Sangamo Biosciences Point Richmond Technical Center Richmond, California 94804
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INTRODUCTION |
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In this review, we address some textbook schematic-inspired misconceptions about the structure of the nucleosome and of chromatin that are relevant to transcriptional control and elaborate on the structural impediments created by chromatin for access to DNA by nonhistone factors. We highlight mechanisms whereby such NHRs as the glucocorticoid, estrogen, and thyroid hormone receptors overcome this impediment and present a hypothetical scenario for gene activation by NHRs in vivo. Various types of chromatin disruption and modification phenomena that occur in vivo and the chromatin-modifying machines thought to be responsible for such modifications are described. We list the evidence implicating such machines in NHR function and conclude by reviewing recent data that illuminate the remarkably dynamic nature of in vivo gene regulation by NHRs.
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THE MISREPRESENTED NUCLEOSOME |
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1. From a structural standpoint, the histone octamer is not a spoola
more adequate analogy would be to imagine DNA wound around a Rubiks
cube: a dynamic, pliable, internally nonhomogeneous particle (4). All
four core histones, H2A, H2B, H3, and H4, contain a distinctive lysine-
and arginine-rich COOH domain that forms a histone fold motif (an
extended central -helix flanked on either side by a loop and two
shorter
-helices). Histones H3 and H4 heterodimerize via a handshake
interaction of their histone fold motifs, and this dimer then
associates with a second such entity to form an H3/H4 tetramer.
In vivo, this particle is deposited onto nascent DNA by
dedicated molecular chaperones (5, 6) and binds to approximately 130 bp
of DNA. The resulting seminucleosome has interesting properties (7)
immediately relevant to transcriptional regulation by NHRs (see below)
and normally exists only on newly replicated DNA (i.e. when
proliferating cells are in S phase). Histones H2A and H2B are added, by
a distinct set of chaperones, to the DNA-bound
(H3/H4)2, thus completing the assembly process
and compacting approximately 146 bp of DNA (in the nucleosome, H2A and
H2B also interact via a handshake). The x-ray crystal structure of the
core histone octamer has been solved (8) and reveals the extended
network of electrostatic and, surprisingly, hydrophobic interactions
between the DNA and the octamer.
2. The junction between (H3/H4)2 and (H2A/H2B)2 is a delicate one, a feature most likely exploited not only in assembly (6), but also, possibly, in the disruption of chromatin by regulators (9). The consequences of such disruptions are not immediately apparent from thread on a spool drawings because they obscure the extraordinary structural stress that B-form DNA undergoes when assembled onto the histone octamer. The 1.7 turns made by DNA in a nucleosome feature the sharpest curvature of the DNA backbone that is thermodynamically feasible, coupled with an underwinding of the double helix (10); thus, the DNA is not complacently wound on the spoolit is extensively bent and twisted out of its familiar B-form shape. Thus, while the union of DNA and core histones in a structurally intact nucleosome is a stable one, it is easy to conceptualize how disruptions or modifications of the histone octamer that destabilize the protein-DNA contacts offer the DNA a window of opportunity to spring back into a less torsionally stressful conformation, a phenomenon with regulatory consequences (see below).
3. Limitations of experimental technique as well as an understandable
desire to present simplified drawings routinely omit from the
nucleosome one of its least understood and most important structural
features: the NH2-terminal tails of the core
histones. External to the histone-fold domain that lies inside the DNA
superhelix, the histone tails account for approximately 25% of the
overall protein mass of the nucleosome and are exceedingly lysine rich.
While known to reside outside of the core nucleosome, they are,
unfortunately, invisible to current x-ray crystallographic analysis
(8). Thus, while nothing is known about the tails secondary
structure, two things are certain: the tails are quite long and in
fully extended form could, potentially, project far beyond the core
particle (11); in addition, they contain a total of 44 lysine residues
per nucleosome (the -NH2 group of lysine has a
pKa of 10.8, i.e. at physiological pH,
the average nucleosome is surrounded by 43.99 charged lysine
side-chains). In a tail-centric view of the nucleosome, therefore, it
resembles an octopus, with eight long tentacles/tails enveloping the
DNA wound around its body in an web of positive charge.
As discovered in the 1960s (12), however, this charge can be
neutralized via acetylation of the lysine residue:
R-(CH2)3-NH3+
+ AcCoA
R-(CH2)3-NH-CO-CH3
Some 40 yr later, we know that every eukaryotic taxon that has been
studied contains a vast number of enzymes, histone acetyltransferases
(HATs), capable of performing this reaction, and have extensive data
connecting such enzymesand their functional antagonists, histone
deacetylases (HDACs)to every aspect of genomic function (13),
including transcriptional control effected by NHRs (3). The invisible
tails, therefore, are quite functionally conspicuous, as discussed in
detail below.
Before describing how NHRs make use of chromatin modification and disruption to effect gene control, it is useful to briefly consider how these regulators are able to interact with their target loci in the context of mature (i.e. unremodeled) chromatin.
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ACCESS GRANTED: HOW NHRs ASSOCIATE WITH CHROMATIN |
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Two technical terms are commonly used to describe the exact way in
which a particular DNA stretch is assembled into nucleosomes. The core
histone octamer forms the most stable contacts with approximately 146
bp of DNA; the addition of linker histone compacts an additional
approximately 15 bp, and about 20 bp of DNA are free (not associated
with core histones). This internucleosomal gap (also known as "linker
DNA") creates an inherent aperiodicity in the way the genome is bound
into chromatin: some sequences are associated with core histones, and
others are located in the linker. The term "translational
positioning" is used to describe where exactly the gaps are in a
particular locus. For example, the following 520-bp segment stretch of
DNA (each letter corresponds to 20 bp) ABCDEFGHIJKLMNOPQRSTUVWXYZ can
form nucleosomes in many different translational frames (DNA bound to
histones is double underlined, while DNA in the linker is in
boldface):
It is important to note that while some loci in the genome are associated with a specific translational frame of histone octamer occupancy, on many loci nucleosomes assume relatively random translational positions (14, 15). For our hypothetical alphabet locus, however, it is clear that frame 1 is a welcoming environment for a regulator seeking to bind sites B and K, but frames 2 and 3 are not (unless, of course, the nucleosome is no obstacle to the regulator; see below). There is direct evidence that a significant number of transcriptional regulators that operate in eukaryotic genomes cannot associate with their binding sites in the context of a mature nucleosome: such, for instance, is the case for TATA box-binding protein (TBP) (16), where the requirement for a significant structural distortion in the width of the DNA minor groove effected by TBP binding (17) is incompatible with the structure of the DNA in the nucleosome. A less severe impairment is presented by the well studied Zn-finger regulator TFIIIA (18, 19): it cannot bind to its site if the translational frame puts it in the center of a nucleosome (e.g. site J in frame 2 above), can stably bind if the DNA is in a linker (site J, frame 3) and can bind with reduced affinity if the DNA is on the edge of the nucleosome (site J, frame 1).
Even if the translational frame is roughly the same on two different
chromosomes, the exact way in which the DNA is wound around the
histones also has major consequences for the capacity of nonhistone
regulators to access their sites. Consider the following fragment of
the Xenopus laevis TRßA gene promoter (20):
The underlined portion of this DNA is a
classical thyroid hormone receptor response element (TRE): two
AGGA/TCA half-sites
(boldface) spaced by 4 bp and arranged in the form of a
direct repeat, with the left half-site bound by the retinoid X receptor
(RXR), and the right half-site bound by the thyroid hormone receptor
(TR)(21). The inevitable consequence of winding DNA around a histone
octamer is that the major groove side of certain sequences will face
toward the histones, and some will face toward the solution, thus
yielding a particular rotational frame, i.e. a specific
orientation of sequences in the double helix relative to the octamer.
Here, for example, is the TRßA sequence in three distinct rotational
frames (sequences located in a major groove rotated toward solution are
underlined, while sequences located in a major groove that
is rotated toward the histones are not; the TRE half-sites are in
boldface):
A remarkable property of many NHRs is their ability to stably bind nucleosomal DNA in vitro and in vivo in a manner largely irrespective of translational frame; thus, quite unlike TBP or TFIIIA, NHRs experience only a modest reduction in affinity for their response elements when they are associated with core histones. For example, glucocorticoid receptor (GR) can associate with its response element on the mouse mammary tumor virus long terminal repeat (MMTV LTR) presented in multiple translational frames in vitro and in vivo (22, 23, 24); an important point, however, is that the rotational frame has to be such that the core residues required for GR binding to DNA are facing toward solution (25); for instance, frames 1 and 3 above expose the response element to the receptor, while frame 2 rotates it toward the histones.
When a favorable rotational frame exists, an association of the NHR with its response element both in vitro and in vivo has been demonstrated for GR action on the MMTV LTR (22), for TR on the TRßA promoter (26, 27), for estrogen receptor (ER) on the pS2 promoter (28), and for retinoic acid receptor (RAR) on the RARß promoter (29, 30). It is thought that the Zn-finger DNA-binding domain of one NHR molecule invades the major groove (31) that lies exposed on the surface of the octamer, and the second molecule of the receptor in the dimer tolerates some inevitable distortion of base-protein contacts both in the case of homodimers for class I NHRs (31) and heterodimers in the case of class II NHRs (21). It is not surprising, therefore, that the known structural anisotropy of the DNA within the nucleosome (10) leads to distinct effects of altering rotational phasing of GR response elements on GR binding when the location of the response element is shifted within the nucleosome (25); it is quite likely that the two neighboring double helices of DNA that come into proximity over segments of the nucleosome also contribute to such effects via steric hindrance.
The past 2 yr have seen the recapitulation of transcriptional control by GR (32), progesterone receptor (PR) (33), ER (34), vitamin D receptor (VDR) (35), and RAR (36) in various in vitro systems with purified receptor on chromatin templates. While these systems are not fully biochemically defined Drosophila embryo extract is used to assemble chromatin, and HeLa nuclear extract is added to endow the system with transcriptional competencythey represent an exciting and important advance in the field and will undoubtedly allow a fine-resolution dissection of receptor action on chromatin (see below), including the initial step of receptor interaction with a nucleosomal template.
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THE HOUSE THAT NHRs BUILD |
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It is unlikely that a general scenario can be envisaged that will account for the behavior of all NHR-regulated genes. A partial synthesis, however, would be helpful before a discussion of the major partners of the NHRs in transcriptional control. The model presented here is based on biochemical studies from a large number of laboratories (3, 42) and from structural and functional studies on the budding yeast HO endonuclease gene (43, 44), the MMTV LTR (2, 32, 45, 46, 47), the mouse serum albumin gene (48, 49, 50), and the Xenopus TRßA gene (27, 51).
1. The receptor accesses its binding site in the chromatinized promoter either due to an existing favorable rotational frame (see above), due to intrinsic nucleosome mobility relative to the DNA that stochastically presents such a frame to the receptor (52), due to the action of a factor that enforces the maintenance of a particular rotational frame within a given locus (49), or in the aftermath of DNA replication when chromatin is incompletely assembled and the nascent DNA is in an accessible conformation (5, 6, 53).
2. Certain chromatin-bound class II NHRs, when in the unliganded state, target corepressor complexes that effect transcriptional repression through deacetylation of chromatin (42); unliganded TR also exploits an auxiliary repression pathway with an undetermined mechanistic foundation that is not HDAC dependent (54).
3. Upon addition of ligand, the NHR recruits an ATP-dependent chromatin remodeling complex that actively reorganizes histone-DNA contacts in the vicinity of the receptor binding site. On a macroscopic level, such targeting leads to the generation of a stretch of chromatin ("DNAse I hypersensitive site") in which the DNA is significantly more accessible to non-histone-regulatory factors than DNA in mature chromatin.
4. The liganded receptor shuttles on and off the DNA (47) while the remodeled state persists, in part, perhaps, due to the binding of additional regulatory factors within the DNAse I-hypersensitive site that exert stimulatory effects on the basal transcriptional machinery. The liganded receptor, as well as these additional nonreceptor factors, both target complexes that possess HAT activity; this recruitment stimulates transcription.
5. In addition to HAT-containing complexes, NHRs also target large coactivator complexes that engage the basal transcriptional machinery (35, 39) and are also involved in transcriptional activation by non-NHR regulators (55).
We will presently review recent progress in our understanding of some of these steps of gene regulation by various NHRs.
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WHAT IS WITHOUT Sin3: REPRESSION REVISITED |
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A physiological rationalization of such receptor behavior lies in the known biological function of thyroid hormone (58, 59) and various retinoids (60) as inducers of cell cycle arrest and differentiation. It is thought that unliganded TR/RXR and RAR/RXR silence genes that are required for execution of this program, and that ligand causes a relief of transcriptional repression and a concomitant drastic change in cell phenotype; interestingly, vitamin D and its receptor, VDR, have a well established analogous role in such processes as osteogenesis (61), but unliganded VDR is not known to act as a transcriptional repressor. An attractive evolutionary parallel exists with the major NHR in insects, the ecdysone receptor, that effects an analogous function in insect metamorphosis (62). The target genes in vertebrates are largely unknown, with a few exceptions (63), but evidence from studies of mutated versions of TR and RAR found in various leukemias are fully consistent with this working model (64, 65).
Unliganded TR and RAR act as potent transcriptional repressors on model promoters (65), and studies with in vitro systems demonstrated that such action could occur on naked DNA templates (66): for instance, unliganded TR efficiently interferes with preinitiation complex formation (67). An alternate or complementary pathway for repression by TR and RAR was proposed when yeast two-hybrid assays identified two large, related polypeptides termed N-CoR (nuclear receptor corepressor) (68, 69) and SMRT (70). Their mechanism of action remained obscure until work from several laboratories (71, 72) offered the provocative finding that the transcriptional repressor Mad/Max associates with the histone deacetylase RPD3 (also called HDAC1) via the adapter molecule Sin3. This observation provided a direct connection between targeted transcriptional repression and the modification of chromatin.
It is useful to note that a correlative relationship between levels of acetylation and transcriptional activity of specific loci had by then been well established: for instance, active domains of chromatin were known to be hyperacetylated (73, 74, 75), while the inactive X in human females was known to be hypoacetylated (76). Thus, it was tempting to speculate that the targeted deacetylation of chromatin could contribute to transcriptional repression in mammals; evidence that N-CoR (77) and SMRT (78) could interact with Sin3, and that Sin3 could associate with HDAC1/RPD3 led to a model in which a hypothetical complex of the corepressor with Sin3-HDAC1 was recruited by the unliganded NHR to specific loci in the genome.
It remained unclear, however, what fraction of cellular N-CoR was associated in vivo with Sin3A, whether the HDAC activity targeted by N-CoR was due to HDAC1/RPD3 or some other HDAC, or whether the hypothetical N-CoR-Sin3-HDAC1 complex can associate in vivo or in vitro with unliganded NHRs (77). A similar state of uncertainty existed with regard to transcriptional repression effected by SMRT; most significantly, it was unknown whether endogenous SMRT associates with Sin3 or HDAC in vivo (78). It also remained to be demonstrated that unliganded NHRs recruit HDAC activity to target promoters; for instance, while the HDAC inhibitor TSA was shown to up-regulate transcriptional activity of a promoter repressed by unliganded RAR/RXR (78), this up-regulation was equally robust in the presence of all-trans retinoic acid (78), i.e. after the receptor released both the corepressor and the attending hypothetical HDAC. These findings were consistent with the notion that TSA effects on transcription were unrelated to the hypothetical HDAC targeted by RAR/RXR (78). To further complicate matters, stable occupancy of target promoters by unliganded RAR/RXR heterodimers has been very difficult to detect in vivo (79); while explanations for such action in absentia are currently lacking, hit-and-run targeting of corepressor action via transient occupancy of target promoters by RAR may be invoked.
Recent data (37, 54) directly argue against the involvement of a hypothetical (SMRT/N-CoR)-Sin3-HDAC1/RPD3 complex in transcriptional repression by unliganded NHRs. For instance, the application of two different biochemical purification strategies to characterize an endogenous SMRT-containing complex in HeLa cells yielded the identical observation that Sin3 or HDAC1 fail to associate with this corepressor (37). The 2% of endogenous SMRT and N-CoR that are not contained in this complex are not associated with Sin3-HDAC1, either (80). Similarly, targeting of N-CoR and HDAC activity to unliganded TR and its mutated derivative, the oncoprotein v-ErbA, occurs without an association with Sin3 and HDAC1 (54). Thus, at the present time, the existence of an in vivo complex between Sin3-HDAC1 and any NHR corepressors or a role of this hypothetical entity in NHR function both await experimental support.
What, then, are the corepressors partners, and are any of them HDACs?
Recent biochemical analysis (37, 81) describes a large ( 2 mDa)
SMRT-containing complex in HeLa cells. Remarkably robust [it is
reported to remain stable in 0.5 M NaCl (37)], this entity
contains multiple polypeptides, including histone deacetylase 3
(HDAC3). An association between HDAC3 and N-CoR/SMRT was also
discovered in Xenopus oocytes (54, 81) as well as in a
distinct biochemical purification scheme applied to HeLa cells (82).
Several HDACs other than HDAC1 have been biochemically connected to
N-CoR and SMRT (80, 83), but no evidence is available as to whether
these associations are relevant to NHR function.
In contrast, the novel Sin-free (SMRT/N-CoR)-HDAC3 complex has been implicated in repression by NHRs by several lines of evidence. In human cells, one of its integral components, the histone-binding protein TBL1 (see below), potentiates transcriptional repression by Gal4-TRLBD when overexpressed (37). Importantly, an allelic version of TR that fails to recruit corepressors (84) also fails to respond to TBL1 overexpression (37). In Xenopus, unliganded TR associates with N-CoR-HDAC3, and treatment with thyroid hormone both relieves transcriptional repression effected by TR and eliminates any detectable association between the receptor and N-CoR or HDAC3 (54). Finally, microinjection of purified antibodies directed against HDAC3 or against N-CoR partly alleviates repression effected by unliganded TR in Xenopus oocytes (81); importantly, such treatment failed to affect basal transcription driven by the reporter construct used, directly linking antibody action to receptor-dependent phenomena.
Any speculation on the significance of targeting of HDAC3, rather than HDAC1, to promoters repressed by unliganded NHRs is complicated by the paucity of information on functional distinctions between these HDACs; their numbers are growing faster than our insight into the selective advantage reaped by the cell from possessing multiple HDACs. In interesting contrast to the HATs, which exhibit a wide spectrum of histone tail lysine specificity for action (see below), HDACs target all lysines with seemingly equal efficiency. The choice by unliganded TR of HDAC3 in particular is an interesting one, however, because this enzyme was reported to be more efficient than its sister protein HDAC1/RPD3 in deacetylating histones assembled into nucleosomes (85). Such capacity may have functional utility for TR when it associates with nascent DNA in the aftermath of DNA replication fork passage [a common occurrence, considering that unliganded TR maintains a variety of cell types in the proliferative state (64, 86)]. On such newly replicated loci, chromatin is gradually assembled de novo by molecular chaperones that deposit histones onto DNA in hyperacetylated form. The newborn hyperacetylated nucleosomes are converted into a mature, deacetylated state over about 30 min by unknown HDACs; on loci bound by unliganded TR, however, such deacetylationand attending transcriptional repressioncould be accelerated by N-CoR-targeted HDAC3.
Several lines of evidence indicate, however, that transcriptional repression by unliganded TR is mediated via both an HDAC-dependent as well as an -independent pathway (54); other eukaryotic transcriptional repressors, for instance, Ikaros (87, 88), display similar properties. An important challenge for future experiments will be to provide a currently lacking experimental connection between in vitro data that TR can directly interfere with basal transcription machinery function (67) and in vivo behavior of the receptor. An additional significant, and currently unaddressed, gap in the literature is a lack of evidence that transcriptional repression by NHRs leads to histone tail deacetylation over target promoters; at present, such data are only available for transcriptional repressors that operate in budding yeast (89, 90). While chromatin immunoprecipitation (91) is an exceptionally powerful and useful technique that has yielded profound insights on various aspects of genomic function in budding yeast (43, 92, 93, 94), it is unclear whether the size of mammalian genomes and the relatively small changes in levels of histone acetylation over target promoters are likely to collude in preventing the acquisition of a robust and interpretable dataset on this issue.
A final possibility that deserves investigation is that unliganded TR and RAR drive transcriptional repression via pathways not previously considered; it is possible, for instance, that some structural feature of the DNA-bound unliganded NHR leads to the relocalization of the entire locus into a transcriptionally inactive nuclear compartment (95). While an earlier hypothesis (96) that unliganded TR targets a ubiquitous chromatin remodeling machine termed Mi-2/NURD (97) lacks both biochemical (54) and functional (96) support, recent work offered a novel, and unexpected, connection between unliganded TR and repression-related chromatin assembly, when a histone-binding protein called transducin ß-like 1 (TBL1) was identified as a stable component of a large SMRT- or N-CoR-containing complex (37, 81). This observation is exciting for two reasons. First, mutations in TBL1 have been implicated in late-onset sensorineural deafness in human patients (98), and strong genetic evidence implicates TRß in auditory cortex development in mice (99). Second, from a mechanistic standpoint, TBL1 has the intriguing capacity to bind histone H3; a distant relative of human TBL1, the budding yeast transcriptional repressor Tup1p, exerts its function, in part, via directing the assembly of a repressive nucleosomal array (100, 101). It is tempting to speculate, therefore, that when bound to target loci in proliferating cells, where chromatin is repeatedly destroyed by DNA replication, and spurious gene activation is therefore much more likely than in quiescent cells, unliganded TR sustains transcriptional repression by non-chromatin-based pathways (54), as well as promoting the assembly of repressive chromatin both by interacting with the histones via the TBL1 moiety of the corepressor complex (37) and accelerating their deacetylation via HDAC3 action (37, 54).
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LENDING A HELPING TAIL: HISTONES AND TRANSCRIPTIONAL ACTIVATION BY NHRs |
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This complexity results from two circumstances. Extraordinary progress in the biochemical characterization of transcriptional cofactors implicated enough of them in NHR signaling to take up a five-page table in a review published in this journal earlier this year (3). For students of chromatin, it is comforting to see that the eukaryotic nucleus is populated with chromatin-modifiying and -remodeling machines, but such abundance does not allow itself easily to being confined into parsimonious models. In particular, there continues to be a significant gap between our understanding of biochemical activities of such machines in vitro and our knowledge of in vivo structural and functional consequences of their presumed action. In addition, we have evidence that the actual assembly of target promoters into chromatin potentiates and attenuates the response to NHR action, but the mechanistic underpinnings of such a synergy between chromatin and the receptors are emerging only very slowly.
Recent reviews describe in detail the various types of chromatin disruption and modification that occur in vivo (11) and also the coactivators that abet NHR signaling by effecting such alterations in chromatin structure (3, 42, 102). We will discuss these issues briefly, focusing on specific examples of NHR action on chromatin templates.
It is helpful to make a distinction between the two major types of chromatin structure disruption that occur in vivo and are known to be associated with transcriptional activation effected by liganded NHRs. The first type is often called "chromatin remodeling" and refers to a dramatic, localized alteration in the fiber of chromatin in which a particular nucleosome, or several adjacent nucleosomes, undergo a receptor-controlled structural change (2). The end result is the creation of a stretch of DNA that is significantly more accessible to nucleases (e.g. DNAse I); hence, its technical name, "DNAse I hypersensitive site." It is quite likely, although a formal demonstration in vivo is currently lacking, that such remodeling effected by liganded NHRs occurs via the recruitment of large ATP-utilizing complex. In the case of GR, the best candidate is a multisubunit complex called SWI/SNF (pronounced "switch-sniff") (45, 102, 103). In budding yeast, powerful genetic data indicate that SWI/SNF controls gene expression via a chromatin-based pathway (104, 105), and chromatin remodeling at specific promoters is known to be SWI/SNF-dependent (106, 107, 108). In vertebrates, however, it is not known whether remodeling at any NHR-regulated promoter in vivo requires SWI/SNF. In purified form, the SWI/SNF complex has a variety of ATP-dependent activities, including the rearrangement of purified nucleosomes to potentiate regulatory factor access to DNA (16, 109, 110), and the induction of histone octamer sliding relative to the DNA (111). It is unclear how the remodeled and mobilized nucleosomes observed in vitro relate in structure to the entity that is revealed in vivo as a DNAse I-hypersensitive site; thus, while many NHRs are known to effect such remodeling (2, 112), their biochemical accomplices in such action remain obscure, a gap that well deserves to be addressed.
Biochemical analysis of eukaryotic nuclei yielded a large number of other ATP-dependent chromatin remodeling machines (38, 113), many of which contain a core catalytic subunit called "imitation switch" (ISWI); as of August, 2000, however, there is no evidence connecting these complexes to gene regulation in vivo in organisms other than budding yeast (and even in yeast, targeted action of any ISWI-containing complex at a specific promoter has not been demonstrated), although PR and ISWI have been reported to synergize in vitro (114).
Whatever the specific ATP-dependent complex that effects such remodeling in vivo, it is useful to note that such a nucleosome-scale disruption is not a formal prerequisite for gene activation and that specific promoters can undergo significant transcriptional up-regulation without concomitant remodeling (e.g. Refs. 112, 115); some hormonally responsive promoters, for example, the ER-regulated pS2 gene (28), do not undergo a macroscopic change in chromatin structure upon ligand-driven transcriptional activation. A further mystery involves the actual fate of the histone octamer during remodeling; while original models proposed that the octamer is physically evicted, subsequent in vitro studies showed that much of the DNA remains in some contact with the octamer (116) and that transcription factors and histones can share the same remodeled nucleosome (117). Analysis of chromatin structure in vivo suggested that specific remodeled loci can remain associated with histones (48), and similar observations were reported for RAR- and GR-regulated genes (22, 29). It is possible that the persistence of the octamer in the vicinity of the receptor-binding site is related to negative feedback mechanisms of attenuating transcriptional response to hormone (see below), since the receptor shuttles on and off the DNA (47), and since continued maintenance of DNA occupancy by a nonhistone factor is required to exclude histone octamers from its binding site (118).
To complicate matters somewhat beyond our current ability to put
forward rational explanations, a meticulous high-resolution analysis of
chromatin structure at the MMTV LTR led to the revision of two
previously held concepts. Earlier studies indicated that this promoter
is assembled into an array of translationally and rotationally
positioned nucleosomes, one of which (nucleosome B) presents on its
surface the glucocorticoid response element (GRE) to the liganded
receptor. Subsequent mapping demonstrated that, in fact, chromatin in
this region is assembled into multiple translational frames, but that
some frames are more common than others (also known as
"frequency-biased distribution"; see Fig. 1) (119); thus, previous mapping data
indicated the existence of a very specific chromatin organization
because the methods used generated data that averaged across a
heterogeneous sample (a common complication). It is possible that
in vivo, nucleosome mobility, whether intrinsic or
facilitated (52, 118, 120), can lead to transition from one frame to
another, and that the receptor then selects those templates that at a
particular point in time are found in a correct rotational frame.
Interestingly, a high-resolution mapping study on GR interaction
with the MMTV LTR in Xenopus oocytes (121) demonstrated the
ability of liganded GR to actively reorganize chromatin not only around
its binding site, but elsewhere in the promoter; thus, GR can
enforce a particular translational frame on the templates
that it encounters, a property thought to be important in the
regulation of mammalian genes by the winged-helix transcription factor
HNF3 (48, 49, 50, 122). It will be of great interest to investigate whether
such active reorganization is simply a matter of freezing chromatin in
a particular frame via a bookend mechanism, or if liganded GR does,
indeed, target a remodeling machine that can effect the active
organization of chromatin into a particular frame.
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The second type of chromatin structure disruption that occurs in vivo is commonly referred to as "chromatin modification": the core histones are the targets of many posttranslational covalent modifications (123), the most prominent of which is the acetylation of the lysines effected by HATs and reversed by HDACs (13, 124). For certain transcriptional activators in budding yeast, powerful data exist that formally prove a causative role between targeted histone acetylation and transcriptional activation: for instance, the HAT Gcn5p is required for activated transcription of specific genes, this requirement is dependent on the biochemical activity of Gcn5p as a HAT, Gcn5p action leads to the hyperacetylation of histones at target promoters, and the abolition of charged lysines in histone tails via their mutation to a noncharged amino acid relieves the requirement for Gcn5p in transcriptional activation (125, 126). It is important to note that the firm positive correlation between extent of acetylation and level of transcriptional activity does not always hold: for example, treatment with HDAC inhibitors has been, rather counterintuitively, shown to repress transcriptional activation driven by liganded GR on the MMTV LTR (127, 128), and genetic data from yeast and Drosophila also point to an unexpected role for HDAC in gene activation (129). In most cases, however, deacetylation correlates with repression, and hyperacetylation correlates with activation.
Mammalian genomes contain a large number of HATs (130), and a variety of biochemical evidence (e.g., two-hybrid interactions, GST pull-down assays with in vitro synthesized proteins, coimmunoprecipitations, etc.) connect many of these enzymes to signaling by liganded NHRs (3). In vivo evidence for a role of specific HATs in transcriptional activation effected by NHRs is, inevitably, less robust, with some exceptions. For example, the clinical syndrome of resistance to thyroid hormone in human patients (131) results from mutations in the ligand binding domain of TRß. Biochemical analysis indicated that many of these mutations impair the receptors capacity to target such HAT coactivators as steroid receptor coactivator-1 (SRC-1) (132, 133). In powerful support of these observations, genetic ablation of the gene for SRC-1 in mice was shown to lead to thyroid hormone resistance in the knockout animals (134). SRC-1 was shown to potentiate PR signaling in an in vitro system with chromatin templates, and such action was obviated by use of the HDAC inhibitor trichostatin A (33); it will be of great interest to determine whether the targeting of coactivator HATs by liganded NHRs in such in vitro systems leads to targeted hyperacetylation of chromatin on a scale similar to that effected by chimeric activators that target yeast coactivators to in vitro assembled chromatin (135). This will be particularly important because of a mysterious discrepancy between the known capacity of such NHR HAT coactivators as CBP/p300 (136) to efficiently hyperacetylate both histones H3 and H4 (137) with the observation that in vivo, certain NHR-regulated promoters undergo a change in the acetylation status of histone H4, while such changes in histone H3 acetylation are rather insignificant (138). It is possible that HATs other than CBP/p300 potentiate NHR signaling at those promoters, or that the acetylation status of histone H4 over those promoters is affected not only by NHR-targeted HATs, but other cell-wide effects of NHR ligands [for instance, H4 acetylation is expected to increase in proliferating cells (139)]. In addition, many HATs are known to have nonhistone targets (13), including transcription factors, other HATs, and components of the basal transcription machinery; it is quite possible that changes in acetylation status of such polypeptides also account for some of the transcriptional effects of liganded NHRs.
The wealth of biochemical evidence connecting the multitude of HATs to liganded NHRs is not, unfortunately, paralleled by insight of how hyperacetylation of chromatin leads to transcriptional activation; to some extent this is due to a lack of structural information about the NH2-terminal histone tails (8). In vitro data have indicated that hyperacetylation of chromatin facilitates transcription elongation by RNA polymerase (120), and recent evidence from budding yeast offers very strong support to this notion (140, 141). Since liganded NHRs are not known to move along their target genes with RNA polymerase, however, it is unlikely that the HATs they are presumed to target affect transcriptional elongation. Several other hypotheses have been put forward and confirmed through in vitro studies, most prominently, that a hyperacetylated nucleosome is more accessible to binding by nonhistone transcriptional regulators (19, 142). It has been difficult until very recently to investigate this issue in vivo, in part because NHRs not only target HATs, but also ATP-remodeling machines; the latter also promote accessibility of DNA to nonhistone regulators, which makes a dissection of the two pathways contribution to overall accessibility very difficult.
Recent data concerning ER action in vitro and in
vivo on the pS2 promoter (28) provide a novel line of support for
the hypothesis of acetylation-driven "facilitated access." Robustly
responsive to estradiol, this promoter contains an ER binding site that
is located at the edge of a translationally positioned nucleosome, and
a TATA box bound at the edge of another positioned nucleosome
approximately 380 bp downstream (i.e. exactly two nucleosome
lengths away; see Fig. 2); the stable and
unique translational frame of these two nucleosomes is likely due to
intrinsic positioning properties of the DNA sequence (28). Liganded ER
was shown to directly engage its ERE on the surface of the octamer and,
without mediating a significant alteration in histone-DNA contacts, to
stimulate transcription of the pS2 gene (28). Considering that
large-scale remodeling similar to that seen over the MMTV LTR does not
occur on this promoter, an important question was the mechanism of such
stimulation. Chromatin immunoprecipitation assays demonstrated that
treatment with estradiol led to hyperacetylation of histones H3 and H4
within both nucleosomes in vivo; concomitant with this
chromatin modification, TBP binding was observed over the TATA element
(143). Importantly, and in full agreement with earlier observations
(16), while TBP could not access its binding site in the promoter when
it was assembled in vitro into mature, deacetylated
chromatin (143), an identical in vitro binding assay
performed with hyperacetylated chromatin showed robust binding of TBP
to the pS2 TATA box on the surface of a hyperacetylated,
translationally positioned nucleosome (143). Such potentiation of
binding occurred on a highly purified chromatin template, thereby
excluding the possibility that contaminating nonhistone components are
responsible for enhanced TBP access (143). These data extend earlier
in vitro observations (19, 142) and offer a comforting
parallel to data obtained in budding yeast (144, 145), according to
which transcriptional activators increase TBP occupancy of target
promoters in vivo.
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It is useful to appreciate, however, that none of the disruption,
remodeling, or modification events (summarized in Table 1) that have been observed in
vivo or in vitro during NHR action result in the
generation of naked DNA. In fact, there are many well documented cases
[e.g. the action of ER on the vitellogenin B1 (146) and the
pS2 (28) promoters, of TR on the TRßA promoter (27, 51), GR on the
MMTV LTR (147)] in which the assembly of target DNA into chromatin
potentiates NHR function (Fig. 2
); importantly, this potentiation
occurs not only by expanding the range within which transcriptional
levels vary, but also by endowing the system with regulatory properties
that cannot be recapitulated on naked DNA (148). An excellent example
is the regulation of the MMTV LTR by GR and other factors; on
transiently transfected (i.e. not physiologically
chromatinized) templates, such cellular factors as NF1 and Oct-1 can
gain promiscuous access to the promoter (46). On endogenous
chromatinized templates, however, such access, and the attending subtle
alterations in promoter activity, are unequivocally dependent on GR
(149, 150).
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IN LIVING COLOR: NHRs IN ACTION |
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It is nontrivial to study the dynamics of the interaction of NHRs with their templates: a cell population large enough to generate experimental signal will be asynchronous with respect to transcriptional state of their template, and while it has been possible to partly circumvent this problem in elegant in vitro experiments (34), transcription in an intact mammalian cell does not lend itself to synchronization easily. In addition, most experimental techniques that investigate protein-DNA interactions (genomic footprinting with DNAse I, in vivo footprinting with DMS, chromatin immunoprecipitation, exonuclease III boundary measurements, etc.) all generate signal that represents an averaged sample of a potentially heterogenous population: for instance, the occupancy of >50% of target molecules by a nonhistone factor is sufficient to generate a genomic or in vivo footprint; similarly, accurately quantitating results from chromatin immunoprecipitation experiments to meaningfully estimate the percentage of templates bound by a protein at a given time is a nontrivial challenge that has yet to be adequately met.
In a technical tour de force, it has recently become
possible to visualize, on a timescale of seconds, the dynamics of
NHR-DNA interactions in chromatin of living cells (47). The significant
technical obstacle of light microscope detection limits was overcome by
creating a cell line that contains, in integrated form, approximately
200 reiterated copies of a 9-kb reporter construct in which the MMTV
LTR is fused to the v-ras gene (152); this approach using
lac repressor and operator sequences has been successfully
used to track chromosome behavior in vivo (153, 154). A
clone for a chimeric NHRGR fused to green fluorescent protein
(GFP)was then introduced into the cells genome under the control of
an inducible promoter; this experimental setup allowed precise control
over the onset of NHR expression and, via the use of GR agonists, over
the intracellular location of GR once it is expressed. Laser confocal
microscopy in dexamethasone-treated cells revealed a large ( 12
µM) intensely fluorescent structure in the
nucleus, whose location corresponded precisely to the site of
v-ras transcription as gauged by in situ
hybridization.
The rate of exchange between GR bound to its response elements and GR elsewhere in the nucleus was then measured using two techniques that have been very successfully used to investigate the dynamics of membrane-based phenomena in cell biology: fluorescence recovery after photobleaching (FRAP) (155, 156, 157) and fluorescence loss in photobleaching (FLIP) (158). In FRAP, a laser beam is pointed at a defined location in the living nucleus (in this case, the 1.8 Mb GR-bound array) and thereby bleaches GFPs fluorescence for a considerable length of time. Recovery of fluorescence in the bleached spot can only occur if nonbleached molecules invade the target area from elsewhere in the nucleus; by definition, the rate of exchange is equal to the rate of recovery. In remarkable contradiction to common-sense notions of stable target site occupancy by NHRs, recovery of fluorescence occurred as rapidly as it could be measured, and in 510 sec following bleaching of all the GR molecules bound to the entire 1.8 Mbp array, these nonfluorescent NHR molecules were replaced by non-bleached GFP-GR (47). These data strongly suggest that liganded, DNA-bound GR undergoes extremely rapid "ejection" and reassociation with its DNA template in a hit-and-run mechanism of transcriptional activation (159). Equally powerful evidence to that effect was obtained using FLIP: GR located elsewhere in the nucleus was bleached, and subsequently, over approximately 60 sec, invaded the 1.8-Mb MMTV array.
This highly unexpected and convincing dataset sheds new light on
observations earlier obtained by K. Nasmyth and colleagues (43) on the
sequence of events that occur during transcriptional activation of the
budding yeast HO endonuclease gene. Genetic observations indicated
that, in full functional analogy to regulation by GR in mammals, the
activation of this gene is dependent on a DNA-bound activator (Swi5p),
the recruitment of a chromatin remodeling machine (SWI/SNF), and the
subsequent targeting of a HAT complex (SAGA). Remarkably, chromatin
immunoprecipitation assays in synchronized cells indicated that the
activator, Swi5p, resides on the HO endonuclease promoter for a very
brief period of time (510 min) and disappears from the DNA at
approximately the same time as it recruits SWI/SNF. Most significantly,
SWI/SNF residency at the promoter then continues for at least 1 h;
thus, epigenetic memory of activator occupancy persists long after it
has been ejected.
What is the mechanism of this ejection and what functional benefit can
it bring to the cell? No data exist to directly address either issue;
by way of speculation, it is possible that ATP-driven remodeling action
of SWI/SNF displaces from the DNA the very activator that recruits it,
via direct action on the receptor-DNA contacts, or on the histones
underlying the receptor-bound nucleosome. On the TRßA promoter in
Xenopus, for instance, unliganded TR binds to multiple
nonconsensus TREs (Fig. 2), and such binding is potentiated by
chromatin assembly (27), perhaps though the structural distortion of
chromatinized DNA that makes TREs more palatable to the receptor.
Addition of ligand leads to the targeted disruption of chromatin around
the receptor binding sites (27, 112) and is expected to therefore
create a template that has less affinity for the receptor
(i.e. lead to receptor ejection). Data obtained with an
in vitro system that recapitulates regulation by purified GR
on a chromatin template indicate that in the absence of ATP, GR can
stably bind, but not remodel, chromatin over the promoter (32). The
addition of ATP leads to chromatin remodeling coupled with an increased
accessibility of DNA over the receptor binding sites (32), in full
agreement with the hit-and-run model for GR function.
An obvious suggestion for the functional utility of such a drifter model of NHR action is in the attenuation of hormonal response. The MMTV LTR becomes refractory to dexamethasone treatment after prolonged exposure to ligand; it is possible that the continuous active elimination of GR from the DNA is partly responsible for this phenomenon. In addition, TR is less stable in the presence of ligand than in its absence (54, 160), which is known to be a consequence of liganded TR being preferentially targeted by the ubiquitin-proteasome protein degradation pathway (160), an example of a general phenomenon of activator degradation by proteolysis (161). It is possible, for instance, that such liganded NHR ejection from the DNA as demonstrated by studies of G. Hager and co-workers contributes to a more efficient targeting of NHRs for degradation and leads to a more effective attenuation of response to hormone.
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CONCLUSION |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Goldmark et al. (Mol Cell 103:423433) demonstrate by genetic analysis of ISWI in budding yeast that in vivo it functions as a transcriptional corepressor for Ume6p to effect silencing of meiosis-specific genes in a pathway parallel to that driven by Sin3p-Rpd3p. These are the first data on ISWI action at a specific promoter in vivo; they offer an important perspective on in vitro studies proposing that mammalian ISWI is a transcriptional coactivator for such NHRs as PR (Di Croce et al., Mol Cell 4:45) and RAR (Dilworth et al., Mol Cell 6:1049).
Received for publication August 16, 2000. Revision received October 3, 2000. Accepted for publication October 16, 2000.
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REFERENCES |
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