Epigenome profiling has led to the paradigm that promoters of active genes are decorated with H3K4me3 and H3K9ac marks. To explore the epigenome of Plasmodium falciparum asexual stages, we performed MS analysis of histone modifications and found a general preponderance of H3/H4 acetylation and H3K4me3. ChIPon-chip profiling of H3, H3K4me3, H3K9me3, and H3K9ac from asynchronous parasites revealed an extensively euchromatic epigenome with heterochromatin restricted to variant surface antigen gene families (VSA) and a number of genes hitherto unlinked to VSA. Remarkably, the vast majority of the genome shows an unexpected pattern of enrichment of H3K4me3 and H3K9ac. Analysis of synchronized parasites revealed significant developmental stage specificity of the epigenome. In rings, H3K4me3 and H3K9ac are homogenous across the genes marking active and inactive genes equally, whereas in schizonts, they are enriched at the 5 end of active genes. This study reveals an unforeseen and unique plasticity in the use of the epigenetic marks and implies the presence of distinct epigenetic pathways in gene silencing/activation throughout the erythrocytic cycle.chromatin ͉ epigenetics ͉ malaria P lasmodium falciparum, the protozoan parasite causing malaria, exhibits a complex life cycle characterized by invasion of different cell types and hosts. During the Ϸ48 h of the intraerythrocytic cycle, a merozoite invades a red blood cell (RBC) and develops into the ring stage, which is followed by the trophozoite stage. Nuclear division marks the beginning of the schizont stage, which results in the formation of up to 32 merozoites that can invade new RBCs (1). Global analysis of transcription (2, 3) and protein expression (4, 5) of the parasite have revealed a high level of coordination in gene expression during the different stages of the life cycle. The absence of chromosomal clustering among genes with similar transitory expression profiles indicates that genes are regulated individually.

Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show that small interfering RNA inhibition of SET8 expression leads to decreased cell proliferation and accumulation of cells in S phase. This is accompanied by DNA double-strand break (DSB) induction and recruitment of the DNA repair proteins replication protein A, Rad51, and 53BP1 to damaged regions. SET8 depletion causes DNA damage specifically during replication, which induces a Chk1-mediated S-phase checkpoint. Furthermore, we find that SET8 interacts with proliferating cell nuclear antigen through a conserved motif, and SET8 is required for DNA replication fork progression. Finally, codepletion of Rad51, an important homologous recombination repair protein, abrogates the DNA damage after SET8 depletion. Overall, we show that SET8 is essential for genomic stability in mammalian cells and that decreased expression of SET8 results in DNA damage and Chk1-dependent S-phase arrest.

Post-translational modifications (PTMs) of histone tails play a key role in epigenetic regulation of gene expression in a range of organisms from yeast to human; however, little is known about histone proteins from the parasite that causes malaria in humans, Plasmodium falciparum. We characterized P. falciparum histone PTMs using advanced mass spectrometry driven proteomics. Acid-extracted proteins were resolved in SDS-PAGE, in-gel trypsin digested, and analyzed by reversed-phase LC-MS/MS. Through the combination of Q-TOF and LTQ-FT mass spectrometry we obtained high mass accuracy of both precursor and fragment ions, which is a prerequisite for high-confidence identifications of multisite peptide modifications. We utilize MS/MS fragment marker ions to validate the identification of histone modifications and report the m/z 143 ion as a novel MS/MS marker ion for monomethylated lysine. We identified all known P. falciparum histones and mapped 44 different modifications, providing a comprehensive view of epigenetic marks in the parasite. Interestingly, the parasite exhibits a histone modification pattern that is distinct from its human host. A general preponderance for modifications associated with a transcriptionally permissive state was observed. Additionally, a novel differentiation in the modification pattern of the two histone H2B variants (H2B and H2Bv) was observed, suggesting divergent functions of the two H2B variants in the parasite. Taken together, our results provide a first comprehensive map of histone modifications in P. falciparum and highlight the utility of tandem MS for detailed analysis of peptides containing multiple PTMs.

Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecular function and the spatial-temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and biomolecular structures.

Secretory preproteins carry signal peptides fused amino-terminally to mature domains. They are post-translationally targeted to cross the plasma membrane in non-folded states with the help of translocases, and fold only at their final destinations. The mechanism of this process of postponed folding is unknown, but is generally attributed to signal peptides and chaperones. We herein demonstrate that, during targeting, most mature domains maintain loosely packed folding intermediates. These largely soluble states are signal peptide independent and essential for translocase recognition. These intermediates are promoted by mature domain features: residue composition, elevated disorder, and reduced hydrophobicity. Consequently, a mature domain folds slower than its cytoplasmic structural homolog. Some mature domains could not evolve stable, loose intermediates, and hence depend on signal peptides for slow folding to the detriment of solubility. These unique features of secretory proteins impact our understanding of protein trafficking, folding, and aggregation, and thus place them in a distinct class.

Protein and peptide mass analysis and amino acid sequencing by mass spectrometry is widely used for identification and annotation of post-translational modifications (PTMs) in proteins. Modification-specific mass increments, neutral losses or diagnostic fragment ions in peptide mass spectra provide direct evidence for the presence of post-translational modifications, such as phosphorylation, acetylation, methylation or glycosylation. However, the commonly used database search engines are not always practical for exhaustive searches for multiple modifications and concomitant missed proteolytic cleavage sites in large-scale proteomic datasets, since the search space is dramatically expanded. We present a formal definition of the problem of searching databases with tandem mass spectra of peptides that are partially (sub-stoichiometrically) modified. In addition, an improved search algorithm and peptide scoring scheme that includes modification specific ion information from MS/MS spectra was implemented and tested using the Virtual Expert Mass Spectrometrist (VEMS) software. A set of 2825 peptide MS/MS spectra were searched with 16 variable modifications and 6 missed cleavages. The scoring scheme returned a large set of post-translationally modified peptides including precise information on modification type and position. The scoring scheme was able to extract and distinguish the near-isobaric modifications of trimethylation and acetylation of lysine residues based on the presence and absence of diagnostic neutral losses and immonium ions. In addition, the VEMS software contains a range of new features for analysis of mass spectrometry data obtained in large-scale proteomic experiments. Windows binaries are available at http://www.yass.sdu.dk/.

Degradation of the histone H4 methyltransferase SET8, which regulates chromosome compaction and genomic integrity, is regulated by the CRL4(CDT2) ubiquitin ligase to facilitate DNA replication and repair.

Given the complexity of the mammalian proteome, high-resolution separation technologies are required to achieve comprehensive proteome coverage and to enhance the detection of low-abundance proteins. Among several technologies, Multidimensional Protein Identification Technology (MudPIT) enables the on-line separation of highly complex peptide mixtures directly coupled with mass spectrometry-based identification. Here, we present a variation of the traditional MudPIT protocol, combining highly sensitive chromatography using a nanoflow liquid chromatography system (nano-LC) with a two-dimensional precolumn in a vented column setup. When compared to the traditional MudPIT approach, this nanoflow variation demonstrated better firstphase separation leading to more proteins being characterized while using rather simple instrumentation and a protocol that requires less time and very little technical expertise to perform.