Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns*

Abstract

Chromatin is a highly dynamic structure that must respond to different stimuli in order to orchestrate all DNA-dependent processes. Post translational modifications (PTMs) 1 of histones play a major role in regulation of chromatin functionality. Evidence is emerging that not only "classical" histone PTMs, such as methylation, acetylation, and phosphorylation at distinct residues, but also proteolytic processing of nucleosome proteins, known as "histone clipping," can be involved in regulation of key cellular processes, such as transcriptional regulation, cell differentiation, and senescence (1-7).Clipping of the histone H3 N-terminal tail was reported to be associated with gene activation in yeast. Santos-Rosa et al. demonstrated a serine protease activity in S. cerevisiae that cleaves histone H3 after residue Ala21 (A21) during sporulation and stationary phase (1). H3 clipping took place specifically within the promoters of sporulation-induced genes following the induction of transcription and prior to histone eviction from these DNA regions. Prevention of H3 N-tail cleavage by amino acid substitution at the endoproteinase recognition site (H3 Q19A, L20A) abolished expression of these genes, indicating that H3 clipping is essential for productive transcription.The biological significance of histone clipping in higher eukaryotes is not yet understood but also appears to be related to functional commitment by the cell. Duncan et al. demonstrated that histone H3 is proteolytically cleaved by the enzyme Cathepsin L1 (CTSL1) at several sites between residues A21 and S28 during mouse ESC differentiation (5). The "in vitro" proteolytic activity of CTSL1 was found to be dependent on the H3 N-tail PTM status. H3K27me2 increased From the ‡Department

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

“…The results of several recent studies involving top-down analyses of histones highlight the complexity of the histone proteome as well as important biological implications that are not easily captured unless the proteoforms are analyzed intact (17)(18)(19)(20). Although these results are very promising, this approach is not routinely utilized because of the many challenges with which it is associated.…”

Epiproteomics: quantitative analysis of histone marks and codes by mass spectrometry

“…The advantages go beyond the aforementioned readout of co-occurring, multiple modifications. Two very recent Middle/Top Down studies have identified unexpected results quite readily: the clipping of histone tails [48] and the hypermethylated trivalent marks in a histone methyltransferase overexpression system [7 •• ]. However, more challenges arise from dissecting the increasing number of isobaric species in Middle/Top Down, which demands innovations in MS3 or other approach, such as internal fragmentation [49] to distinguish them.…”

Middle-down proteomics: a still unexploited resource for chromatin biology

“…To our knowledge, the first publication is the work of Taverna et al where they identified correlations between K4 methylation and other site acetylation on the histone H3 of Tetrahymena thermophila [42]. Other models have been investigated, like Toxoplasma gondii [72], Caenorhabditis elegans [73], Neurospora crassa [74] and 3D spheroids cell cultures [75]. In this last publication, the authors characterized the PTM composition of histones that underwent biological proteolytic cleavage, a.k.a.…”