Biochemical and functional differences of chromatin assembled replication-coupled or independent in Xenopus laevis egg extracts.

Two main chromatin assembly pathways ensure the proper transmission of chromatin organization and chromatin-based information throughout the cell cycle. A replication-dependent (RD) pathway that couples chromatin assembly to DNA synthesis and a replication-independent (RI) pathway. Whether these pathways contribute to the establishment of chromatin domains like heterochromatin or euchromatin by introducing modifications on histones or modulating chromatin structure remains unknown. Using Xenopus laevis egg extracts we monitored RD and RI chromatin assembly on single-stranded and double-stranded DNA templates. Even though RD assembly proceeded faster than RI assembly the histone content and saturation level with nucleosomes were similar. Despite these comparable topological features, the hydrodynamic behavior of both chromatin species in sucrose gradient centrifugation clearly differed. The RD assembled chromatin ran at lower sucrose concentrations than the RI created chromatin suggesting different compaction levels of the generated chromatin fibers. AFM studies confirmed that both chromatin species formed discrete higher order structures. Next, we analyzed the underlying biochemical composition of the RD and RI derived chromatin. Mass spectrometry analysis of chromatin-associated proteins revealed distinct subsets of proteins associated either with both or one of the pathways. For example, the histone variant H3.3 was exclusively incorporated during RI assembly. In contrast, the variants H3.2 and H2A.Z were confirmed as components of both chromatin species. Within the RI assembly pathway we detected pronounced methylation of H3K4 and monomethylation of H4K20. None of these methyl marks was found on the RD assembled material. The installation of the methyl marks did not seem to depend on each other. PR-Set7, the H4K20 monomethyltransferase, methylated chromatin independent of H3K4 methylation. Furthermore, PR-Set7 was not directly targeted to chromatin by HIRA, the key chaperone during RI assembly. Instead, using immunoprecipitation we found that HIRA is complexed with an H3K4 methyltransferase in the egg extract. The observed biochemical differences raised the question whether the transcriptional and thus functional competence of both chromatin species were also different. Transcriptional analysis of purified chromatin revealed that the RI assembly pathway resulted in transcriptional active chromatin whereas the RD generated chromatin was refractory to transcription. Blocking methylation with SAH led to a change in RI assembled chromatin structure and to a reduction of its transcriptional activity demonstrating the influence of these methyl marks for the transcriptional read-out. In summary, we could demonstrate that the RD and RI chromatin assembly pathway generate chromatin species that differ on the structural, biochemical and functional level. Whereas the de novo assembly during replication produces a silenced chromatin fiber, RI assembled chromatin is transcriptional active. Since RI assembly occurs outside of S-phase we believe that it thereby enables the cell to restore the transcriptional competence of certain genomic regions after S-phase.