The role of histone acetylation and its involvement in the regulation of transcription has long been a topic of research in cell and molecular biology labs. Recent studies have revealed the role of histone acetylation in other important processes regulating the structure and function of chromatin, and hence, the eukaryotic genome. The process of organizing the millions (billions in the case of humans) of base pairs of genetic material in the eukaryotic nucleus has profound effects on DNA-dependent events, such as transcription, recombination, replication and repair. DNA is organized by its incorporation into chromatin, the basic subunit of chromatin being the nucleosome. A nucleosome is composed of 147 base pairs of DNA coiled around an octamer of histone proteins, two molecules each of histone H2A, H2B, H3 and H4. Histone H1 associates with chromatin outside the core octamer unit and regulates higher order chromatin structure.
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Chromatin and chromosomes undergo dramatic and dynamic changes in organization in response to a myriad of cellular signals. Chromosomes condense and relax during the cell division process. Damaged DNA adopts a unique structure that facilitates repair. Critical to cell function, most of the genome must remain in a transcriptionally silent state, save for specific combinations of genes, which vary significantly between cell types. These processes must be tightly regulated to maintain the integrity of the genome and the proper function of the cell.
Multiple mechanisms exist within the nucleus that allow the function and organization of the genome to be dynamically regulated, responding rapidly to different signals or inputs. Most notably, though, a large body of evidence dating back forty years and more has accumulated to indicate that post-translational modification of histones is crucial to all genome-based activity (see Kouzarides, 2007). The recruitment of transcriptional regulators and chromosomal proteins brings along enzymes that modify (by either addition or removal) specific functional groups on histones, and these dynamic addition and subtraction events have profound effects on the structure and function of chromatin.
Histone Acetylation and Transcription
The most widely studied histone modification is acetylation. Histones are covalently modified at the epsilon-amino group of conserved lysines by a class of enzymes called histone acetyltransferases (HATs). HATs come in two flavors, cytoplasmic and nuclear. The cytoplasmic HATs (e.g. Hat1) acetylate histones prior to nuclear localization and chromatin assembly, whereas the nuclear HATs acetylate histones in a manner associated with transcription and other DNAdependent processes. Some of the earliest observations made by Allfrey and colleagues linked histone acetylation to transcription (Allfrey et al., 1964).
A large amount of descriptive and correlational evidence accumulated in the thirty years after this initial work, until the identification of the first nuclear histone acetyltransferase, Tetrahymena p55, provided the first mechanistic link between histone acetylation and transcriptional activation (Brownell et al., 1996). The yeast homologue of p55, Gcn5, had previously been identified as an important transcriptional co-activator, a protein recruited to gene promoters prior to transcription and necessary for activation. Further solidifying the connection between histone acetylation and transcription, it was determined that the HAT activity of Gcn5 is required for its ability to activate transcription (Candau et al., 1997; Kuo et al., 1998). Biochemical analysis of a variety of proteins and protein complexes involved in transcriptional activation led to the identification of a large number of histone acetyltransferases, strengthening the hypothesis that histone acetylation is tightly connected to (and likely part and parcel of) transcriptional activation (reviewed in Roth and Denu, 2001).
Additional support for this hypothesis came from the opposite side of the histone acetylation coin, histone deacetylation. Contemporaneous with the identification of Gcn5 as the first HAT, the mammalian homologue of a known transcriptional repressor from yeast, Rpd3, was identified as the first enzyme that removes epsilon-acetylation from histones, a histone deacetylase, or HDAC (Taunton et al., 1996). Running parallel to the advances made with HATs, a wide variety of HDACs and HDAC-containing protein complexes were identified and linked to the repression of transcription (reviewed Yang and Seto, 2003).
While the connection between histone acetylation and transcriptional activation has been known for some time, it has only recently been established that histone acetylation is involved in the process of transcriptional elongation. Whereas promoters of active genes are highly acetylated, the coding regions of genes need to be maintained in a hypoacetylated state to prevent the aberrant initiation of transcription. A histone methyltransferase (Set2) that travels with the elongating RNA polymerase marks the coding sequence with a unique modification, histone H3 methylated at lysine 36. A histone deacetylase complex (Rpd3S) is recruited and specifically deacetylates coding region chromatin through the recognition of lysine 36 by the chromodomain protein, Eaf3, a subunit of Rpd3S (Carrozza et al., 2005; reviewed in Lee and Shilatifard, 2007).
Lys 16: An Acetylation Site with a Unique Molecular Identity
With regard to transcriptional activation, the acetylation of specific lysines appears to be less important than the overall acetylation level of histone proteins on the whole. There appears to be a functional redundancy between lysine residues, with no single lysine being more or less critical than the next. Indeed, HAT enzymes exhibit broad substrate specificity, although are apparently limited by which histone they are capable of acetylating. The exception to this rule is lysine 16 of histone H4, the acetylation and deacetylation of which have specific roles in important transcriptional processes (reviewed in Shia et al., 2006).
In yeast, acetylation of histone H3 at lysine 16 is required to maintain the boundaries between euchromatin (open and transcriptionally competent regions of chromatin) and heterochromatin (closed and transcriptionally inert). In Drosophila, lysine 16 acetylation is involved in the process of dosage compensation, the coordinate upregulation of genes on the single male X chromosome that equalizes gene expression with that of genes on the two X chromosomes in females. In humans, the loss of lysine 16 acetylation appears to be a hallmark of specific types of cancer. The common thread between these findings may be found in the results of a recent study employing a unique biochemical method to study histone acetylation. Using a peptide-ligation method to synthesize recombinant chromatin acetylated homogeneously, it was determined that acetylation of lysine 16 inhibits the formation of higher-order chromatin. This result suggests that the unique role played by lysine 16 is to serve as a central regulator of chromatin structure (Shogren-Knaak et al., 2006).
Genome Integrity, DNA Replication & DNA Damage Repair
There is a recent and growing body of evidence linking histone acetylation to DNA replication and repair. One of the first studies suggesting a link between acetylation and DNA repair came from a genetic study of histone H4 acetylation in yeast. A yeast strain containing mutations in the four amino-terminal lysines of histone H4 has severely compromised genome integrity, leading to continual activation of the DNA damage repair checkpoint (Megee et al., 1995). A more recent study showed that this same yeast strain exhibits a dramatically reduced ability to repair double-strand breaks. It was determined that the histone acetyltransferase complex NuA4 was recruited to the site of double-strand breaks. Efficient repair of DNA damage was shown to require the catalytic subunit of the NuA4, Esa1, a HAT enzyme that acetylates histone H4. Thus, not only are proper levels of histone H4 acetylation required for maintaining genome integrity, it appears that acetylation of histone H4 participates directly in one or more steps required to repair broken chromatin (Bird et al., 2002).
The involvement of histone acetylation in DNA damage repair extends to mammals as well. The mammalian Esa1 homologue, TIP60, is involved with many aspects of the DNA damage repair pathway (reviewed in Squatrito et al., 2006). In addition to a direct role in the repair of DNA damage similar to that of Esa1 (Murr et al., 2006), TIP60 is also involved in the activation of the DNA damage repair pathway through the acetylation and activation of the ATM kinase (Sun et al., 2005). TIP60 also acetylates the DNA damage-specific histone variant, H2AX, in response to DNA damage. Acetylation of H2AX leads to its subsequent ubiquitylation and remodeling of chromatin near the break, facilitating DNA repair (Ikura et al., 2007). The fascinating connection between histone acetylation and DNA damage repair has been made even more tantalizing by the identification of the importance of a single acetylation site in the process. This same single acetylation event is also required for DNA replication and chromatin assembly, which are associated with replicationcoupled DNA repair. Li and colleagues found that acetylation of lysine 56 in yeast plays a specific role in chromatin assembly, dependant upon chromatin assembly factor-1 (CAF-1), a histone chaperone complex (Li et al., 2008). It was determined that acetylation of lysine 56 increases the affinity of CAF-1 for histone H3, thus improving the efficiency of chromatin assembly.
In a related study, it was found that acetylation of lysine 56 (and downstream chromatin assembly) is linked to proper cell cycle re-entry subsequent to double-strand DNA break repair (Chen et al., 2008). The histone chaperone Asf1 (acting at a point before CAF-1) stimulates the acetylation of histone H3 at lysine 56 prior to its transfer to the nucleus and incorporation into chromatin. Lysine 56 acetylation is required for the timely deactivation of the DNA damage checkpoint after DNA repair is complete, allowing cells to re-enter the cell cycle. This suggests that the presence of H3 acetylated at lysine 56 is a hallmark of the properly re-assembled chromatin at the site of a newly repaired double-strand break, allowing the inactivation of the DNA damage checkpoint. However, in separate studies it was determined that unregulated lysine 56 acetylation is deleterious to cells and leads to spontaneous DNA damage (Celic et al., 2008; Haldar and Kamakaka, 2008).
It is possible that this is also the case in mammals as well as in yeast, as hyperacetylation of histones leads to the activation of the ATM DNA damage checkpoint protein (Bakkenist and Kastan, 2003). In addition to the specific role of acetylation at lysine 56 of histone H3 to facilitate chromatin assembly and serve as a marker for newly repaired chromatin, global acetylation levels must be kept at an intermediate, highly regulated level in order to maintain proper genome structure.
Involved as it is in the mechanism of DNA replication and chromatin assembly, there is evidence that histone acetylation also plays a role in the timing of DNA replication and replication origin activity. In general, increases in histone acetylation of chromatin surrounding an origin of replication tend to cause the origin to initiate replication earlier, compared to when the origin is within hypoacetylated chromatin (reviewed in Weinreich et al., 2004). Origins located near or within heterochromatin replicate late, but this timing can be accelerated by artificially increasing the acetylation state of histones near the origin. As origin firing occurs throughout S-phase, rather than clustered at one time point, it is likely that individual origin firing is influenced by the local chromatin environment surrounding the origin (Vogelauer et al., 2002).
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