Abstract
BackgroundHistone 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.ResultsTo gain insights into the mechanisms of SUV420H2 recruitment at heterochromatin, we applied a tandem affinity purification approach coupled to mass spectrometry. We identified heterochromatin proteins HP1 as main interacting partners. The regions responsible for the binding were mapped to the heterochromatic targeting module of SUV420H2 and HP1 chromoshadow domain. We studied the dynamic properties of SUV420H2 and the HP1 in living cells using fluorescence recovery after photobleaching. Our results showed that HP1 proteins are highly mobile with different dynamics during the cell cycle, whereas SUV420H2 remains strongly bound to pericentric heterochromatin. An 88 amino-acids region of SUV420H2, the heterochromatic targeting module, recapitulates both, HP1 binding and strong association to heterochromatin.ConclusionFRAP experiments reveal that in contrast to HP1, SUV420H2 is strongly associated to pericentric heterochromatin. Then, the fraction of SUV420H2 captured and characterized by TAP/MS is a soluble fraction which may be in a stable association with HP1. Consequently, SUV420H2 may be recruited to heterochromatin in association with HP1, and stably maintained at its heterochromatin sites in an HP1-independent fashion.
Highlights
Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions
In order to gain insight into the mechanisms involved in the recruitment of SUV420H enzymes at heterochromatin and identify their interacting partners, we applied the tandem affinity purification (TAP) technology coupled to tandem mass spectrometry (MS/MS), since this approach has proven to efficiently allow the characterization of protein complexes from different cells in culture or organisms [20]
The SUV420H family of histone methyltransferases catalyzes trimethylation of histone H4K20 which is characteristic to pericentric heterochromatin
Summary
Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. Histone C- and N-terminal tails are flexible, protrude from the nucleosome octamer structure, and are subjected to post-translational modifications, including acetylation, methylation, phosphorylation, ubiquitination or sumoylation Among these modifications, histone lysine methylation patterns have been associated with distinct chromatin states and are proposed to be major epigenetic marks that could extend the information potential of the genetic code by fixing the chromatin organization in a heritable manner (for a review [2]). H3K9me marks placed by SUV39H activities stabilize heterochromatin protein 1 (HP1) binding at heterochromatin [10,11], and HP1 proteins would recruit the histone methyltransferases SUV420H2 and SUV420H1 which in turn, trimethylate H4K20 [5,12,13] At present, it is unclear whether SUV420H histone methyltransferases interact only temporally with chromatin to methylate H4K20 or participate in a more stable multiprotein complex together with HP1 or other chromatin proteins to support a stable heterochromatin structure
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