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S phase (Synthesis phase) is the phase of the cell cycle in which DNA is replicated, occurring between G1 phase and G2 phase. [1] Since accurate duplication of the genome is critical to successful cell division, the processes that occur during S-phase are tightly regulated and widely conserved.
Entry into S-phase is controlled by the G1 restriction point (R), which commits cells to the remainder of the cell-cycle if there is adequate nutrients and growth signaling. [2] This transition is essentially irreversible; after passing the restriction point, the cell will progress through S-phase even if environmental conditions become unfavorable. [2]
Accordingly, entry into S-phase is controlled by molecular pathways that facilitate a rapid, unidirectional shift in cell state. In yeast, for instance, cell growth induces accumulation of Cln3 cyclin, which complexes with the cyclin dependent kinase CDK2. [3] The Cln3-CDK2 complex promotes transcription of S-phase genes by inactivating the transcriptional repressor Whi5. [3] Since upregulation of S-phase genes drive further suppression of Whi5, this pathway creates a positive feedback loop that fully commits cells to S-phase gene expression. [3]
A remarkably similar regulatory scheme exists in mammalian cells. [3] Mitogenic signals received throughout G1-phase cause gradual accumulation of cyclin D, which complexes with CDK4/6. [3] Active cyclin D-CDK4/6 complex induces release of E2F transcription factor, which in turn initiates expression of S-phase genes. [3] Several E2F target genes promote further release of E2F, creating a positive feedback loop similar to the one found in yeast. [3]
Throughout M phase and G1 phase, cells assemble inactive pre-replication complexes (pre-RC) on replication origins distributed throughout the genome. [4] During S-phase, the cell converts pre-RCs into active replication forks to initiate DNA replication. [4] This process depends on the kinase activity of Cdc7 and various S-phase CDKs, both of which are upregulated upon S-phase entry. [4]
Activation of the pre-RC is a closely regulated and highly sequential process. After Cdc7 and S-phase CDKs phosphorylate their respective substrates, a second set of replicative factors associate with the pre-RC. [4] Stable association encourages MCM helicase to unwind a small stretch of parental DNA into two strands of ssDNA, which in turn recruits replication protein A (RPA), an ssDNA binding protein. [4] RPA recruitment primes the replication fork for loading of replicative DNA polymerases and PCNA sliding clamps. [4] Loading of these factors completes the active replication fork and initiates synthesis of new DNA.
Complete replication fork assembly and activation only occurs on a small subset of replication origins. All eukaryotes possess many more replication origins than strictly needed during one cycle of DNA replication. [5] Redundant origins may increase the flexibility of DNA replication, allowing cells to control the rate of DNA synthesis and respond to replication stress. [5]
Since new DNA must be packaged into nucleosomes to function properly, synthesis of canonical (non-variant) histone proteins occurs alongside DNA replication. During early S-phase, the cyclin E-Cdk2 complex phosphorylates NPAT, a nuclear coactivator of histone transcription. [6] NPAT is activated by phosphorylation and recruits the Tip60 chromatin remodeling complex to the promoters of histone genes. [6] Tip60 activity removes inhibitory chromatin structures and drives a three to ten-fold increase in transcription rate. [1] [6]
In addition to increasing transcription of histone genes, S-phase entry also regulates histone production at the RNA level. Instead of polyadenylated tails, canonical histone transcripts possess a conserved 3` stem loop motif that selective binds to Stem Loop Binding Protein (SLBP). [7] SLBP binding is required for efficient processing, export, and translation of histone mRNAs, allowing it to function as a highly sensitive biochemical "switch". [7] During S-phase, accumulation of SLBP acts together with NPAT to drastically increase the efficiency of histone production. [7] However, once S-phase ends, both SLBP and bound RNA are rapidly degraded. [8] This immediately halts histone production and prevents a toxic buildup of free histones. [9]
Free histones produced by the cell during S-phase are rapidly incorporated into new nucleosomes. This process is closely tied to the replication fork, occurring immediately in “front” and “behind” the replication complex. Translocation of MCM helicase along the leading strand disrupts parental nucleosome octamers, resulting in the release of H3-H4 and H2A-H2B subunits. [10] Reassembly of nucleosomes behind the replication fork is mediated by chromatin assembly factors (CAFs) that are loosely associated with replication proteins. [4] [11] Though not fully understood, the reassembly does not appear to utilize the semi-conservative scheme seen in DNA replication. [11] Labeling experiments indicate that nucleosome duplication is predominantly conservative. [11] [10] The paternal H3-H4 core nucleosome remains completely segregated from newly synthesized H3-H4, resulting in the formation of nucleosomes that either contain exclusively old H3-H4 or exclusively new H3-H4. [10] [11] “Old” and “new” histones are assigned to each daughter strand semi-randomly, resulting in equal division of regulatory modifications. [10]
Immediately after division, each daughter chromatid only possesses half the epigenetic modifications present in the paternal chromatid. [10] The cell must use this partial set of instructions to re-establish functional chromatin domains before entering mitosis.
For large genomic regions, inheritance of old H3-H4 nucleosomes is sufficient for accurate re-establishment of chromatin domains. [10] Polycomb Repressive Complex 2 (PRC2) and several other histone-modifying complexes can "copy" modifications present on old histones onto new histones. [10] This process amplifies epigenetic marks and counters the dilutive effect of nucleosome duplication. [10]
However, for small domains approaching the size of individual genes, old nucleosomes are spread too thinly for accurate propagation of histone modifications. [10] In these regions, chromatin structure is probably controlled by incorporation of histone variants during nucleosome reassembly. [10] The close correlation seen between H3.3/H2A.Z and transcriptionally active regions lends support to this proposed mechanism. [10] Unfortunately, a causal relationship has yet to be proven. [10]
During S-phase, the cell continuously scrutinizes its genome for abnormalities. Detection of DNA damage induces activation of three canonical S-phase "checkpoint pathways" that delay or arrest further cell cycle progression: [12]
In addition to these canonical checkpoints, recent evidence suggests that abnormalities in histone supply and nucleosome assembly can also alter S-phase progression. [14] Depletion of free histones in Drosophila cells dramatically prolongs S-phase and causes permanent arrest in G2-phase. [14] This unique arrest phenotype is not associated with activation of canonical DNA damage pathways, indicating that nucleosome assembly and histone supply may be scrutinized by a novel S-phase checkpoint. [14]
The cell cycle, or cell-division cycle, is the series of events that take place in a cell that causes it to divide into two daughter cells. These events include the duplication of its DNA and some of its organelles, and subsequently the partitioning of its cytoplasm, chromosomes and other components into two daughter cells in a process called cell division.
In biology, histones are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn are wrapped into 30-nanometer fibers that form tightly packed chromatin. Histones prevent DNA from becoming tangled and protect it from DNA damage. In addition, histones play important roles in gene regulation and DNA replication. Without histones, unwound DNA in chromosomes would be very long. For example, each human cell has about 1.8 meters of DNA if completely stretched out; however, when wound about histones, this length is reduced to about 90 micrometers (0.09 mm) of 30 nm diameter chromatin fibers.
A nucleosome is the basic structural unit of DNA packaging in eukaryotes. The structure of a nucleosome consists of a segment of DNA wound around eight histone proteins and resembles thread wrapped around a spool. The nucleosome is the fundamental subunit of chromatin. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a histone octamer. Each histone octamer is composed of two copies each of the histone proteins H2A, H2B, H3, and H4.
Histone acetyltransferases (HATs) are enzymes that acetylate conserved lysine amino acids on histone proteins by transferring an acetyl group from acetyl-CoA to form ε-N-acetyllysine. DNA is wrapped around histones, and, by transferring an acetyl group to the histones, genes can be turned on and off. In general, histone acetylation increases gene expression.
E2F is a group of genes that encodes a family of transcription factors (TF) in higher eukaryotes. Three of them are activators: E2F1, 2 and E2F3a. Six others act as suppressors: E2F3b, E2F4-8. All of them are involved in the cell cycle regulation and synthesis of DNA in mammalian cells. E2Fs as TFs bind to the TTTCCCGC consensus binding site in the target promoter sequence.
Histone H4 is one of the five main histone proteins involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and a long N-terminal tail, H4 is involved with the structure of the nucleosome of the 'beads on a string' organization. Histone proteins are highly post-translationally modified. Covalently bonded modifications include acetylation and methylation of the N-terminal tails. These modifications may alter expression of genes located on DNA associated with its parent histone octamer. Histone H4 is an important protein in the structure and function of chromatin, where its sequence variants and variable modification states are thought to play a role in the dynamic and long term regulation of genes.
Histone H2B is one of the 5 main histone proteins involved in the structure of chromatin in eukaryotic cells. Featuring a main globular domain and long N-terminal and C-terminal tails, H2B is involved with the structure of the nucleosomes.
Histone methylation is a process by which methyl groups are transferred to amino acids of histone proteins that make up nucleosomes, which the DNA double helix wraps around to form chromosomes. Methylation of histones can either increase or decrease transcription of genes, depending on which amino acids in the histones are methylated, and how many methyl groups are attached. Methylation events that weaken chemical attractions between histone tails and DNA increase transcription because they enable the DNA to uncoil from nucleosomes so that transcription factor proteins and RNA polymerase can access the DNA. This process is critical for the regulation of gene expression that allows different cells to express different genes.
The G1/S transition is a stage in the cell cycle at the boundary between the G1 phase, in which the cell grows, and the S phase, during which DNA is replicated. It is governed by cell cycle checkpoints to ensure cell cycle integrity and the subsequent S phase can pause in response to improperly or partially replicated DNA. During this transition the cell makes decisions to become quiescent, differentiate, make DNA repairs, or proliferate based on environmental cues and molecular signaling inputs. The G1/S transition occurs late in G1 and the absence or improper application of this highly regulated checkpoint can lead to cellular transformation and disease states such as cancer.
Eukaryotic DNA replication is a conserved mechanism that restricts DNA replication to once per cell cycle. Eukaryotic DNA replication of chromosomal DNA is central for the duplication of a cell and is necessary for the maintenance of the eukaryotic genome.
Histone acetylation and deacetylation are the processes by which the lysine residues within the N-terminal tail protruding from the histone core of the nucleosome are acetylated and deacetylated as part of gene regulation.
Chromatin remodeling is the dynamic modification of chromatin architecture to allow access of condensed genomic DNA to the regulatory transcription machinery proteins, and thereby control gene expression. Such remodeling is principally carried out by 1) covalent histone modifications by specific enzymes, e.g., histone acetyltransferases (HATs), deacetylases, methyltransferases, and kinases, and 2) ATP-dependent chromatin remodeling complexes which either move, eject or restructure nucleosomes. Besides actively regulating gene expression, dynamic remodeling of chromatin imparts an epigenetic regulatory role in several key biological processes, egg cells DNA replication and repair; apoptosis; chromosome segregation as well as development and pluripotency. Aberrations in chromatin remodeling proteins are found to be associated with human diseases, including cancer. Targeting chromatin remodeling pathways is currently evolving as a major therapeutic strategy in the treatment of several cancers.
Histone H4 is a protein that in humans is encoded by the HIST4H4 gene.
Histone H4 is a protein that in humans is encoded by the HIST2H4A gene.
Histone H4 is a protein that in humans is encoded by the HIST1H4A gene.
FACT is a heterodimeric protein complex that affects eukaryotic RNA polymerase II transcription elongation both in vitro and in vivo. It was discovered in 1998 as a factor purified from human cells that was essential for productive, in vitro Pol II transcription on a chromatinized DNA template.
The retinoblastoma protein is a tumor suppressor protein that is dysfunctional in several major cancers. One function of pRb is to prevent excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide. When the cell is ready to divide, pRb is phosphorylated, inactivating it, and the cell cycle is allowed to progress. It is also a recruiter of several chromatin remodeling enzymes such as methylases and acetylases.
Memory is commonly referred to as the ability to encode, store, retain and subsequently recall information and past experiences in the human brain. This process involves many proteins, one of which is the Histone-binding protein RbAp48, encoded by the RBBP4 gene in humans.
Chromatin assembly factor-1 (CAF-1) is a protein complex — including Chaf1a (p150), Chaf1b (p60), and p48 subunits in humans, or Cac1, Cac2, and Cac3, respectively, in yeast— that assembles histone tetramers onto replicating DNA during the S phase of the cell cycle.
H4K20me is an epigenetic modification to the DNA packaging protein Histone H4. It is a mark that indicates the mono-methylation at the 20th lysine residue of the histone H4 protein. This mark can be di- and tri-methylated. It is critical for genome integrity including DNA damage repair, DNA replication and chromatin compaction.