Breaking the Histone Code With Quantitative Mass Spectrometry

Laura-Mae P Britton; Michelle Gonzales-Cope; Barry M Zee; Benjamin A Garcia

Disclosures

Expert Rev Proteomics. 2011;8(5):631-643. 

In This Article

Abstract and Introduction

Abstract

Histone post-translational modifications (PTMs) comprise one of the most intricate nuclear signaling networks that govern gene expression in a long-term and dynamic fashion. These PTMs are considered to be 'epigenetic' or heritable from one cell generation to the next and help establish genomic expression patterns. While much of the analyses of histones have historically been performed using site-specific antibodies, these methods are replete with technical obstacles (i.e., cross-reactivity and epitope occlusion). Mass spectrometry-based proteomics has begun to play a significant role in the interrogation of histone PTMs, revealing many new aspects of these modifications that cannot be easily determined with standard biological approaches. Here, we review the accomplishments of mass spectrometry in the histone field, and outline the future roadblocks that must be overcome for mass spectrometry-based proteomics to become the method of choice for chromatin biologists.

Introduction

The study of chromatin biology has evolved to reveal another layer of control over the cellular processes of transcription, cellular division, differentiation and cellular repair. Once deemed a solely structural element of chromatin, the nucleosome has emerged as a crucial controller of cellular fate. In eukaryotes, this basic repeating unit of chromatin is comprised of approximately 147 bp of DNA wrapped around an octamer of the highly conserved core histone proteins (H2A, H2B, H3 and H4).[1] The amino-terminal domains of these proteins project outward from the core particle and are accessible to proteases. Interestingly, these tail domains act as the sites of a myriad of covalent post-translational modifications (PTMs), many of which have been linked directly to the transcriptional output of the genome. Thus far, these PTMS include modifications on lysine residues such as: acetylation, ubiquitination and mono-, di- and tri-methylation; mono- and asymmetric or symmetric di-methylation on arginine residues; and phosphorylation on serine and threonine residues. Specific modified residues, such H3 lysine 9 acetylation (H3K9ac), H3 serine 10 phosphorylation (H3S10ph) and H3 lysine 27 methylation (H3K27me), serve as target epitopes for chromatin remodeling machinery, as well as gene repression and/or activation complexes.[1] However, these marks are usually found in series or in combinations. Out of these observations was born the 'histone code hypothesis', which states that "distinct histone modifications, on one or more N-terminal tails, act sequentially or in combination to form a 'histone code' that is read by other proteins (containing specialized binding domains) to bring about distinct downstream events".[2,3] Histone PTMs, along with DNA methylation and small noncoding RNA, collectively make up mechanisms referred to as 'epigenetic' controls, which are believed to affect gene expression patterns and phenotype in a heritable manner.

In recent decades, epigenetics has come to the forefront of clinical investigation as researchers are faced with the obstacle of identifying nongenetic components that display the same phenotypical disease state as several previously characterized genetic conditions. Technological advances in the field of genetics and medicine have unveiled new genetic bases for hundreds of human diseases. For a number of these disorders, such as Prader–Willi and Angelman sister syndromes, Beckwith–Wiedemann Syndrome and Rett Syndrome, the phenotypic variability from patient to patient is drastic even though the underlying genetic mutations are well defined.[4–12] Deregulation of controlled cellular pathways is a hallmark of tumorigenesis and epigenetic signatures, such as altered distribution of 5-methylcytosine DNA modifications and hypermethylated gene promoters, have become distinct markers of cancer. Changes in histone modification patterns have also gained attention for possible roles in various types of cancer. For example, misdirected targeting of histone acetyltransferases and histone deacetylases have been found in several types of leukemias.[13,14] Loss of histone H4K20me3 and H4K16ac have been determined to be hallmarks of all common cancers (both in cancer cell lines and primary tumors),[15] and these modification losses were found to appear early, accumulating during tumorigenesis. Changes in global levels of individual histone H4 and H3 acetylation and methylation have also been associated with prostate cancer and these changes were indicative of clinical results.[16] In addition, increased histone kinase activity has been shown to be associated with colorectal cancer, and the list of malignancies correlated with significant changes in histone PTM patterns during specific disease states continues to grow, as recently shown in leukemogenesis.[17] Last, small molecule inhibitors of histone deacetylases are currently in various phases of clinical trials for treating several forms of cancer.[18] The continued comprehensive hybridization of epigenetic and genetic approaches to understanding cancer formation and development could lead to personalized treatment regimens for chemoresistant cancer patients as opposed to blanket treatments.

Nevertheless, while data continue to accumulate regarding disruption of histone modification patterns and links to human disease, the precise epigenetic mechanisms possibly underlying these diseases are not yet fully understood. These observations have made a strong case for the initiation of an international Human Epigenome Project on the scale of the Human Genome Project, and organization of this possible effort by the Alliance for the Human Epigenome and Disease and the Epigenome Network of Excellence has begun.[19] The Alliance for the Human Epigenome and Disease, backed by the American Association for Cancer Research, has very recently published a rough formal plan to begin indexing specific histone PTMs in a defined subset of selected 'reference epigenomes' that could provide a reference standard to which disease states could be compared.[19] The NIH has also committed over US$100 million over the next several years to fund and accelerate epigenetics/epigenomics research. Understanding the epigenetic changes in normal human development and disease has far-reaching implications, which would make immediate impacts in many fields ranging from stem cell biology to neuroscience, and could serve as a novel means of disease-targeted therapeutic intervention. Therefore, as can be imagined, robust methods to identify and measure the abundance levels of epigenetic marks such as histone PTMs are of tremendous importance.

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