Monomethylation of Lysine 20 on Histone H4 Facilitates Chromatin Maturation

Abstract

Histone modifications play an important role in shaping chromatin structure. Here, we describe the use of an in vitro chromatin assembly system from Drosophila embryo extracts to investigate the dynamic changes of histone modifications subsequent to histone deposition. In accordance with what has been observed in vivo, we find a deacetylation of the initially diacetylated isoform of histone H4, which is dependent on chromatin assembly. Immediately after deposition of the histones onto DNA, H4 is monomethylated at K20, which is required for an efficient deacetylation of the H4 molecule. H4K20 methylation-dependent dl(3)MBT association with chromatin and the identification of a dl(3)MBT-dRPD3 complex suggest that a deacetylase is specifically recruited to the monomethylated substrate through interaction with dl(3)MBT. Our data demonstrate that histone modifications are added and removed during chromatin assembly in a highly regulated manner.All DNA in a eukaryotic cell is assembled into chromatin to fit it into the restricted nuclear space and to organize the genome (29, 56). As the DNA content of a cell doubles during S-phase of the cell cycle, the cell has to provide sufficient histone molecules to package the newly replicated DNA into chromatin. This is achieved mainly by a coupling of histone and DNA synthesis (37). The progression of the DNA replication machinery disrupts the nucleosome in front of the replication fork, which is then reassembled onto the newly synthesized DNA strands in a random manner (15,20). The remaining gaps are subsequently filled up with newly translated histone molecules, leading to a mosaic pattern of new and old histone octamers (2, 21). This process is assisted by the action of nuclear chaperones, which bind to the histones before deposition (14, 34). The newly deposited histones are more loosely associated with the DNA than the bulk histones and mature slowly into a more stable chromatin structure. Assembly is achieved via an ordered deposition of H3 and H4, followed by the binding of H2A/H2B dimers and finally the interaction of the linker histone H1 with the chromatin fiber (19)(20)(21)(22)49).Posttranslational modifications of histone molecules are generally considered to play an important role during the establishment and maintenance of chromatin structures. The combination of histone modifications has been proposed to constitute a "histone code" (23, 55), which is involved in the maintenance of epigenetic information. However, it is unclear how histone modifications are replicated during cellular division and DNA repair when newly translated histones are deposited onto the DNA.Modification marks can be stably maintained through multiple mitotic divisions (3,23,30,55). However, for most histone modifications, the molecular mechanisms that regulate this maintenance have so far remained elusive. A key aspect of this maintenance or reestablishment of modification patterns is the precise copying of histone modification patterns from the parental histone to the newly synthesized on...

Histone H4 Lysine 20 (H4K20) Methylation, Expanding the Signaling Potential of the Proteome One Methyl Moiety at a Time

“…In this study, mass spectrometry was used to determine the order of H4 PTMs during deposition of histones on DNA templates in the context of Drosophila extracts. Mono-methylation of H4K20 was found to follow acetylation events and to be required for subsequent de-acetylation, implicating H4K20me1 in chromatin assembly via marking the fully assembled nucleosome after histone deposition (79). An unbiased middle-down proteomics was also used to correlate the status of H4K20 methylation relative to the differential potency of cells (80).…”

A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin

“…Nucleosome-depleted regions (NDRs) are found surrounding the transcribed units, and the 5 in yeast at the TSS, and they are often associated with histone modifications related to high nucleosome turnover: H4 acetylation (Scharf et al 2009b), H3K4 methylation (Kraushaar et al 2013), and/or the histone variant H2A.Z (Guillemette et al 2005;Zhang et al 2005;Jin and Felsenfeld 2007). Nucleosome turnover in promoter regions is directly affected by ATP-dependent chromatin remodelers such as Ino80, which binds to the 5 ′ NDR and promotes turnover of TSS nucleosomes (Yen et al 2013).…”

A Quantitative Proteomic Analysis of In Vitro Assembled Chromatin

“…We have shown in the past that TSA treatment results in a moderate increase of histone acetylation on assembled chromatin in contrast to what is observed in tissue culture cells where TSA treatment results in a strong hyperacetylation of histones (53)(54)(55). During in vitro assembly, the histones are deposited in a preacetylated form, which will not get deacetylated when TSA is present (41). TSA treatment has been shown to prevent the formation of transcriptionally repressed chromatin (39) in Xenopus oocytes and lead to an altered pattern of DNA replication origin activity in human tissue culture cells (40).…”

“…S1D and S1E). It is worth mentioning that the presence of TSA does not result in a histone hyperacetylation, which is usually observed in tissue culture cells but induces a moderate increase in the acetylation because of a failure to efficiently remove the acetylation pattern present on histones before assembly (41). The reason for this is unclear but it is probably because of a general lack of site specific histone acetyltransferases in early embryos (34).…”

“…By choosing three different time points, we were able to describe the kinetics of the chromatin assembly in vitro at an (41,42) or the coordinated binding and release of chromatin binding factors (41,43,44), the proteomic composition of in vitro and in vivo assembled chromatin has not been compared so far. The recent advancements toward a complete proteomic description of in vivo chromatin assembly (13,26) have now made such comparisons feasible.…”