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Histone variants at the transcription start-site Tatiana A. Soboleva*, Maxim Nekrasov*, Daniel P. Ryan*, and David J. Tremethick The John Curtin School of Medical Research, The Australian National University, PO Box 334, Canberra, ACT 2601, Australia
The function of a eukaryotic cell crucially depends on accurate gene transcription to ensure the right genes are expressed whereas unrequired genes are repressed. Therefore, arguably, one of the most important regions in the genome is the transcription start-site (TSS) of protein-coding and non-coding genes. Until recently, understanding the mechanisms that define the location of the TSS and how it is created has largely focused on the role of DNA sequence-specific transcription factors. However, within the nucleus of a eukaryotic cell, transcription occurs in a highly compacted nucleosomal environment, and it is becoming clear that accessibility of the TSS is a key controlling step in transcriptional regulation. It has traditionally been thought that transcription can only proceed once the nucleosomes at the TSS have been evicted. New work suggests otherwise, however, and the focus of this review is to challenge this belief. Nucleosome occupancy at promoters The recruitment of general transcription factors and RNA polymerase II (Pol II) to the TSS occurs within the context of a highly compressed DNA template in the form of chromatin. Chromatin is built from nucleosomes, the universal repeating protein–DNA complex in all eukaryotic cells. Each nucleosome comprises two tight superhelical turns of DNA wrapped around a disk-shaped protein assembly of eight histone molecules (two molecules each of histone H2A, H2B, H3, and H4). Nucleosomes are connected by short linker DNA segments (20–50 bp) to form long arrays, which undergo short-range intra- and longrange inter-nucleosomal interactions to form largely disorganized chromatin fibers [1]. There are at least two structural levels by which chromatin can inhibit the association of the general transcription machinery with the TSS. At the first level, a single nucleosome located at the TSS can create a formidable barrier to the binding of general transcription factors and the large Pol II enzyme [2–4]. At the second level, the TSS can be rendered even more inaccessible by being buried deep within a chromatin fiber [5,6]. Nevertheless, an intriguing property of chromatin is that
Corresponding author: Tremethick, D.J. ([email protected]). Keywords: chromatin; histone variants; transcription start site; transcriptional regulation; unstable nucleosomes; nucleosome mapping. * These authors contributed equally to this work. 0168-9525/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tig.2014.03.002
the loss of only a single nucleosome within an array can inhibit chromatin compaction at a local level [7]. Genome-wide mapping of nucleosomes for a variety of eukaryotes has provided an unprecedented amount of new information concerning how nucleosomes are organized along the DNA, particularly at functionally important regions such as promoters, and how this nucleosome arrangement changes prior to and following transcriptional activation. Such promoter mapping studies in human cells [8–10], as well as for many other eukaryotes [11–15], revealed an apparent nucleosome-free region (NFR) at an active TSS followed by a strongly positioned ‘+1’ nucleosome immediately downstream of the TSS. The size and location of the NFR is dependent upon the class of promoter (Box 1), and does not remain constant because it can change during the course of the cell cycle [16,17]. The strongly positioned +1 nucleosome at an active promoter is then followed downstream by a uniform or phased arrangement of nucleosomes into the gene body. This general genome-wide phasing of nucleosomes is directional Glossary ATP-dependent chromatin remodeling activity: an enzymatic activity that hydrolyzes ATP to facilitate access of nucleosomal DNA by locally disrupting DNA–histone contacts, moving the histone octamer to a different DNA position, evicting or replacing different histone subtypes, or completely displacing the histone octamer from the DNA. CCCTC-binding factor protein (CTCF): a zinc-finger protein that binds to the core sequence CCCTC. Its major role is to regulate the 3D structure of chromatin by bringing together distant binding sites thus forming chromatin loops to regulate gene activity. It also demarcates the boundaries between active and inactive chromatin. CpG island: a sequence of DNA is described as a CpG island when the CG dinucleotide is over-represented compared to the genomic average; 40–70% of human promoters contain a CpG island. DNase I hypersensitive site: a short region of chromatin that is characterized by a dramatically enhanced sensitivity to DNA cleavage by the nuclease DNase I. Such sites are viewed as accessible regions of chromatin and are created by the binding of transcription factors, but it is also important to note that the DNase I enzyme recognizes sequence-dependent structural variations of the DNA double helix (and therefore does not necessarily indicate that the site is more accessible). Mapping of DNase I hypersensitive sites is a commonly used method to identify gene regulatory elements. Histone post-translational modifications: all histones undergo post-translational modifications whereby specific amino acid residues, particularly in histone tails, become chemically modified. These modifications, in a combinatorial manner, can affect histone–DNA interactions, histone–histone interactions, and the affinity for other proteins that regulate chromatin function. Specific histone modifications are linked to either an active or silenced chromatin state. RNA polymerase II (Pol II) pausing: in metazoans Pol II can initiate transcription but stall or pause 25–30 bp downstream of the TSS at the boundary of the +1 nucleosome. This pausing appears to be a common rate-limiting step in transcription and, importantly, the transition of Pol II from a paused to a productive elongating state represents a key regulatory step in the transcription process.
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Review Box 1. Dichotomy of Pol II promoters Complex eukaryotic Pol II promoters can be classified according to their patterns of transcription initiation, which is defined by the location and the number of TSSs utilized. These promoter-specific differences in transcription initiation are mirrored by significant changes to their nucleosome organization. There are three classes of TSSs, broad (where initiation events span a region from 30 to over 100 nt), sharp (usually a single TSS), and an intermediate class that display similarities and differences between these two classes [79,80]. The broad class of TSSs usually contain a CpG island is usually associated with non-tissue specific genes such as constitutive housekeeping genes, and is also associated with low transcriptional plasticity [79,80]. The sharp class by contrast, often contains only a single major TSS, and is associated with tissue-specific genes and/or with inducible genes that respond to extracellular stimuli [79,80]. Therefore this class is associated with greater transcriptional plasticity. Promoters in this class contain a TATA box (consensus sequence TATAAA), which is located 30 nt upstream of the TSS. These two different classes of promoters also display major differences at the chromatin level. The broad/CpG island class displays a static chromatin architecture with a broad and strong apparent NFR at their TSSs followed by well-positioned nucleosomes in the transcribed regions. By contrast, the chromatin architecture of the low CpG/sharp TSS class displays a more dynamic nucleosome occupancy with a less pronounced NFR. The broad/CpG island class also tends to be more enriched with H2A.Z compared to the sharp class [79,80]. For both classes of promoters it is the overall abundance of promoter nucleosomes that is correlated with transcription levels, whereas their respective patterns of nucleosome occupancy are important for proper transcriptional regulation. The low CpG/sharp, but not the high CpG/broad promoter class, is also more dependent upon chromatin remodeling activities for transcriptional activation [79,80]. More recently, a study identified a third class of Pol II promoters that has a combination of features of both the broad and sharp classes [80]. This same study also concluded that the relationship between promoter classes and chromatin architecture is far more complex than originally thought, and involves many other factors including core promoter sequence features and insulator preferences and, thus, cannot be simply explained by just the presence or absence of a CpG island alone.
and apparently dependent upon transcription because it is not observed upstream of the TSS [8,9,12,18]. It is known, however, that specific positioning of nucleosomes does occur on the promoter of particular individual genes to regulate transcription factor access [15,19–22]. A repressed gene, by contrast, lacks this phasing of nucleosomes and, as expected, the TSS is covered by nucleosomes [8,9,12]. Therefore, transcriptional activation has been proposed to involve nucleosome eviction from the TSS followed by the phasing of downstream nucleosomes. Several different mechanisms have been proposed to explain the strong positioning of the +1 nucleosome and the subsequent ordered arrangement of downstream nucleosomes. In budding yeast poly (dA:dT) tracts at the TSS may act as a nucleosome ‘repulsion’ element to help position the +1 nucleosome [18,23]. In more complex eukaryotes it has been proposed that the Pol II enzyme that is either poised (see Glossary) or stalled at the TSS can act as a barrier to position the +1 nucleosome [8,9,12]. In turn, the highly positioned +1 nucleosome may itself passively align downstream nucleosomes at regular intervals, most likely aided by ATP-dependent chromatin remodeling activities [24] and/or by continued rounds of transcriptional elongation 2
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[8,9,12,18,25]. Interestingly, in budding yeast the TSS resides 13 bp within the 50 border of the +1 nucleosome [14,26], whereas in humans the 50 end maps 40 or 10 bp downstream of the TSS of active or inactive genes, respectively [8]. Further, a detailed comparison of genome-wide nucleosome positioning at the TSS between the distantly related budding and fission yeasts revealed that different mechanisms for nucleosome positioning are utilized, arguing for a surprising divergence of nucleosome-positioning mechanisms during the evolution of eukaryotic genomes [12]. An important aspect in interpreting nucleosome-mapping studies in mice and humans is that most maps produced thus far are only ‘average’ patterns of nucleosome occupancy of many genes, unlike the high-resolution nucleosome maps that have been produced for yeast (e.g., see [17,27]). This obviously is because genomes of complex eukaryotes are considerably larger than those of yeasts, and current high-throughput DNA sequencing technologies cannot produce the required nucleosome coverage in a cost-effective manner. Furthermore, if the starting material is a heterogeneous mixture of cell types, then the average maps are not only averaged across genes but also represent an average for the different cell types. Nevertheless, recent studies have produced high-coverage human maps [9,10], and new technical developments are beginning to overcome this limitation [28]. The picture that is emerging is that, at an individual gene level, the nucleosome organization is much more complex and gene specific than the average pattern described above [28]. This may not be surprising given the dichotomy of promoters (Box 1). H2A.Z (H2A histone family member Z) regulates transcription in multiple and competing ways H2A.Z, encoded by two genes in humans, H2A.Z.1 and H2A.Z.2 (H2AFZ and H2AFV) [29], is an evolutionarily conserved, and metazoan essential, histone variant of the H2A class. H2A.Z has been implicated in a diverse range of functions including transcriptional activation and repression (both in plants and animals) [20,21,24,30–38], suppression of antisense transcription [39], DNA repair [40], and chromosome stability, cohesion, and segregation [40– 43]. In mammals it is crucial for early development [44] and for the regulation of gene expression during embryonic and extraembryonic stem cell differentiation [34,41,42]. Adding to the complexity of its function, an alternatively spliced form of H2A.Z.2 (H2A.Z.2.2) exists in humans, which is highly enriched in the brain [43]. A particularly striking observation is that, for all eukaryotes examined, the special +1 nucleosome positioned immediately downstream of the NFR is marked by the incorporation of H2A.Z (Figure 1A) [14,17,23, 26,44]. Most commonly, this +1 H2A.Z-containing nucleosome is associated with transcriptionally poised genes and genes that are expressed at a low to a moderate level [17,37,45,46]. In budding yeast it is also found on inactive promoters [23]. A second H2A.Z-containing nucleosome can also be located immediately upstream of the NFR (the –2 position, Figure 1A; note that in simple eukaryotes this nucleosome has been referred to as the –1 nucleosome, see
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Figure 1. Models for different types of chromatin configurations at the TSS. Depending upon the physiological context and stage of the cell cycle, there are at least four different types of nucleosomal arrangements at the transcription start-site (TSS). Heterotypic H2A.Z/H2A nucleosomes have been detected at the TSS for genes active in trophoblast stem cells at G1 phase (A) but not at the S and M phases of the cell cycle (B). Because the locations of H2A.Z/H2A and H3.3/H2A.Z appear to be the same in trophoblast stem cells and Hela cells, we propose that heterotypic H2A.Z/H2A nucleosomes at the TSS also contain histone H3.3. The presence of a heterotypic H2A.Z/H2A nucleosome at the TSS may be a common feature of many higher eukaryotic genes because it is also found at particular genes active during mouse spermatogenesis (C). However, despite having a H2A.Z/H2A nucleosome at the TSS for genes active in the testis, the histone composition of its +1 nucleosome differs from that of a +1 nucleosome in mouse trophoblast stem cells because it is heterotypic rather than being homotypic. A fourth type of TSS chromatin structure exists, which is displayed in the mouse testis, where H2A.Lap 1 rather than H2A.Z occupies the TSS of an active promoter (D). Further, when H2A.Lap1 is present at the TSS, the +1 nucleosome is a canonical H2A/H2A and not a homotypic H2A.Z/H2A–containing nucleosome. Note that in simple eukaryotes, nucleosome positions –2 and –1 are referred to positions –1 and 0, respectively (e.g., see [81]).
Figure 1 legend) such that the TSS is flanked on both sides by H2A.Z. Although this H2A.Z–TSS–H2A.Z chromatin organization appears to be common in both lower and higher eukaryotes (with the exception of Drosophila [14]), promoter-type (Box 1) and cell type-specific differences occur. For example, H2A.Z is absent from the +1 nucleosome for many genes active in the mouse testis (see below and Figures 1D and 2) [47]. A major unanswered question is – what is the function of H2A.Z at the +1 nucleosome? In both budding and fission yeasts the presence of H2A.Z is negatively correlated with transcription [38,45,48], whereas in humans and mice it is positively correlated with transcriptional activity (for low to moderately expressed genes) [17,46]. This major difference appears to reflect when H2A.Z is incorporated into the +1 nucleosome. In yeast, H2A.Z is deposited when the promoter is in a repressed or basal state [23,38,48]. Following transcriptional activation and productive elongation by RNA Pol II, H2A.Z is displaced from the +1 nucleosome, thus yielding
a negative correlation between H2A.Z and transcriptional activity. By contrast, a positive correlation for humans and mice implies that H2A.Z is deposited during the transcriptional activation process (most likely immediately before or concomitantly with Pol II). However, analogous to the situation in yeast, as the transcription rate increases H2A.Z is displaced from the +1 nucleosome, thereby yielding a negative correlation [17,46]. These observations raise the possibility that the formation of two well-positioned H2A.Z nucleosomes, flanking the TSS, helps to create a NFR rendering the TSS accessible to Pol II. This hypothesis is supported by the observation that the recruitment of human Pol II to promoters is inhibited globally by knockdown of H2A.Z expression [33]. However, studies in budding yeast have shown that it is the prior formation of the NFR that actually promotes H2A.Z deposition rather than the other way around [49]. Taken together, this suggests that H2A.Z may have a more direct role in recruiting Pol II [33]. 3
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Gene expression, RNA metabolic processes, spermiogenesis H3.1/H3.3 /
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Figure 2. During the same stages of spermatogenesis the transcription start-sites (TSS) of active genes can selectively contain either a H2A.Z–H2A heterotypic- or a H2A.Lap1-containing nucleosome. (A) Shown is the relative level of mRNA expression of H2A.Z and H2A.Lap1 during different stages of mouse spermatogenesis [46,82]. H2A.Z, blue; H2A.Lap1, red. The final developmental stage of the testis is shown under the graph: 10 day old (do) testis, spermatogonia (Sg); 12 do testis, early spermatocyte (pre-pachytene Early Spc); 19 do testis, pachytene spermatocyte (Late Spc); 24 do testis, early round spermatids (Early RS); 28 do testis, late round spermatids (Late RS). Total transcriptional activity is indicated above the graph as a black line, with the wider black area showing the highest level of gene transcription between the late pachytene and late round spermatid stages when H2A.Lap1 is expressed. (B) A model describing how H2A.Z and H2A.Lap1 contribute to the regulation of TSS chromatin organization during spermatogenesis. Throughout spermatogenesis, active genes involved in general metabolic processes contain a heterotypic H2A.Z/H2A nucleosome at their TSS [27]. When H2A.Lap1 is expressed, it is specifically targeted to those genes involved in gene expression and RNA metabolic processes replacing H2A.Z at the TSS [27]. H2A.Lap1 is also targeted to genes that were previously silent but specifically activated during spermiogenesis (which begins at day 24). Some of these activated genes are located on the inactive X chromosome [47]. Abbreviations: chr., chromosome; Nuc, nucleosome.
The yeast multisubunit SWR (Swi2/Snf2-related) complex [SRCAP (Snf2-related CREB activator protein) and p400 in humans] is responsible for replacing H2A with H2A.Z in nucleosomes, a process that requires ATP [50– 52]. SWR1 prefers to bind to long nucleosome-free DNA and it is proposed that this preference can explain why H2A.Z incorporation follows the formation of a NFR, given the view that the NFR is a region free of histones [53,54]. Intriguingly, the INO80 (inositol requiring 80) complex, whose enzyme activity is responsible for removing H2A.Z from nucleosomes, is also believed to be targeted to the 4
+1 H2A.Z nucleosome by recognizing the adjacent NFR [54]. Therefore, this cycle of reciprocal H2A.Z–H2A exchange at the +1 nucleosome catalyzed by SWR1 and INO80, could potentially regulate transcription elongation through the +1 nucleosome by influencing its stability and dynamics. In higher eukaryotes, additional players may be involved in the H2A.Z–H2A exchange process. Most recently, ANP32E (acidic nuclear phosphoprotein 32 kilodalton e) was identified as a novel H2A.Z chaperone that can specifically remove H2A.Z from the nucleosome. In its absence there is a genome-wide accumulation of H2A.Z particularly
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Box 2. Histone variants H3.3 and H2A.Bbd Histone variants are non-allelic forms of the canonical histones that vary in their primary sequence. They are expressed at low levels compared to their conventional counterparts (
Review
Histone variants at the transcription start-site Tatiana A. Soboleva*, Maxim Nekrasov*, Daniel P. Ryan*, and David J. Tremethick The John Curtin School of Medical Research, The Australian National University, PO Box 334, Canberra, ACT 2601, Australia
The function of a eukaryotic cell crucially depends on accurate gene transcription to ensure the right genes are expressed whereas unrequired genes are repressed. Therefore, arguably, one of the most important regions in the genome is the transcription start-site (TSS) of protein-coding and non-coding genes. Until recently, understanding the mechanisms that define the location of the TSS and how it is created has largely focused on the role of DNA sequence-specific transcription factors. However, within the nucleus of a eukaryotic cell, transcription occurs in a highly compacted nucleosomal environment, and it is becoming clear that accessibility of the TSS is a key controlling step in transcriptional regulation. It has traditionally been thought that transcription can only proceed once the nucleosomes at the TSS have been evicted. New work suggests otherwise, however, and the focus of this review is to challenge this belief. Nucleosome occupancy at promoters The recruitment of general transcription factors and RNA polymerase II (Pol II) to the TSS occurs within the context of a highly compressed DNA template in the form of chromatin. Chromatin is built from nucleosomes, the universal repeating protein–DNA complex in all eukaryotic cells. Each nucleosome comprises two tight superhelical turns of DNA wrapped around a disk-shaped protein assembly of eight histone molecules (two molecules each of histone H2A, H2B, H3, and H4). Nucleosomes are connected by short linker DNA segments (20–50 bp) to form long arrays, which undergo short-range intra- and longrange inter-nucleosomal interactions to form largely disorganized chromatin fibers [1]. There are at least two structural levels by which chromatin can inhibit the association of the general transcription machinery with the TSS. At the first level, a single nucleosome located at the TSS can create a formidable barrier to the binding of general transcription factors and the large Pol II enzyme [2–4]. At the second level, the TSS can be rendered even more inaccessible by being buried deep within a chromatin fiber [5,6]. Nevertheless, an intriguing property of chromatin is that
Corresponding author: Tremethick, D.J. ([email protected]). Keywords: chromatin; histone variants; transcription start site; transcriptional regulation; unstable nucleosomes; nucleosome mapping. * These authors contributed equally to this work. 0168-9525/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tig.2014.03.002
the loss of only a single nucleosome within an array can inhibit chromatin compaction at a local level [7]. Genome-wide mapping of nucleosomes for a variety of eukaryotes has provided an unprecedented amount of new information concerning how nucleosomes are organized along the DNA, particularly at functionally important regions such as promoters, and how this nucleosome arrangement changes prior to and following transcriptional activation. Such promoter mapping studies in human cells [8–10], as well as for many other eukaryotes [11–15], revealed an apparent nucleosome-free region (NFR) at an active TSS followed by a strongly positioned ‘+1’ nucleosome immediately downstream of the TSS. The size and location of the NFR is dependent upon the class of promoter (Box 1), and does not remain constant because it can change during the course of the cell cycle [16,17]. The strongly positioned +1 nucleosome at an active promoter is then followed downstream by a uniform or phased arrangement of nucleosomes into the gene body. This general genome-wide phasing of nucleosomes is directional Glossary ATP-dependent chromatin remodeling activity: an enzymatic activity that hydrolyzes ATP to facilitate access of nucleosomal DNA by locally disrupting DNA–histone contacts, moving the histone octamer to a different DNA position, evicting or replacing different histone subtypes, or completely displacing the histone octamer from the DNA. CCCTC-binding factor protein (CTCF): a zinc-finger protein that binds to the core sequence CCCTC. Its major role is to regulate the 3D structure of chromatin by bringing together distant binding sites thus forming chromatin loops to regulate gene activity. It also demarcates the boundaries between active and inactive chromatin. CpG island: a sequence of DNA is described as a CpG island when the CG dinucleotide is over-represented compared to the genomic average; 40–70% of human promoters contain a CpG island. DNase I hypersensitive site: a short region of chromatin that is characterized by a dramatically enhanced sensitivity to DNA cleavage by the nuclease DNase I. Such sites are viewed as accessible regions of chromatin and are created by the binding of transcription factors, but it is also important to note that the DNase I enzyme recognizes sequence-dependent structural variations of the DNA double helix (and therefore does not necessarily indicate that the site is more accessible). Mapping of DNase I hypersensitive sites is a commonly used method to identify gene regulatory elements. Histone post-translational modifications: all histones undergo post-translational modifications whereby specific amino acid residues, particularly in histone tails, become chemically modified. These modifications, in a combinatorial manner, can affect histone–DNA interactions, histone–histone interactions, and the affinity for other proteins that regulate chromatin function. Specific histone modifications are linked to either an active or silenced chromatin state. RNA polymerase II (Pol II) pausing: in metazoans Pol II can initiate transcription but stall or pause 25–30 bp downstream of the TSS at the boundary of the +1 nucleosome. This pausing appears to be a common rate-limiting step in transcription and, importantly, the transition of Pol II from a paused to a productive elongating state represents a key regulatory step in the transcription process.
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Review Box 1. Dichotomy of Pol II promoters Complex eukaryotic Pol II promoters can be classified according to their patterns of transcription initiation, which is defined by the location and the number of TSSs utilized. These promoter-specific differences in transcription initiation are mirrored by significant changes to their nucleosome organization. There are three classes of TSSs, broad (where initiation events span a region from 30 to over 100 nt), sharp (usually a single TSS), and an intermediate class that display similarities and differences between these two classes [79,80]. The broad class of TSSs usually contain a CpG island is usually associated with non-tissue specific genes such as constitutive housekeeping genes, and is also associated with low transcriptional plasticity [79,80]. The sharp class by contrast, often contains only a single major TSS, and is associated with tissue-specific genes and/or with inducible genes that respond to extracellular stimuli [79,80]. Therefore this class is associated with greater transcriptional plasticity. Promoters in this class contain a TATA box (consensus sequence TATAAA), which is located 30 nt upstream of the TSS. These two different classes of promoters also display major differences at the chromatin level. The broad/CpG island class displays a static chromatin architecture with a broad and strong apparent NFR at their TSSs followed by well-positioned nucleosomes in the transcribed regions. By contrast, the chromatin architecture of the low CpG/sharp TSS class displays a more dynamic nucleosome occupancy with a less pronounced NFR. The broad/CpG island class also tends to be more enriched with H2A.Z compared to the sharp class [79,80]. For both classes of promoters it is the overall abundance of promoter nucleosomes that is correlated with transcription levels, whereas their respective patterns of nucleosome occupancy are important for proper transcriptional regulation. The low CpG/sharp, but not the high CpG/broad promoter class, is also more dependent upon chromatin remodeling activities for transcriptional activation [79,80]. More recently, a study identified a third class of Pol II promoters that has a combination of features of both the broad and sharp classes [80]. This same study also concluded that the relationship between promoter classes and chromatin architecture is far more complex than originally thought, and involves many other factors including core promoter sequence features and insulator preferences and, thus, cannot be simply explained by just the presence or absence of a CpG island alone.
and apparently dependent upon transcription because it is not observed upstream of the TSS [8,9,12,18]. It is known, however, that specific positioning of nucleosomes does occur on the promoter of particular individual genes to regulate transcription factor access [15,19–22]. A repressed gene, by contrast, lacks this phasing of nucleosomes and, as expected, the TSS is covered by nucleosomes [8,9,12]. Therefore, transcriptional activation has been proposed to involve nucleosome eviction from the TSS followed by the phasing of downstream nucleosomes. Several different mechanisms have been proposed to explain the strong positioning of the +1 nucleosome and the subsequent ordered arrangement of downstream nucleosomes. In budding yeast poly (dA:dT) tracts at the TSS may act as a nucleosome ‘repulsion’ element to help position the +1 nucleosome [18,23]. In more complex eukaryotes it has been proposed that the Pol II enzyme that is either poised (see Glossary) or stalled at the TSS can act as a barrier to position the +1 nucleosome [8,9,12]. In turn, the highly positioned +1 nucleosome may itself passively align downstream nucleosomes at regular intervals, most likely aided by ATP-dependent chromatin remodeling activities [24] and/or by continued rounds of transcriptional elongation 2
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[8,9,12,18,25]. Interestingly, in budding yeast the TSS resides 13 bp within the 50 border of the +1 nucleosome [14,26], whereas in humans the 50 end maps 40 or 10 bp downstream of the TSS of active or inactive genes, respectively [8]. Further, a detailed comparison of genome-wide nucleosome positioning at the TSS between the distantly related budding and fission yeasts revealed that different mechanisms for nucleosome positioning are utilized, arguing for a surprising divergence of nucleosome-positioning mechanisms during the evolution of eukaryotic genomes [12]. An important aspect in interpreting nucleosome-mapping studies in mice and humans is that most maps produced thus far are only ‘average’ patterns of nucleosome occupancy of many genes, unlike the high-resolution nucleosome maps that have been produced for yeast (e.g., see [17,27]). This obviously is because genomes of complex eukaryotes are considerably larger than those of yeasts, and current high-throughput DNA sequencing technologies cannot produce the required nucleosome coverage in a cost-effective manner. Furthermore, if the starting material is a heterogeneous mixture of cell types, then the average maps are not only averaged across genes but also represent an average for the different cell types. Nevertheless, recent studies have produced high-coverage human maps [9,10], and new technical developments are beginning to overcome this limitation [28]. The picture that is emerging is that, at an individual gene level, the nucleosome organization is much more complex and gene specific than the average pattern described above [28]. This may not be surprising given the dichotomy of promoters (Box 1). H2A.Z (H2A histone family member Z) regulates transcription in multiple and competing ways H2A.Z, encoded by two genes in humans, H2A.Z.1 and H2A.Z.2 (H2AFZ and H2AFV) [29], is an evolutionarily conserved, and metazoan essential, histone variant of the H2A class. H2A.Z has been implicated in a diverse range of functions including transcriptional activation and repression (both in plants and animals) [20,21,24,30–38], suppression of antisense transcription [39], DNA repair [40], and chromosome stability, cohesion, and segregation [40– 43]. In mammals it is crucial for early development [44] and for the regulation of gene expression during embryonic and extraembryonic stem cell differentiation [34,41,42]. Adding to the complexity of its function, an alternatively spliced form of H2A.Z.2 (H2A.Z.2.2) exists in humans, which is highly enriched in the brain [43]. A particularly striking observation is that, for all eukaryotes examined, the special +1 nucleosome positioned immediately downstream of the NFR is marked by the incorporation of H2A.Z (Figure 1A) [14,17,23, 26,44]. Most commonly, this +1 H2A.Z-containing nucleosome is associated with transcriptionally poised genes and genes that are expressed at a low to a moderate level [17,37,45,46]. In budding yeast it is also found on inactive promoters [23]. A second H2A.Z-containing nucleosome can also be located immediately upstream of the NFR (the –2 position, Figure 1A; note that in simple eukaryotes this nucleosome has been referred to as the –1 nucleosome, see
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A Z ?
A Z
–2 2 Nuc
+1 Nuc
A Z –240
(C) Mouse tess
H3.1/H3.3
–240
–50 0
+135
––2 2 Nuc
–1 1 Nuc
+1 Nuc
Z Z
L L
A A
Other histones
(D) Mouse tess
–240
–50 0
TSS
+135
Distance from f TSS (bp) ( p) TRENDS in Genetics
Figure 1. Models for different types of chromatin configurations at the TSS. Depending upon the physiological context and stage of the cell cycle, there are at least four different types of nucleosomal arrangements at the transcription start-site (TSS). Heterotypic H2A.Z/H2A nucleosomes have been detected at the TSS for genes active in trophoblast stem cells at G1 phase (A) but not at the S and M phases of the cell cycle (B). Because the locations of H2A.Z/H2A and H3.3/H2A.Z appear to be the same in trophoblast stem cells and Hela cells, we propose that heterotypic H2A.Z/H2A nucleosomes at the TSS also contain histone H3.3. The presence of a heterotypic H2A.Z/H2A nucleosome at the TSS may be a common feature of many higher eukaryotic genes because it is also found at particular genes active during mouse spermatogenesis (C). However, despite having a H2A.Z/H2A nucleosome at the TSS for genes active in the testis, the histone composition of its +1 nucleosome differs from that of a +1 nucleosome in mouse trophoblast stem cells because it is heterotypic rather than being homotypic. A fourth type of TSS chromatin structure exists, which is displayed in the mouse testis, where H2A.Lap 1 rather than H2A.Z occupies the TSS of an active promoter (D). Further, when H2A.Lap1 is present at the TSS, the +1 nucleosome is a canonical H2A/H2A and not a homotypic H2A.Z/H2A–containing nucleosome. Note that in simple eukaryotes, nucleosome positions –2 and –1 are referred to positions –1 and 0, respectively (e.g., see [81]).
Figure 1 legend) such that the TSS is flanked on both sides by H2A.Z. Although this H2A.Z–TSS–H2A.Z chromatin organization appears to be common in both lower and higher eukaryotes (with the exception of Drosophila [14]), promoter-type (Box 1) and cell type-specific differences occur. For example, H2A.Z is absent from the +1 nucleosome for many genes active in the mouse testis (see below and Figures 1D and 2) [47]. A major unanswered question is – what is the function of H2A.Z at the +1 nucleosome? In both budding and fission yeasts the presence of H2A.Z is negatively correlated with transcription [38,45,48], whereas in humans and mice it is positively correlated with transcriptional activity (for low to moderately expressed genes) [17,46]. This major difference appears to reflect when H2A.Z is incorporated into the +1 nucleosome. In yeast, H2A.Z is deposited when the promoter is in a repressed or basal state [23,38,48]. Following transcriptional activation and productive elongation by RNA Pol II, H2A.Z is displaced from the +1 nucleosome, thus yielding
a negative correlation between H2A.Z and transcriptional activity. By contrast, a positive correlation for humans and mice implies that H2A.Z is deposited during the transcriptional activation process (most likely immediately before or concomitantly with Pol II). However, analogous to the situation in yeast, as the transcription rate increases H2A.Z is displaced from the +1 nucleosome, thereby yielding a negative correlation [17,46]. These observations raise the possibility that the formation of two well-positioned H2A.Z nucleosomes, flanking the TSS, helps to create a NFR rendering the TSS accessible to Pol II. This hypothesis is supported by the observation that the recruitment of human Pol II to promoters is inhibited globally by knockdown of H2A.Z expression [33]. However, studies in budding yeast have shown that it is the prior formation of the NFR that actually promotes H2A.Z deposition rather than the other way around [49]. Taken together, this suggests that H2A.Z may have a more direct role in recruiting Pol II [33]. 3
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Transcriponal acvity
(A)
Key:
Relave mRNA expression
H2A.Z H2A.Lap1
10-d Sg
12-d Early Spc
19-d Late Spc
(B)
24-d Early RS
28-d 28 d Late RS
General metabolic processes –2 Nuc
H3.1/H3.3 /
Z Z
–1 Nuc
+1 Nuc
A Z
A Z
? –240
–50 0
+135
–2 Nuc
Z Z
–1 Nuc N
+1 Nuc
–2 Nuc
–1 Nuc
+1 Nuc
A Z ?
A Z
Z Z
L L
A A
+135
–240
–50 0
–240 Key:
A
Z
L
H2A
H2A.Z
H2A.Lap1
Other histones
–50 0
X chr. gene acvaon
Gene expression, RNA metabolic processes, spermiogenesis H3.1/H3.3 /
+135
TSS TRENDS in Genetics
Figure 2. During the same stages of spermatogenesis the transcription start-sites (TSS) of active genes can selectively contain either a H2A.Z–H2A heterotypic- or a H2A.Lap1-containing nucleosome. (A) Shown is the relative level of mRNA expression of H2A.Z and H2A.Lap1 during different stages of mouse spermatogenesis [46,82]. H2A.Z, blue; H2A.Lap1, red. The final developmental stage of the testis is shown under the graph: 10 day old (do) testis, spermatogonia (Sg); 12 do testis, early spermatocyte (pre-pachytene Early Spc); 19 do testis, pachytene spermatocyte (Late Spc); 24 do testis, early round spermatids (Early RS); 28 do testis, late round spermatids (Late RS). Total transcriptional activity is indicated above the graph as a black line, with the wider black area showing the highest level of gene transcription between the late pachytene and late round spermatid stages when H2A.Lap1 is expressed. (B) A model describing how H2A.Z and H2A.Lap1 contribute to the regulation of TSS chromatin organization during spermatogenesis. Throughout spermatogenesis, active genes involved in general metabolic processes contain a heterotypic H2A.Z/H2A nucleosome at their TSS [27]. When H2A.Lap1 is expressed, it is specifically targeted to those genes involved in gene expression and RNA metabolic processes replacing H2A.Z at the TSS [27]. H2A.Lap1 is also targeted to genes that were previously silent but specifically activated during spermiogenesis (which begins at day 24). Some of these activated genes are located on the inactive X chromosome [47]. Abbreviations: chr., chromosome; Nuc, nucleosome.
The yeast multisubunit SWR (Swi2/Snf2-related) complex [SRCAP (Snf2-related CREB activator protein) and p400 in humans] is responsible for replacing H2A with H2A.Z in nucleosomes, a process that requires ATP [50– 52]. SWR1 prefers to bind to long nucleosome-free DNA and it is proposed that this preference can explain why H2A.Z incorporation follows the formation of a NFR, given the view that the NFR is a region free of histones [53,54]. Intriguingly, the INO80 (inositol requiring 80) complex, whose enzyme activity is responsible for removing H2A.Z from nucleosomes, is also believed to be targeted to the 4
+1 H2A.Z nucleosome by recognizing the adjacent NFR [54]. Therefore, this cycle of reciprocal H2A.Z–H2A exchange at the +1 nucleosome catalyzed by SWR1 and INO80, could potentially regulate transcription elongation through the +1 nucleosome by influencing its stability and dynamics. In higher eukaryotes, additional players may be involved in the H2A.Z–H2A exchange process. Most recently, ANP32E (acidic nuclear phosphoprotein 32 kilodalton e) was identified as a novel H2A.Z chaperone that can specifically remove H2A.Z from the nucleosome. In its absence there is a genome-wide accumulation of H2A.Z particularly
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Box 2. Histone variants H3.3 and H2A.Bbd Histone variants are non-allelic forms of the canonical histones that vary in their primary sequence. They are expressed at low levels compared to their conventional counterparts (
