Epigenetics has become a fundamental scientific discipline with various implications for biology and medicine. Epigenetic marks, mostly DNA methylation and histone post-translational modifications (PTMs), play important roles in chromatin structure and function. Accurate quantification of these marks is an ongoing challenge due to the variety of modifications and their wide dynamic range of abundance. Here we present EpiProfile 2.0, an extended version of our 2015 software (v1.0), for accurate quantification of histone peptides based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. EpiProfile 2.0 is now optimized for data-independent acquisition through the use of precursor and fragment extracted ion chromatography to accurately determine the chromatographic profile and to discriminate isobaric forms of peptides. The software uses an intelligent retention time prediction trained on the analyzed samples to enable accurate peak detection. EpiProfile 2.0 supports label-free and isotopic labeling, different organisms, known sequence mutations in diseases, different derivatization strategies, and unusual PTMs (such as acyl-derived modifications). In summary, EpiProfile 2.0 is a universal and accurate platform for the quantification of histone marks via LC-MS/MS. Being the first software of its kind, we anticipate that EpiProfile 2.0 will play a fundamental role in epigenetic studies relevant to biology and translational medicine. EpiProfile is freely available at https://github.com/zfyuan/EpiProfile2.0_Family .

Diseases associated with mitochondrial DNA (mtDNA) mutations are highly variable in phenotype, in large part because of differences in the percentage of normal and mutant mtDNAs (heteroplasmy) present within the cell. For example, increasing heteroplasmy levels of the mtDNA tRNALeu(UUR) nucleotide (nt) 3243A > G mutation result successively in diabetes, neuromuscular degenerative disease, and perinatal lethality. These phenotypes are associated with differences in mitochondrial function and nuclear DNA (nDNA) gene expression, which are recapitulated in cybrid cell lines with different percentages of m.3243G mutant mtDNAs. Using metabolic tracing, histone mass spectrometry, and NADH fluorescence lifetime imaging microscopy in these cells, we now show that increasing levels of this single mtDNA mutation cause profound changes in the nuclear epigenome. At high heteroplasmy, mitochondrially derived acetyl-CoA levels decrease causing decreased histone H4 acetylation, with glutamine-derived acetyl-CoA compensating when glucose-derived acetyl-CoA is limiting. In contrast, α-ketoglutarate levels increase at midlevel heteroplasmy and are inversely correlated with histone H3 methylation. Inhibition of mitochondrial protein synthesis induces acetylation and methylation changes, and restoration of mitochondrial function reverses these effects. mtDNA heteroplasmy also affects mitochondrial NAD+/NADH ratio, which correlates with nuclear histone acetylation, whereas nuclear NAD+/NADH ratio correlates with changes in nDNA and mtDNA transcription. Thus, mutations in the mtDNA cause distinct metabolic and epigenomic changes at different heteroplasmy levels, potentially explaining transcriptional and phenotypic variability of mitochondrial disease.

After transcription, a messenger RNA (mRNA) is further post‐transcriptionally regulated by several features including RNA secondary structure and covalent RNA modifications (specifically N 6 ‐methyladenosine, m 6 A). Both RNA secondary structure and m 6 A have been demonstrated to regulate mRNA stability and translation and have been independently linked to plant responses to soil salinity levels. However, the effect of m 6 A on regulating RNA secondary structure and the combinatorial interplay between these two RNA features during salt stress response has yet to be studied. Here, we globally identify RNA‐protein interactions and RNA secondary structure during systemic salt stress. This analysis reveals that RNA secondary structure changes significantly during salt stress, and that it is independent of global changes in RNA‐protein interactions. Conversely, we find that m 6 A is anti‐correlated with RNA secondary structure in a condition‐dependent manner, with salt‐specific m 6 A correlated with a decrease in mRNA secondary structure during salt stress. Taken together, we suggest that salt‐specific m 6 A deposition and the associated loss of RNA secondary structure results in increases in mRNA stability for transcripts encoding abiotic stress response proteins and ultimately increases in protein levels from these stabilized transcripts. In total, our comprehensive analyses reveal important post‐transcriptional regulatory mechanisms involved in plant long‐term salt stress response and adaptation.

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation.

Functional epigenetic regulation occurs by dynamic modification of chromatin, including genetic material (i.e. DNA methylation), histone proteins, and other nuclear proteins. Due to the highly complex nature of the histone code, mass spectrometry (MS) has become the leading technique in identification of single and combinatorial histone modifications. MS has now overcome antibody based strategies due to its automation, high resolution and accurate quantitation. Moreover, multiple approaches to analysis have been developed for global quantitation of post-translational modifications (PTMs), including large-scale characterization of modification co-existence (middle-down and top-down proteomics), which is not currently possible with any other biochemical strategy. Recently, our group and others have simplified and increased the effectiveness of analyzing histone PTMs by improving multiple MS methods and data analysis tools. This review provides an overview of the major achievements in the analysis of histone PTMs using MS with a focus on the most recent improvements. We speculate that the workflow for histone analysis at its state-of-the-art is highly reliable in terms of identification and quantitation accuracy, and it has the potential to become a routine method for systems biology thanks to the possibility of integrating histone MS results with genomics and proteomics datasets.