Understanding nucleosome dynamics and their links to gene expression and DNA replication

Abstract

Advances in genomics technology have provided the means to probe myriad chromatin interactions at unprecedented spatial and temporal resolution. This has led to a profound understanding of nucleosome organization within the genome, revealing that nucleosomes are highly dynamic. Nucleosome dynamics are governed by a complex interplay of histone composition, histone post-translational modifications, nucleosome occupancy and positioning within chromatin, which are influenced by numerous regulatory factors, including general regulatory factors, chromatin remodellers, chaperones and polymerases. It is now known that these dynamics regulate diverse cellular processes ranging from gene transcription to DNA replication and repair.

Figure 1. Nucleosome organization as a combination of nucleosome occupancy and positioning

a | Nucleosomes can be found at conserved distances from genomic loci such as the transcription start site (TSS) and origins of replication. The blue lines represent the typical occupancy and positioning of nucleosomes relative to common genomic positions. The peaks and valleys correspond to locations of high and low nucleosome occupancy, respectively. The width and narrowness of the peaks correspond to the relative positioning of those nucleosomes. The arrows correspond to predicted dyads. Just downstream of the nucleosome-free region (NFR) (in the direction of transcription) a well-positioned nucleosome, termed ‘+1’ is present. The +1 nucleosome serves as the downstream border of the NFR. A well-positioned −1 nucleosome forms the upstream border of the NFR. Nucleosomes form well-positioned arrays downstream of the +1 nucleosome into the bodies of genes. However, this positioning dissipates further into gene bodies, becoming ‘fuzzy’. Origins of replication are also characterized by well-positioned nucleosomes that flank NFRs, which encompass an autonomous replicating sequence (ARS) consensus motif. b | Nucleosome occupancy is defined as the probability of a nucleosomes being present over a specific genomic region within a population of cells and is often measured in sequencing-based experiments by the number of aligned sequencing reads mapped to this region. Nucleosome positioning is defined as the probability of a nucleosome reference point (for example, a dyad) being at a specific genomic coordinate relative to surrounding coordinates. Good nucleosome positioning can be biologically interpreted as a nucleosome dyad occurring at the same genomic coordinate every time it is present. Poor positioning or ‘fuzziness’ can be interpreted as a nucleosome dyad occupying a range of positions within the same general footprint of an entire nucleosome. Nucleosome occupancy and positioning are independent metrics of nucleosome organization.

Figure 2. Nucleosome occupancy as a function of histone turnover

a | The Swr1 remodelling complex (SWR-C) targets the well-positioned +1 and −1 nucleosomes flanking nucleosome-free regions (NFRs) of active promoters, which typically contain the H2A.Z variant of the H2A histone^,. SWR-C mediates the exchange of H2A–H2B to H2A.Z–H2B. Subunits of the SWR-C bind within the NFR and assist with positioning the rest of the complex over adjacent nucleosomes. Once positioned, ATP hydrolysis is used to dislodge the resident H2A–H2B histone dimer. Subunits of the SWR-C capture chaperone-bound H2A.Z–H2B and deliver it to the now vacated H2A–H2B site. The reversal of this process may be mediated by INO80 ( REF. 55). INO80-mediated H2A.Z exchange results in increased turnover of the full nucleosome octamer, possibly owing to increased exposure of H3–H4 during H2A.Z–H2B eviction^,. b | In an inducible histone turnover system, newly synthesized histones are typically distinguished from existing histones by a distinct epitope tag (magenta). The zero time point is typically a time point of tagged-histone induction. The rate of incorporation of the epitope tagged-histones into chromatin is then monitored over time.

Figure 3. Determinants of nucleosome positioning

a | Nucleosomal DNA, particularly at +1 and −1 positions with respect to transcription start sites (TSSs), is modestly enriched with AA, TT, AT, and TA dinucleotides at a 10 bp periodicity and GG, CC, GC, and CG dinucleotides offset by 5 bp from those dinucleotides and also possessing a 10 bp periodicity^,,,,. These periodicities result in the formation of preferred bends in DNA, thereby defining how DNA wraps around the histone octamer. Point 0 represents the dyad with preference for AA, TT, AT and TA dinucleotides, and preference in 10 bp periodicity at 15, 25, 35, 45, 55 and 65 bp from the dyad are represented by the red lines. The GG, CC, GC and CG dinucleotide preferences are represented by the short blue lines at points 10, 20, 30, 40, 50, 60 and 70 bp from the dyad. b | The position of DNA relative to the histone octamer defines its rotational setting. DNA sequences possessing the nucleotide periodicities shown in part a possess a strong rotational setting, which means that they typically wrap around the octamer in a preferred orientation. However, for a single rotational setting, multiple translational settings can exist, typically in 10 bp intervals, reflecting the nucleotide preference periodicities in the DNA. c | The switch/sucrose non-fermenting (SWI/SNF) chromatin remodelling complex helps to regulate various cellular processes, including stress responses, such as heat shock. It may do so by altering the accessibility of promoter DNA to regulatory proteins and the transcription machinery (contributing to both activation and repression of transcription)^,. d | The highly abundant and essential remodelling the structure of chromatin (RSC) complex acts on poly(dA:dT) tracts, which are common in Saccharomyces cerevisiae promoters. RSC regulates the size of the nucleosome-free region in these promoters by repositioning and/or evicting nucleosomes located near poly(dA:dT) tracts in an ATP-dependent manner^,,. e | The chromodomain helicase DNA binding 1 (CHD1) remodeller produces regularly spaced nucleosome arrays in vitro using ATP. The targeting of nucleosome remodelling activity of CHD1 is suspected to be linked to transcription^,, which explains its apparent lack of DNA-binding specificity in genome-wide in vitro nucleosome assembly assays. f | In S. cerevisiae the imitation SWI (ISWI) family of chromatin remodellers are enriched at the 5′ ends of gene bodies. In vitro experiments have revealed that ISW1a and ISW2 are crucial for establishing proper in vivo nucleosome spacing. Spacing is further improved with the addition of general transcription factors (GRFs), which supports the predicted role of GRFs as anchor points, or boundary elements, to guide the directionality for ISW1a. See also part e. Pol II, RNA polymerase II.

Figure 4. Examination of nucleosome substructures and methods to detect them

a | There is now evidence that besides the canonical histone octamer, a variety of subnucleosomal structures exist. The hexasome comprises a H3–H4 tetramer and a single H2A–H2B dimer, and is a functional intermediate, possibly resulting from the removal a H2A–H2B dimer by RNA polymerase II (Poll II) during transcription or through its intrinsic dynamics imparted by chromatin remodellers. A prenucleosome contains the full complement of the octamer but is wrapped by only 80 bp of DNA. Half-nucleosomes (also known as hemisomes) comprise a single copy of each histone core particle. There is evidence that half-nucleosomes might accumulate at certain regions of DNA. Tetrasomes are H3–H4 tetramers, which exist as possible intermediates during DNA replication (nucleosomes which are partially disassembled ahead of the replication fork and partially reassembled after the fork has passed)^,. b | The extent of micrococcal nuclease (MNase) digestion affects the relative enrichment of nucleosomes at certain positions. Lighter digestion (with lower amounts of MNase) has the potential to enrich for open DNA not bound by nucleosomes, especially when chromatin immunoprecipitation is not used in conjunction. Heavier MNase digestion tends to better resolve true nucleosomes at the potential cost of losing the ability to detect fragile nucleosomes. Independent of size-selection criteria during experimental design, both current sequencing technology and PCR include an intrinsic DNA-size bias for library construction and sequencing cluster formation. c | During transcription, Pol II must transcribe nucleosomal DNA. To facilitate this, nucleosomes are at least partially unravelled during transcription and an H2A–H2B dimer is removed forming a temporary hexasome. The FACT (facilitates chromatin transcription) complex is believed to interact with the free H2A–H2B dimers and assists with the nucleosome disassembly and reassembly^–.

Figure 5. Nucleosome dynamics of transcription at ribosomal protein genes

Ribosomal protein genes are among highly transcribed genes in Saccharomyces cerevisiae although they are silenced immediately when cells are stressed. Repressor/activator protein 1 (Rap1) binds upstream of the transcription start site (TSS) at ribosomal protein genes and forms the anchoring point for binding transcription factors forkhead-like 1 (Fhl1), high mobility group 1 (Hmo1), interacts with forkhead 1 (Ifh1) and split finger protein (Sfp1). Together, these transcription factors enable the assembly of the pre-initiation complex (PIC) at the TSS. Upon heat-shock stress, components of the PIC are evicted and chromatin remodellers are recruited to the +1 nucleosome. Transcription factors are then removed from the promoter, and the +1 nucleosome is then shifted upstream into the promoter region^,. This supports the model whereby the specific position of the +1 nucleosome can contribute to overall gene expression by either allowing or interfering with PIC formation.

Figure 6. Nucleosome dynamics of DNA replication

a | Active origins of replication in Saccharomyces cerevisiae are characterized by an ARS consensus sequence (ACS) located in a nucleosome-depleted region flanked by well-positioned nucleosomes containing the H2A.Z histone variant. Many non-functional ACSs exist in the S. cerevisiae genome and contain occluding nucleosomes. This implicates nucleosome positioning in regulating functional origins of replication. b | The models for H3–H4 tetramer inheritance during replication include the conservative, semi-conservative and dispersive models. The conservative model is defined as the deposition of the entire tetramer on one daughter strand and another newly synthesized H3–H4 tetramer is deposited on another. In the semi-conservative model, the tetramer is broken into its component H3–H4 dimers and each dimer is placed on a daughter strand with a new H3–H4 dimer. The dispersive model is a combination of the conservative and semi-conservative model wherein the tetramer is either maintained or split. Evidence showing certain tetramers evenly split between daughter strands and other tetramers entirely segregated to a single strand suggest a guided dispersive model wherein tetramers are subject to different outcomes depending on their histone variant composition. c | Nucleosomes in gene bodies feature poor positioning immediately after replication, which is likely to be due to some level of random deposition immediately following the replication fork. At highly transcribed genes, this positioning is rapidly re-ordered, leading to even nucleosomal arrays with the +1 nucleosome containing H2A.Z variant, indicating that transcription supports nucleosome positioning. TSS, transcription start site.