Usage of the H3 variants during the S-phase of the cell cycle in Physarum polycephalum.

DNA replication occurring in S-phase is critical for the maintenance of the cell fate from one generation to the next, and requires the duplication of epigenetic information. The integrity of the epigenome is, in part, insured by the recycling of parental histones and de novo deposition of newly synthesized histones. While the histone variants have revealed important functions in epigenetic regulations, the deposition in chromatin during S-phase of newly synthesized histone variants remains unclear. The identification of histone variants of H3 and unique features of Physarum polycephalum provides a powerful system for investigating de novo deposition of newly synthesized histones by tracking the incorporation of exogenous histones within cells. The analyses revealed that the rate of deposition of H3.1 and H3.3 is anticorrelated as S-phase progresses, H3.3 is predominately produced and utilized in early S and dropped throughout S-phase, while H3.1 behaved in the opposite way. Disturbing the expression of H3 variants by siRNAs revealed mutual compensation of histone transcripts. Interestingly, the incorporation of pre-formed constrained histone complexes showed that tetramers of H3/H4 are more efficiently utilized by the cell than dimers. These results support the model whereby the histone variant distribution is established upon replication and new histone deposition.

The Histone Chaperone Network Is Highly Conserved in Physarum polycephalum.

The nucleosome is composed of histones and DNA. Prior to their deposition on chromatin, histones are shielded by specialized and diverse proteins known as histone chaperones. They escort histones during their entire cellular life and ensure their proper incorporation in chromatin. Physarum polycephalum is a Mycetozoan, a clade located at the crown of the eukaryotic tree. We previously found that histones, which are highly conserved between plants and animals, are also highly conserved in Physarum. However, histone chaperones differ significantly between animal and plant kingdoms, and this thus probed us to further study the conservation of histone chaperones in Physarum and their evolution relative to animal and plants. Most of the known histone chaperones and their functional domains are conserved as well as key residues required for histone and chaperone interactions. Physarum is divergent from yeast, plants and animals, but PpHIRA, PpCABIN1 and PpSPT6 are similar in structure to plant orthologues. PpFACT is closely related to the yeast complex, and the Physarum genome encodes the animal-specific APFL chaperone. Furthermore, we performed RNA sequencing to monitor chaperone expression during the cell cycle and uncovered two distinct patterns during S-phase. In summary, our study demonstrates the conserved role of histone chaperones in handling histones in an early-branching eukaryote.

Replication-Coupled Chromatin Remodeling: An Overview of Disassembly and Assembly of Chromatin during Replication.

The doubling of genomic DNA during the S-phase of the cell cycle involves the global remodeling of chromatin at replication forks. The present review focuses on the eviction of nucleosomes in front of the replication forks to facilitate the passage of replication machinery and the mechanism of replication-coupled chromatin assembly behind the replication forks. The recycling of parental histones as well as the nuclear import and the assembly of newly synthesized histones are also discussed with regard to the epigenetic inheritance.

Transient expression of RAD51 in the late G2-phase is required for cell cycle progression in synchronous Physarum cells.

The homologous recombination factor RAD51 is highly conserved. This criterion enabled us to identify a RAD51 ortholog in Physarum polycephalum. We found that the Physarum protein presents a high homology to the human protein and cross-reacted with antibodies directed against the human RAD51. Taking advantage of the natural synchrony of millions of nuclei within a single cell of Physarum, we investigated the fluctuation of the amount of the PpRAD51 throughout the cell cycle. Our results showed that in the late G2-phase, RAD51 was transiently expressed in a large quantity. Furthermore, knocking-down RAD51 in the G2-phase abolished this transient expression before mitosis and affected cell cycle progression. These results support the idea that RAD51 plays a role in the progression of the cell cycle in the late G2-phase.

Replication-independent nucleosome exchange is enhanced by local and specific acetylation of histone H4.

We used a novel single-cell strategy to examine the fate of histones during G(2)-phase. Consistent with previous results, we find that in G(2)-phase, the majority of nuclear histones are assembled into chromatin, whereas a small fraction comprises an unassembled pool. Small increases in the amount of histones within the free pool affect the extent of exchange, suggesting that the free pool is in dynamic equilibrium with chromatin proteins. Unexpectedly, acetylated H4 is preferentially partitioned to the unassembled pool. Although an increase in global histone acetylation did not affect overall nucleosome dynamics, an H4 containing lysine to glutamine substitutions as mimics of acetylation significantly increased the rate of exchange, but did not affect the acetylation state of neighbouring nucleosomes. Interestingly, transcribed regions are particularly predisposed to exchange on incorporation of H4 acetylation mimics compared with surrounding regions. Our results support a model whereby histone acetylation on K8 and K16 specifically marks nucleosomes for eviction, with histones being rapidly deacetylated on reassembly.

Replication-coupled chromatin assembly of newly synthesized histones: distinct functions for the histone tail domains.

The maintenance of the genome during replication requires the assembly of nucleosomes with newly synthesized histones. Achieving the deposition of newly synthesized histones in chromatin implies their transport from the cytoplasm to the nucleus at the replication sites. Several lines of evidence have revealed critical functions of the histone tail domains in these conserved cellular processes. In this review, we discuss the role of the amino termini of the nucleosome building blocks, H2A/H2B and H3/H4, in different model systems. The experimental data showed that H2A/H2B tails and H3/H4 tails display distinct functions in nuclear import and chromatin assembly. Furthermore, we describe recent studies exploiting the unique properties of the slime mold, Physarum polycephalum , that have advanced understanding of the function of the highly conserved replication-dependent diacetylation of H4.

Identification and characterization of histones in Physarum polycephalum evidence a phylogenetic vicinity of Mycetozoans to the animal kingdom.

Physarum polycephalum belongs to Mycetozoans, a phylogenetic clade apart from the animal, plant and fungus kingdoms. Histones are nuclear proteins involved in genome organization and regulation and are among the most evolutionary conserved proteins within eukaryotes. Therefore, this raises the question of their conservation in Physarum and the position of this organism within the eukaryotic phylogenic tree based on histone sequences. We carried out a comprehensive study of histones in Physarum polycephalum using genomic, transcriptomic and molecular data. Our results allowed to identify the different isoforms of the core histones H2A, H2B, H3 and H4 which exhibit strong conservation of amino acid residues previously identified as subject to post-translational modifications. Furthermore, we also identified the linker histone H1, the most divergent histone, and characterized a large number of its PTMs by mass spectrometry. We also performed an in-depth investigation of histone genes and transcript structures. Histone proteins are highly conserved in Physarum and their characterization will contribute to a better understanding of the polyphyletic Mycetozoan group. Our data reinforce that P. polycephalum is evolutionary closer to animals than plants and located at the crown of the eukaryotic tree. Our study provides new insights in the evolutionary history of Physarum and eukaryote lineages.

The core histone N-terminal tail domains negatively regulate binding of transcription factor IIIA to a nucleosome containing a 5S RNA gene via a novel mechanism.

Reconstitution of a DNA fragment containing a 5S RNA gene from Xenopus borealis into a nucleosome greatly restricts binding of the primary 5S transcription factor, TFIIIA. Consistent with transcription experiments using reconstituted templates, removal of the histone tail domains stimulates TFIIIA binding to the 5S nucleosome greater than 100-fold. However, we show that tail removal increases the probability of 5S DNA unwrapping from the core histone surface by only approximately fivefold. Moreover, using site-specific histone-to-DNA cross-linking, we show that TFIIIA binding neither induces nor requires nucleosome movement. Binding studies with COOH-terminal deletion mutants of TFIIIA and 5S nucleosomes reconstituted with native and tailless core histones indicate that the core histone tail domains play a direct role in restricting the binding of TFIIIA. Deletion of only the COOH-terminal transcription activation domain dramatically stimulates TFIIIA binding to the native nucleosome, while further C-terminal deletions or removal of the tail domains does not lead to further increases in TFIIIA binding. We conclude that the unmodified core histone tail domains directly negatively influence TFIIIA binding to the nucleosome in a manner that requires the C-terminal transcription activation domain of TFIIIA. Our data suggest an additional mechanism by which the core histone tail domains regulate the binding of trans-acting factors in chromatin.

Physarum polycephalum for Studying the Function of Histone Modifications In Vivo.

Histone modifications have been widely correlated with genetic activities. However, how these posttranslational modifications affect the dynamics and the structure of chromatin is poorly understood. Here, we describe the incorporation of the exogenous histone proteins into the slime mold Physarum polycephalum, which has been revealed to be a valuable tool for examining different facets of the function histones in chromatin dynamics like replication-coupled chromatin assembly, histone exchange, and nucleosome turnover.

Histones H3 and H4 require their relevant amino-tails for efficient nuclear import and replication-coupled chromatin assembly in vivo.

Concomitant chromatin assembly and DNA duplication is essential for cell survival and genome integrity, and requires newly synthesized histones. Although the N-terminal domains of newly synthesized H3 and H4 present critical functions, their requirement for replication-coupled chromatin assembly is controversial. Using the unique capability of the spontaneous internalization of exogenous proteins in Physarum, we showed that H3 and H4 N-tails present critical functions in nuclear import during the S-phase, but are dispensable for assembly into nucleosomes. However, our data revealed that chromatin assembly in the S-phase of complexes presenting ectopic N-terminal domains occurs by a replication-independent mechanism. We found that replication-dependent chromatin assembly requires an H3/H4 complex with the relevant N-tail domains, suggesting a concomitant recognition of the two histone domains by histone chaperones.

Complement C3 of the innate immune system secreted by muscle adipogenic cells promotes myogenic differentiation.

Myogenic differentiation results in different cell type cooperation, but the molecules involved in the myogenic cell activation remain elusive. Here, we show that muscle-resident pre-adipocytes promote myogenic differentiation through the secretion of factors. Using proteomic and transcriptomic analyses, we identified that proliferative adipogenic lineage cells produce and secrete a key factor of the innate immune system, the complement C3. Cell culture experiments revealed that C3 promotes the differentiation of myogenic progenitors following internalisation of the immune molecule. These data demonstrate that the third component of the complement system, which is a pivotal factor in the immune response to pathogens, is also involved in the differentiation of myogenic progenitor cells.

Nucleosome Dancing at the Tempo of Histone Tail Acetylation.

The impact of histone acetylation on transcription was revealed over 50 years ago by Allfrey and colleagues. However, it took decades for an understanding of the fine mechanism by which this posttranslational modification affects chromatin structure and promotes transcription. Here, we review breakthroughs linking histone tail acetylation, histone dynamics, and transcription. We also discuss the histone exchange during transcription and highlight the important function of a pool of non-chromatinized histones in chromatin dynamics.

H4 replication-dependent diacetylation and Hat1 promote S-phase chromatin assembly in vivo.

While specific posttranslational modification patterns within the H3 and H4 tail domains are associated with the S-phase, their actual functions in replication-dependent chromatin assembly have not yet been defined. Here we used incorporation of trace amounts of recombinant proteins into naturally synchronous macroplasmodia of Physarum polycephalum to examine the function of H3 and H4 tail domains in replication-coupled chromatin assembly. We found that the H3/H4 complex lacking the H4 tail domain was not efficiently recovered in nuclei, whereas depletion of the H3 tail domain did not impede nuclear import but chromatin assembly failed. Furthermore, our results revealed that the proper pattern of acetylation on the H4 tail domain is required for nuclear import and chromatin assembly. This is most likely due to binding of Hat1, as coimmunoprecipitation experiments showed Hat1 associated with predeposition histones in the cytoplasm and with replicating chromatin. These results suggest that the type B histone acetyltransferase assists in shuttling the H3/H4 complex from cytoplasm to the replication forks.

Linker histone phosphorylation regulates global timing of replication origin firing.

Despite the presence of linker histone in all eukaryotes, the primary function(s) of this histone have been difficult to clarify. Knock-out experiments indicate that H1s play a role in regulation of only a small subset of genes but are an essential component in mouse development. Here, we show that linker histone (H1) is involved in the global regulation of DNA replication in Physarum polycephalum. We find that genomic DNA of H1 knock-down cells is more rapidly replicated, an effect due at least in part to disruption of the native timing of replication fork firing. Immunoprecipitation experiments demonstrate that H1 is transiently lost from replicating chromatin via a process facilitated by phosphorylation. Our results suggest that linker histones generate a chromatin environment refractory to replication and that their transient removal via protein phosphorylation during S phase is a critical step in the epigenetic regulation of replication timing.

Histone dynamics during transcription: exchange of H2A/H2B dimers and H3/H4 tetramers during pol II elongation.

Chromatin within eukaryotic cell nuclei accommodates many complex activities that require at least partial disassembly and reassembly of nucleosomes. This disassembly/reassembly is thought to be somewhat localized when associated with processes such as site-specific DNA repair but likely occurs over extended regions during processive processes such as DNA replication or transcription. Here we review data addressing the effect of transcription elongation on nucleosome disassembly/reassembly, specifically focusing on the issue of transcription-dependent exchange of H2A/H2B dimers and H3/H4 tetramers. We suggest a model whereby passage of a polymerase through a nucleosome induces displacement of H2A/H2B dimers with a much higher probability than displacement of H3/H4 tetramers such that the extent of tetramer replacement is relatively low and proportional to polymerase density on any particular gene.

Replication-independent core histone dynamics at transcriptionally active loci in vivo.

We used a novel labeling technique in the naturally synchronous organism Physarum polycephalum to examine the fate of core histones in G2 phase. We find rapid exchange of H2A/H2B dimers with free pools that is greatly diminished by treatment of the cells with alpha-amanitin. This exchange is enhanced in pol II-coding sequences compared with extragenic regions or inactive loci. In contrast, H3/H4 tetramers exhibit far lower levels of exchange in the pol II-transcribed genes tested, suggesting that tetramer exchange occurs via a distinct mechanism. However, we find that transcribed regions of the ribosomal RNA gene loci exhibit rapid exchange of H3/H4 tetramers. Thus, our data show that the majority of the pol II transcription-dependent histone exchange is due to elongation in vivo rather than promoter remodeling or other pol II-dependent alterations in promoter structure and, in contrast to pol I, pol II transcription through nucleosomes in vivo causes facile exchange of both H2A/H2B dimers while allowing conservation of epigenetic "marks" and other post-translational modifications on H3 and H4.

Chromatin in need of a fix: phosphorylation of H2AX connects chromatin to DNA repair.

A bevy of recent reports have firmly established a mechanistic link between a histone posttranslational modification associated with DNA double-strand breaks and recruitment of chromatin-modifying activities. These papers show that in addition to providing signals for transcriptional regulation, specific histone "codes" can coordinate and target multiple activities involved in DNA repair.

Analysis of chromatin assembled in vivo using exogenous histones in Physarum polycephalum.

Histones are involved in the regulation of almost all events within the eukaryotic cell nucleus that utilize DNA as a substrate. We have developed a novel approach for examining the function of histone proteins and specific domains of these proteins in these various nuclear processes, and in particular assembly of chromatin throughout the cell cycle. This approach exploits several unique characteristics of the slime mold Physarum polycephalum, including the natural synchrony of all (approximately 10(8)) nuclei throughout the cell cycle and the ability of this organism to take up exogenous proteins. Here, culture techniques and biochemical procedures for the incorporation of exogenous core histones into Physarum chromatin in vivo are described. The procedures for subsequent verification of the assembly of exogenous proteins into bona fide nucleosomes are also described.