Smith, S. & Stillman, B. Stepwise assembly of chromatin during DNA replication in vitro. EMBO J. 10, 971-980

Cold Spring Harbor Laboratory, NY 11724.
The EMBO Journal (Impact Factor: 10.43). 05/1991; 10(4):971-80.
Source: PubMed


A cell free system that supports replication-dependent chromatin assembly has been used to determine the mechanism of histone deposition during DNA replication. CAF-I, a human cell nuclear factor, promotes chromatin assembly on replicating SV40 DNA in the presence of a crude cytosol replication extract. Biochemical fractionation of the cytosol extract has allowed separation of the chromatin assembly reaction into two steps. During the first step, CAF-I targets the deposition of newly synthesized histones H3 and H4 to the replicating DNA. This reaction is dependent upon and coupled with DNA replication, and utilizes the newly synthesized forms of histones H3 and H4, which unlike bulk histone found in chromatin, do not bind to DNA by themselves. The H3/H4-replicated DNA complex is a stable intermediate which exhibits a micrococcal nuclease resistant structure and can be isolated by sucrose gradient sedimentation. In the second step, this replicated precursor is converted to mature chromatin by the addition of histones H2A and H2B in a reaction that can occur after DNA replication. The requirement for CAF-I in at least the first step of the reaction suggests a level of cellular control for this fundamental process.

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Available from: Bruce W Stillman, May 14, 2014
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    • "CAF-1 and Rtt106 each receive two H3-H4 heterodimers from Asf1, from which they facilitate the formation of the H3-H4 tetramer [17-19]. In the next step, the replication-dependent histone chaperones, such as CAF-1, transfer newly-synthesized (H3-H4)2 tetramers to the newly-replicated DNA [20] (Figure 1). Currently, our understanding of chromatin assembly after DNA replication, described here, is limited to the incorporation of newly-synthesized histones, which carry their own pattern of deposition-specific histone modifications that are rapidly unmodified following chromatin assembly. "
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    ABSTRACT: DNA replication is a highly conserved process that accurately copies the genetic information from one generation to the next. The processes of chromatin disassembly and reassembly during DNA replication also have to be precisely regulated to ensure that the genetic material is compactly packaged to fit into the nucleus while also maintaining the epigenetic information that is carried by the histone proteins bound to the DNA, through cell divisions. Half of the histones that are deposited during replication are from the parental chromatin and carry the parental epigenetic information, while the other half of the histones are newly-synthesized. It has been of growing interest to understand how the parental pattern of epigenetic marks is re-established on the newly-synthesized histones, in a DNA sequence-specific manner, in order to maintain the epigenetic information through cell divisions. In this review we will discuss how histone chaperone proteins precisely coordinate the chromatin assembly process during DNA replication. We also discuss the recent evidence that histone-modifying enzymes, rather than the parental histones, are themselves epigenetic factors that remain associated with the DNA through replication to re-establish the epigenetic information on the newly-assembled chromatin.
    Epigenetics & Chromatin 10/2013; 6(1):32. DOI:10.1186/1756-8935-6-32 · 5.33 Impact Factor
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    • "Chromatin assembly factor 1 (CAF1) is a canonical histone H3/H4 chaperone that mediates replication-coupled nucleosome assembly (Gaillard et al., 1996; Kaufman et al., 1995; Smith and Stillman, 1991; Verreault et al., 1996). CAF1 comprises three evolutionarily conserved subunits, named after their apparent molecular weights. "
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    ABSTRACT: One of the most dramatic forms of chromatin reorganization occurs during spermatogenesis, when the paternal genome is repackaged from a nucleosomal to a protamine-based structure. We assessed the role of the canonical histone chaperone CAF1 in Drosophila spermatogenesis. In this process, CAF1 does not behave as a complex, but its subunits display distinct chromatin dynamics. During histone-to-protamine replacement, CAF1-p180 dissociates from the DNA while CAF1-p75 binds and stays on as a component of sperm chromatin. Association of CAF1-p75 with the paternal genome depends on CAF1-p180 and protamines. Conversely, CAF1-p75 binds protamines and is required for their incorporation into sperm chromatin. Histone removal, however, occurs independently of CAF1 or protamines. Thus, CAF1-p180 and CAF1-p75 function in a temporal hierarchy during sperm chromatin assembly, with CAF1-p75 acting as a protamine-loading factor. These results show that CAF1 subunits mediate the assembly of two fundamentally different forms of chromatin.
    Cell Reports 06/2013; 4(1). DOI:10.1016/j.celrep.2013.06.002 · 8.36 Impact Factor
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    • "In addition, although the transcription of the standard histone octamer components is cell-cycle-dependent [47] and loading of these histones on to newly synthesized daughter DNA strands is carried out by components associated with the replication fork {e.g. CAF-1 (chromatin assembly factor 1) [48]}, there is no evidence that histone H1 loading is synchronized with the cell cycle. Therefore it is possible that the passage of the replication fork serves to dissociate histone H1 from DNA and so prevents stable interaction leading to the formation of heterochromatin (see Figure 2), although to the best of our knowledge this mechanism linking the frequency of S-phase entry to pluripotency has not yet been directly investigated. "
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    ABSTRACT: PSCs (pluripotent stem cells) possess two key properties that have made them the focus of global research efforts in regenerative medicine: they have unlimited expansion potential under conditions which favour their preservation as PSCs and they have the ability to generate all somatic cell types upon differentiation (pluripotency). Conditions have been defined in vitro in which pluripotency is maintained, or else differentiation is favoured and is directed towards specific somatic cell types. However, an unanswered question is whether or not the core cell cycle machinery directly regulates the pluripotency and differentiation properties of PSCs. If so, then manipulation of the cell cycle may represent an additional tool by which in vitro maintenance or differentiation of PSCs may be controlled in regenerative medicine. The present review aims to summarize our current understanding of links between the core cell cycle machinery and the maintenance of pluripotency in ESCs (embryonic stem cells) and iPSCs (induced PSCs).
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