Quantitative Proteomic Analysis of Histone Modifications

1.1. Nucleosome and Chromatin In eukaryotic cells, chromosomal DNA is packaged into a compact structure, chromatin, with the use of four core histones (H2A, H2B, H3, and H4). The fundamental repeating unit of chromatin is the nucleosome, which is composed of an octamer of the core histones, around which ~147 base pairs of DNA are wrapped. Nucleosomes are in turn folded into progressively higher-order structures. Dynamic chromatin remodeling plays a critical role in regulating diverse DNA-based biological processes, such as transcription of RNA, DNA replication, and DNA repair, as well as chromosome condensation and segregation.1 The core histone proteins (not histone octamer) are small (10–20 kDa) and highly basic. They are predominantly globular except for their N-terminal “tails”, which are unstructured and protrude from the surface of the chromatin polymer. Amino acid sequence analysis shows that histone proteins are highly conserved in eukaryotic cells from yeast to human, implying that most amino acid residues, if not all, are likely to be important for structure or function. Indeed, studies among histone variants as well as mutational evidence in cancers suggest that a change of a single amino acid residue can lead to very different biological output and even disease, such as cancer.2 Histone post-translational modification (PTM), or histone mark, in combination with DNA modifications, histone variants, and ATP-dependent protein complex formation, is used by cells to dynamically modulate chromatin structure and function. Because PTMs alter the properties of the substrate amino acid residue, typically more significant than a mutation, they are likely to affect histone structure and therefore function.3 Indeed, PTMs are abundant in histones, especially at their N-terminal tails, and have roles in modulating chromatin dynamics and diverse DNA-templated biological processes (Figure 1).1 Dysregulation of these processes has been intimately associated with the development of diseases such as cancer.4 Figure 1 Structures of histone post-translational modifications. 1.2. Biological Mechanism of Histone PTMs As of this writing, 20 types of histone PTMs had been reported: phosphorylation, acetylation, monomethylation, dimethylation, trimethylation, propionylation, butyrylation, crotonylation, 2-hydroxylisobutyrylation, malonylation, succinylation, glutarylation, formylation, hydroxylation, ubiquitination, SUMOylation, O-GlcNAcylation, ADP-ribosylation, proline isomerization, and citrullination (Figure 1).5 In more recent times, known PTM sites on histones have been identified either by sequence-specific antibodies or by mass spectrometry (MS) methods in an unbiased manner.6 The function and dynamic regulation of these PTMs have been the subject of extensive investigations over the past decade. Histone PTMs are thought to regulate chromatin structure and function by two mechanisms.1a,b First, histone PTMs can directly modulate the packaging of chromatin by either altering the charge state of histones or through inter nucleosomal interactions, thereby regulating chromatin higher-order structure and the access of DNA-binding proteins, such as transcription factors. Additionally, histone PTMs can modify chromatin structure and function either by recruiting PTM-specific binding proteins (also called “readers”) and their associated binding partners (“effector proteins”) or by inhibiting the binding of a protein to the chromatin. PTM-induced changes in protein interactions between chromatin and its binding proteins are in turn translated into biological outcomes.7 Proteins are recruited to histone PTMs through direct binding to specific domains. For example, chromo, Tudor, PHD, MBT, PWWP, WD, ADD, zf-CW, BAH, and CHD domains are all known to bind methyllysine,8 while the bromodomain binds acetyllysine.9 Proteins containing these PTM-specific binding domains may recruit additional protein factors to execute their functions. Alternatively, they may carry enzymatic activities that can further modify chromatin structure and function. Histone marks are known to be critical in regulation of diverse DNA-templated biological processes.1 Interestingly, some of these histone PTMs correlate with transcriptional activation or repression, depending on the types and the locations of the PTMs.1b,10 To execute DNA-templated processes, histone PTMs coordinate the unraveling of chromatin to carry out specific functions. For example, histone lysine acetylation (Kac) typically correlates with transcriptional activation, while lysine deacetylation correlates with transcriptional repression.1b,11 Lysine methylation (Kme) is implicated in both gene activation (H3K4, H3K36, and H3K79) and transcriptional repression (H3K9, H3K27, and H4K20).12 As examples, some monomethylation (e.g., H3K9me1 and H3K27me1) is involved in transcriptional activation, while trimethylation at the same sites (H3K9me3 and H3K27me3) is linked to repression.13 Likewise, some other histone PTMs also correlate with DNA repair (e.g., H2AS129 phosphorylation and H4S1 phosphorylation)14 and replication (e.g., acetylation).15 Dysregulation of each step of histone PTMs, including adding the histone marks by a “writer”, removing the histone mark by an “eraser”, and misinterpretation by a “reader” protein, has shown to be associated with disease, such as cancer.4a,e These histone PTMs are proposed to contribute a “histone code” or “histone language” that dictates the functions of the proteins in gene expression and chromatin dynamics.1a,c,d Addition and removal of histone PTMs are regulated by diverse groups of enzymes that were initially identified in the past decade, but still are being discovered in recent times. These enzymes are responsible for adding (“writing”) or removing (“erasing”) the histone PTM “code”. The resulting histone marks are in turn translated into biological outputs by different mechanisms. Chromatin dynamics are mainly controlled by ATP-dependent chromatin remodeling enzymes/complexes and histone PTMs.16 The “histone code” can facilitate the recruitment of diverse chromatin remodeling enzymes to regulate chromatin dynamics. Conversely, chromatin remodeling enzymes can also influence the histone PTMs.17 For example, an ATP-dependent nucleosome remodeling complex, nucleosome remodelling and deacetylation complex (NuRD), can facilitate the deacetylation of the target histones.18 Some histone PTMs, if not all, are inheritable during cell division and correlate with gene expression. Therefore, histone PTMs are linked with epigenetic phenomena and are generally considered to be a major type of epigenetic marks.19