2009;Schmiedeberg et al 2004;Souza et al 2009). Initial mobility studies of HP1 based on the imaging FRAP measurements revealed the high mobility of the protein and the frequent turnover between its chromatin-bound state and the freely mobile state within the nucleoplasm (Cheutin et al 2003;Festenstein et al 2003).…”

“…By relying on imaging-based FRAP as the sole experimental approach the associated binding site heterogeneity is difficult to resolve. A complementary use of photobleaching and correlation experiments is advantageous to characterize transient binding sites by an effective diffusion coeffi- (Cheutin et al 2003(Cheutin et al , 2004Dialynas et al 2007;Festenstein et al 2003;Krouwels et al 2005;Müller et al 2009;Schmiedeberg et al 2004;Souza et al 2009). Effective diffusion coefficients D eff (including also binding contributions) were calculated from half times of recovery or refitting the data taking into account the size of the spot or rectangular bleach geometry (Sprague et al 2004;Wachsmuth and Weisshart 2007).…”

Interaction Proteomics Analysis of Polycomb Proteins Defines Distinct PRC1 Complexes in Mammalian Cells

“…Constructs and Vectors Engineering-Expression vectors were generated by site-specific recombination using the GATEWAY system (Invitrogen) of PCR-amplified ORFs into TAP-, green fluorescence protein (GFP)-, or hemagglutinin (HA) epitope-tagged Moloney murine leukemia virus-based vectors previously described (43,44). FLAG epitope-tagged expression vectors were engineered by inserting GATEWAY and FLAG epitope cassettes into pSG-5 (Stratagene, LaJolla, CA).…”

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription

“…35 This HTM is an 88 amino-acids region of SUV420H2, responsible for stable association to pericentric heterochromatin. First, pools of L929 cells expressing RFP-CBX2 fusion protein were generated by retroviral transduction.…”

“…TAP-, green fluorescence protein (GFP)-and GAL4 DNA-binding domain (DBD) epitope-tagged expression vectors were previously described in reference 15, 35 and 43. The mCherry sequence was amplified by PCR from pmCherry-C1 (Clontech) and the HTM sequence was amplified from the SUV420H2 cDNA 35 to engineer novel tagged Moloney murine leukemia virus-based vectors. Expression vectors were generated by site-specific recombination using the GATEWAY system (Invitrogen) of PCR-amplified ORFs into these destination vectors.…”