Although chromatin assembly, structure, and function are poorly understood, it is essential to gain a clear understanding of these processes as they are intrinsic to the analyses of human diseases, including many forms of cancer that involve defects in the chromatin utilizing process. What we do know about chromatin assembly following DNA replication is that initiation occurs with the import of newly synthesized histones into the nucleus. A rapid multi-step process immediately follows this event which involves the initial deposition of a heterotetramer of histones H3 and H4, followed by incorporation of two histone H2A and H2B heterodimers to complete the nucleosome. ATP is then required, in addition to other chromatin assembly factors, to establish a regular spacing of assembled nucleosomes. The incorporation of linker histones accompanies the folding of the complete nucleofilament to form the seloniod, or the 30nm fiber. This is followed by further undefined folding events that produce the complete structural organization of chromatin within the nucleus.
Regulation of Chromatin Assembly
The process of assembling DNA into chromatin is regulated by several factors and other defined and undefined events that occur in the cell. Some of the presently defined events include the variation that occurs within the basic constituents involved in chromatin organization, ie: histone variants and modifications. In addition, regulation of chromatin assembly is dependent on whether it occurs during DNA replication or outside of DNA replication. As such, the concept of regulating chromatin assembly must be analyzed with regard to the different events, players, and factors that are involved in each different aspect of chromatin assembly.
Histone variants and modifications - During the first steps of chromatin assembly, incorporation of histones variants and modifications to histones, such as acetylation, have been shown to alter chromatin structure and activity. An intricate theory has been proposed which suggests that there is a genome-wide mechanism for regulation of chromatin structure. This theory proposes that the combination of histone amino-terminal modifications, termed the "histone code," represents a fundamental regulatory mechanism that impacts many chromatin-templated processes. This "histone code" has been hypothesized to function in two ways: as a signal for repression or activation of a specific gene in response to cellular demands and as a marking system that could be used during cellular differentiation to determine specific chromatin states that are inherited in an epigenetic manner. Specifically, the "histone code" predicts that modifications of histone tails may induce interaction affinities for chromatin-associated proteins and that the modifications may be interdependent and generate various combinations on any one nucleosome. In addition, it has been proposed that the properties of higher order chromatin, euchromatin and heterochromatin, may be dependent on the local concentration and combination of differentially modified nucleosomes, as reviewed in. However, the "histone code" has yet to be fully deciphered and is still the topic of intense research.
DNA-replication dependent assembly - The majority of chromatin assembly occurs after DNA replication, which requires that the chromatin structure is briefly disrupted at the replication fork to allow the replication machinery to pass. In fact, it has been established that the entire chromatin structure must be disassembled in order for DNA replication to occur. In the first step, the parental histone proteins are removed, perhaps by being displaced as the replication fork moves through the DNA, followed by the rapid reassembly onto the daughter strands. In order for full assembly of the newly replicated DNA to occur, newly synthesized histones must also be deposited onto the daughter strands. However, in order to fully unravel the chromatin assembly process, the mechanism of how these histones are being deposited and removed of the DNA needs to be addressed.
The mechanism of histone deposition during DNA replication has historically been investigated using in vivo studies that follow the fate of newly synthesized histones that are incorporated into chromatin during DNA replication. To address the mechanisms of chromatin assembly during DNA replication in vitro, a system was developed to mimic what actually occurs in the cell. It was determined that cytosol extracts derived from human cells support complete and authentic replication of SV40 origincontaining plasmid DNA in the presence of purified SV40 T antigen. The cytosol extract, which cannot assemble the DNA on its own, provides all of the cellular proteins required for DNA replication, except for CAF-1 (Chromatin Assembly Factor 1), as well as the four core histones. It is important to note that the cytosolic histones correspond to the newly synthesized histone pool and thus are the precursors required for chromatin assembly. The system was validated by the fact that the assembly proceeded in a step-wise manner and was coupled specifically to replicated DNA.
Chromatin Assembly Proteins
DNA has to be organized and packaged into higher order structures in order to be contained within the nucleus. To achieve this, DNA is coiled around a histone octomer to form a nucleosome, a fundamental building block of chromatin. New chromatin must be assembled during DNA replication and repair. This process is essential in human cells, as in the absence of proper chromatin assembly DNA synthesis cannot be completed. Gene transcription is also directly related to chromatin formation since chromatin structure affects gene activation and repression: heterochromatin is tightly packaged and causes silencing of genes, whereas euchromatin is packaged more loosely and allows gene transcription to occur. Thus, chromatin plays a crucial role in all DNA metabolic processes.
Nucleosomes are made up of two coils (approximately 146 base pairs) of DNA wrapped around four core histones: two H2A/H2B dimers and a H3/H4 tetramer. The four histones have corresponding histone chaperones, or chromatin assembly factors, responsible for neutralizing highly charged histones and depositing them onto DNA. Histones H2A/H2B have two histone chaperones: nucleosome assembly protein 1, NAP-1, a cytoplasmic-nuclear histone transfer factor, which functions during DNA synthesis, and histone regulator A, HIRA (Hir1 and Hir2 in Saccharomyces cerevisiae), proteins that act during transcription. Histone H3/H4 tetramers are deposited onto DNA by chromatin assembly factor 1 (CAF-I) which functions during both DNA replication and repair. Histones H3 and H4 also bind anti-silencing function 1 (Asf1) and form the replication-coupling assembly factor (RCAF) complex, which is thought to synergize with CAF-I. These two latter chromatin assembly factor complexes are the focus of the studies presented in this dissertation.