Histone H1
The function of the neuronal differentiation gene daughterless is required for the proper initiation
of neuronal lineage development in all peripheral nervous system (PNS) lineages following the selection of neuronal precursor cells.
Previous studies have shown that the ubiquitously expressed Da protein is required for the proper
expression of neuronal precursor genes and lineage identity genes in the PNS of Drosophila
embryos. These genes are required for differentiation and cell fate determination in the
developing PNS. These findings, however, do not explain the failure of the nascent PNS precursors to
undergo a normal cell cycle and divide in da mutants. Four genes whose products are required for
various stages of the cell cycle are misexpressed in the PNS of da mutant embryos. Cyclin A, barren, disc proliferation abnormal and Histone H1 transcripts are significantly reduced or undetectable in the precursors of the PNS at stages 11 and 12. Precursors are still present at these stages in da mutants. This suggests that
all aspects of PNS precursor differentiation examined so far are under the transcriptional control of da. Sensory organ precursors lacking Da may fail to express and/or accumulate other factors, such as critical differentiation genes, required for SOP entry into the cell cycle. It should be pointed out that these factors are unlikely to be the thus-far described neuronal precursor genes, as mutations in these genes do not result in any obvious cell cycle defects (Hassan, 1997).
Imitation SWI (ISWI) and other ATP-dependent chromatin-remodeling factors play key roles in transcription and other processes by altering the structure and positioning of nucleosomes. Recent studies have also implicated ISWI in the regulation of higher-order chromatin structure, but its role in this process remains poorly understood. To clarify the role of ISWI in vivo, defects in chromosome structure and gene expression were examined resulting from the loss of Iswi function in Drosophila. Consistent with a broad role in transcriptional regulation, the expression of a large number of genes is altered in Iswi mutant larvae. The expression of a dominant-negative form of ISWI leads to dramatic alterations in higher-order chromatin structure, including the apparent decondensation of both mitotic and polytene chromosomes. The loss of ISWI function does not cause obvious defects in nucleosome assembly, but results in a significant reduction in the level of histone H1 associated with chromatin in vivo. These findings suggest that ISWI plays a global role in chromatin compaction in vivo by promoting the association of the linker histone H1 with chromatin (Corona, 2007; full text of article).
Most studies of ISWI complexes in Drosophila and other organisms have focused on their ability to alter the structure or spacing of nucleosomes, the fundamental unit of chromatin structure. These findings reveal that ISWI also plays a global role in the regulation of higher-order chromatin structure. The Iswi mutations used in this study eliminate the function of multiple chromatin-remodeling complexes, including ACF, NURF, and CHRAC. Which of these complexes are required for the formation of higher-order chromatin structure? Loss of function mutations in Acf1 -- which encodes a subunit protein shared by ACF and CHRAC -- do not cause obvious defects in higher-order chromatin structure. By contrast, loss of function mutations in E(bx) -- which encodes a subunit specific to NURF -- cause male X chromosome defects similar to those observed in Iswi mutants. These findings suggest that ISWI modulates higher-order chromatin structure within the context of NURF, as opposed to ACF or CHRAC (Corona, 2007).
A striking correlation was observed between the severity of the chromosome defects resulting from the loss of ISWI function and the loss of the linker histone H1. This correlation suggests that ISWI regulates higher-order chromatin structure by promoting the association of histone H1 with chromatin. Histone H1 and other linker histones influence higher-order chromatin structure in vitro by stabilizing interactions between nucleosomes and chromatin fibers. Although the ability of histone H1 to promote chromatin compaction in vitro is well established, its function in vivo has been a topic of considerable debate. A protein with biochemical properties reminiscent of linker histones -- HHO1 -- is present in budding yeast; surprisingly, HHO1 is not essential for viability in yeast, and hho1 mutations have little effect on either gene expression or chromatin structure. Genetic studies in Tetrahymena have suggested roles for linker histones in chromatin condensation and gene expression, but the relevance of these studies to histone H1 function in higher eukaryotes remains unclear. Studies of histone H1 function in higher eukaryotes have been complicated by the presence of redundant genes encoding histone H1 or histone H1 subtypes. In spite of these difficulties, recent studies have revealed important roles for histone H1 in chromosome compaction in Xenopus and mice. Thus, the chromosome defects observed in Iswi mutants could easily result from inefficient incorporation of histone H1 into chromatin (Corona, 2007).
How might ISWI promote the association of histone H1 with chromatin? Since ISWI is not required for histone H1 synthesis, ISWI may directly promote the assembly of chromatin containing histone H1 following DNA replication. Recent biochemical studies provide support for this possibility: ACF promotes the ATP-dependent assembly of H1-containing chromatin in vitro. Loss of ACF1 function does not cause obvious changes in chromosome structure, however, suggesting that ACF either does not regulate higher-order chromatin structure in vivo or plays a redundant role in this process. It remains possible that ISWI promotes the assembly of histone-H1-containing chromatin within the context of NURF or another chromatin-remodeling complex (Corona, 2007).
The ability to promote histone H1 assembly is not a common property of all chromatin-remodeling factors, as illustrated by recent biochemical studies of CHD1. Like ACF and other ISWI complexes, the CHD1 ATPase promotes the assembly of regularly spaced nucleosomes in vitro. By contrast, CHD1 does not promote the incorporation of histone H1 during chromatin assembly in vitro. These biochemical studies provide a plausible explanation for why the loss of ISWI function leads to the loss of histone H1 without causing dramatic changes in nucleosome assembly in vivo (Corona, 2007).
In other organisms, depletion of histone H1 leads to a significant decrease in the nucleosome repeat length, presumably because of the failure to efficiently incorporate histone H1 during replication-coupled chromatin assembly. By contrast, the loss of ISWI function in salivary gland nuclei leads to a decrease in the amount of histone H1 associated with chromatin without causing dramatic changes in nucleosome repeat length. It is therefore tempting to speculate that ISWI promotes histone H1 incorporation via a replication-independent process. The association of histone H1 with chromatin is far less stable than that of core histones; histone H1 undergoes dynamic, global exchange throughout the cell cycle. Photobleaching experiments in Tetrahymena and vertebrates have suggested that the majority of histone H1 molecules associated with chromatin are exchanged every few minutes, but little is known about the factors that regulate this process. Based on the current findings, ISWI is an excellent candidate for a factor that regulates the dynamic exchange of histone H1 in vivo. Further work will be necessary to determine whether ISWI promotes histone H1 incorporation via replication-dependent or -independent mechanisms (Corona, 2007).
These findings suggest that acetylation of H4K16 may regulate the association of linker histones with chromatin in vivo. The histone H4 tail is required for the nucleosome-stimulated ATPase activity of ISWI, and for its ability to slide nucleosomes and alter their spacing in vitro. The region of the H4 tail that is critical for ISWI function in vitro is a DNA-bound basic patch (R17H18R19) adjacent to H4K16, the residue that is acetylated by the MOF histone acetyltransferase. The acetylation of H4K16 interferes with the ability of ISWI to interact with the histone H4 tail and alter the spacing of nucleosome arrays in vitro. Consistent with these findings, dosage compensation is necessary and sufficient for the decondensation of the X chromosome in Iswi mutant larvae, and genetic studies have revealed a strong functional antagonism between ISWI and MOF. Thus, H4K16 acetylation may function as a switch that regulates the histone H1 assembly mediated by ISWI (Corona, 2007).
Microarray studies revealed that ISWI is required for the proper expression of a large number of genes. These findings are consistent with numerous studies implicating ISWI in transcriptional regulation in vitro and in vivo. Does ISWI modulate transcription by altering higher-order chromatin structure? It is suspected that ISWI regulates transcription and higher-order chromatin structure via distinct mechanisms, since no obvious correlation is observed between the magnitude of the changes in gene expression and chromosome structure observed in Iswi mutant larvae. This is consistent with genetic studies in other organisms that have revealed that the loss of histone H1 does not cause dramatic changes in gene expression. No correlation was observed between the magnitude of transcriptional derepression and gene size in Iswi mutant larvae, as would be expected if ISWI relieved a general block to transcriptional elongation by Pol II. It should be noted, however, that relatively subtle, but biologically important, changes in gene expression may have escaped detection in our microarray studies. Further work will be necessary to clarify this issue and to determine whether ISWI regulates transcription and higher-order chromatin structure via distinct or related mechanisms (Corona, 2007).
The 100 copies of tandemly arrayed Drosophila linker (H1) and core (H2A/B and H3/H4) histone gene cluster are coordinately regulated during the cell cycle. However, the molecular mechanisms that must allow differential transcription of linker versus core histones prevalent during development remain elusive. This study used fluorescence imaging, biochemistry, and genetics to show that TBP (TATA-box-binding protein)-related factor 2 (TRF2) selectively regulates the TATA-less Histone H1 gene promoter, while TBP/TFIID targets core histone transcription. Importantly, TRF2-depleted polytene chromosomes display severe chromosomal structural defects. This selective usage of TRF2 and TBP provides a novel mechanism to differentially direct transcription within the histone cluster. Moreover, genome-wide chromatin immunoprecipitation (ChIP)-on-chip analyses coupled with RNA interference (RNAi)-mediated functional studies revealed that TRF2 targets several classes of TATA-less promoters of >1000 genes including those driving transcription of essential chromatin organization and protein synthesis genes. These studies establish that TRF2 promoter recognition complexes play a significantly more central role in governing metazoan transcription than previously appreciated (Isogai, 2007).
Core promoters serve as the platform for the assembly of transcription initiation complexes critical for specifying accurate and regulated RNA synthesis. The eukaryotic cellular RNA polymerase II (Pol II) machinery has evolved to recognize multiple core-promoter elements such as the TATA box, Initiator, and DPE. Indeed, studies of metazoan core promoters revealed considerably greater cis-element diversification than previously expected. For example, TATA boxes, which were thought to be the most widely distributed prototypic core-promoter element recognized by the general transcription factor TBP (TATA-box-binding protein)/TFIID (consisting of TBP and TBP-associated factors, TAFs), are found in <20%-30% of annotated promoters in Drosophila and human. Instead, the majority of core promoters fall into various distinct TATA-less categories. Consistent with diversified core-promoter structures, recent studies identified a family of TBP-related factors (TRFs), but their potential core-promoter recognition functions have remained elusive (Isogai, 2007).
Metazoan cells have been found to use a diversified set of TBP-related molecules that display altered DNA-binding specificities. In Drosophila, TRFs have been implicated in promoter-selective transcription for both Pol II and Pol III gene promoters. However, a comprehensive analysis of TRFs in promoter-selective recognition of Pol II core promoters has not been performed. Earlier studies found that a multisubunit TRF2-containing complex includes the transcription factor DREF and is involved in targeting a subset of promoters containing the DNA replication-related element (DRE). The PCNA gene promoter contains such a DRE and represents a novel tandem core-promoter class composed of two distinct transcriptional start sites, each of which appears to be subject to regulation either by the TRF2/DREF complex or TBP/TAFs. While TRF2 recruitment to the core promoter via DREF may account for a subset of TRF2-dependent promoters, TRF2 is also found in complexes lacking DREF. For example, TRF2 and DREF display only a limited set of overlapping sites in Drosophila Schneider cells visualized by immunofluorescence staining, suggesting that TRF2 may be playing multiple roles -- some in conjunction with DREF and others independent of DREF. It was therefore surmised that there may be additional important TRF2 target promoters that remained uncharacterized (Isogai, 2007).
In order to gain a more comprehensive map of potential TRF2-dependent promoters, a genome-wide analysis of TRF2 recognition sites was conducted both by polytene chromosome staining as well as chromatin immunoprecipitation (ChIP) coupled with high-density tiling microarray detection (ChIP-on-chip). These approaches have revealed several important target genes that illustrate how TRF2 is used as an alternative core-promoter recognition factor. First, biochemical and genetic evidence is provided that two distinct sets of core-promoter recognition factors are responsible for directing transcription of the nucleosome core histone genes (H2A/B and H3/H4) and the linker histone H1. Genome-wide ChIP-on-chip analysis revealed that TRF2 recognizes and binds in vivo to a large number of TATA-less core promoters. Importantly, a majority of these TATA-less promoters are selectively recognized by TRF2, but not by TBP. Moreover, with salivary gland-specific depletion of TRF2, it was found that TRF2 participates in regulation of chromatin organization and cell growth, by controlling Histone H1 and ribosomal protein gene expression. Taken together, these data establish that TRF2 is responsible for differentially recognizing and regulating a subset of TATA-less promoters that have shed the requirement for TBP through the usage of novel core-promoter structures. Remarkably, even coordinately expressed gene clusters such as the histone complex have evolved mechanisms to be differentially regulated by alternative core-promoter recognition machinery (Isogai, 2007).
In Drosophila, the five histone genes are found in a cluster that is tandemly amplified ~100 times. Despite the need to coordinate histone gene expression during the cell cycle, the ratio of linker and core histones can vary dramatically within each cell, among different tissues, and during embryonic development. This observation suggested that Histone H1 gene expression may be differentially regulated relative to the patterns of core histone gene transcription. A genome-wide survey of TRF2 target sites uncovered the finding that the histone gene cluster contains both TBP and TRF2 recognition sites. Most strikingly, these two core-promoter recognition factors are segregated within the histone cluster with TBP targeted to the core histone (H2A/B, H3, and H4) promoters, while TRF2 selectively directs transcription of the linker histone H1. This finding reveals a novel mechanism in which Histone H1 gene expression may be differentially regulated relative to the patterns of core histone gene transcription (Isogai, 2007).
The finding that a TRF2-containing preinitiation complex is responsible for Histone H1 expression while the prototypic TBP/TFIID complex directs transcription of the core histones suggests that the expression of the linker histone H1 and core histones must be uncoupled under certain circumstances, possibly in a developmental-specific and cell type-specific manner. The analysis of TRF2-depleted salivary gland polytene chromosomes suggests that this is indeed the case. Remarkably, the polytene chromosomes in TRF2-deficient cells exhibited severe defects in chromosome organization and structure reminiscent of the failure to form 30-nm fibers in H1-depleted chromatin. Given that the Drosophila genome encodes only one H1 subtype compared with five to six in mammals, it is interesting that the H1 knockdown via TRF2 depletion resulted in a severely altered chromatin structure, which represents another in vivo evidence that histone H1 is indeed linked to organization of chromatin structure. Importantly, these TRF2-depleted cells appear to specifically down-regulate Histone H1 mRNA while leaving core histone transcripts intact. These findings suggest that TRF2 must serve as a key component of the transcriptional initiation complex evolved to differentially control linker histone versus core histone expression (Isogai, 2007).
Transcription of nonpolyadenylated histone genes appears to be associated with a specific nuclear body, the histone locus body (HLB), through a physical coupling between the HLB and the histone gene cluster locus. The HLB is loaded with RNA synthesis and processing machinery, possibly serving as a "factory" for histone mRNA production. Thus, in order to rapidly produce histone transcripts during embryogenesis, Drosophila appears to have adapted an elegant strategy that involves tandemly amplified gene cassettes sequestered within a distinct nuclear address (the HLB). Interestingly, it appears that only specific subsets of transcription factors are deposited in the HLB. For example, among the three TBP paralogs in Drosophila (TBP, TRF1, and TRF2), only TRF2 and TBP that are used for linker and core histone transcription, in addition to Pol II, are 'preloaded' within the HLB, perhaps to facilitate rapid as well as differential linker versus core histone transcript production. Therefore, the histone gene cluster presents an important paradigm wherein a distinct nuclear body loaded with specific transcriptional as well as post-transcriptional machinery becomes dedicated to the purpose of coordinately and differentially regulating five essential genes (Isogai, 2007).
High-resolution genome mapping of TRF2 recognition sites using the ChIP-on-chip platform has revealed >1000 novel binding sites, with 80% distinct from and 20% overlapping with TBP-binding sites. These results suggest that the TRF2-dependent and TBP-independent Histone H1 promoter is not an exception. Indeed, the H1 case may represent a more general case for how TRF2 can serve as an alternative core-promoter recognition factor at many Pol II genes. A comprehensive and detailed sequence motif analysis of the Drosophila genome revealed that TRF2-bound promoters significantly lack TATA boxes, while the TATA box is tightly correlated with TBP-binding sites. Instead, TRF2 appears to selectively recognize promoters containing other distinct core-promoter elements such as Motif 1, DRE, and Motif 7. In addition, functional analysis of transcripts derived from TRF2-depleted salivary glands confirmed that TRF2 activity is indeed required for directing these TRF2 target promoters. Thus, the genome-wide analysis significantly strengthens the emerging picture that TRF2 likely evolved to recognize and regulate a large class of TATA-less core promoters (Isogai, 2007).
One question concerning TRF2 function in promoter recognition is whether TRF2, like TBP, can directly recognize and bind to a distinct core-promoter element. TRF2 is likely to possess very different DNA-binding specificities from TBP since the amino acid residues critical for TATA-box recognition have been altered in TRF2 (Dantonel, 1999; Ohbayashi, 1999; Rabenstein, 1999). However, all attempts to experimentally identify a direct TRF2-binding sequence have thus far failed. Similarly, the most recent computational efforts using TRF2 ChIP-on-chip data sets failed to identify any strong consensus core-promoter motifs comparable with the prototypic TATA box with its approximately minus 30-bp location relative to the start of transcription. Instead, motifs such as the DRE and other uncharacterized elements were identified with no set common position relative to the transcriptional start site. These findings are, however, consistent with previous studies in which TRF2 failed to bind the core promoter by itself. Instead, it appears that TRF2 recruitment to at least a subset of core promoters relies on specific interactions between TRF2 and various other sequence-specific DNA-binding proteins, such as DREF. However, unlike previous studies, the genome-wide survey of TRF2- and TBP-binding sites in Drosophila revealed a considerably more comprehensive picture of how TRF2 may be used as an alternative core-promoter recognition factor. Importantly, mixing and matching various enhancer-binding factors (i.e., sequence-specific DNA-binding factors) and alternative core-promoter recognition factors (i.e., TFIID vs. TRF2) appears to be a powerful and perhaps common strategy for metazoan organisms to diversify transcriptional outputs (Isogai, 2007).
The genome-wide ChIP-on-chip analysis also provides strong evidence that metazoan organisms make much more use of tandem core promoters containing both TFIID and TRF2 recognition sites than might have been anticipated. Whether or not this type of dual core-promoter structure represents a case of redundant pathways or is subject to selective and differential regulation of downstream targets remains unclear. Interestingly, two previously characterized TRF2 targets (PCNA and DNApolα180) appear unaffected when TRF2 is depleted in salivary glands, possibly due to the ability of such dual core promoters to use alternative transcription complexes. Thus, the possibility that TRF2 may be used in lieu of TBP/TFIID to diversify transcriptional outputs in response to specific signals cannot be ruled out. It would be of interest for future studies to determine how these two distinct core-promoter recognition factors TBP/TRF2 operating at dual tandem promoters may be coordinated. Are these core-promoter recognition complexes at tandem core promoters recruited by common or distinct activator proteins? Since salivary gland depletion of TRF2 protein resulted in developmental defects, TRF2 may be necessary to selectively up-regulate genes required for specific developmental pathways (Isogai, 2007).
The identification of direct TRF2 target genes in the present study has revealed a striking link between TRF2 and specific biological processes such as chromatin organization and protein synthesis. Since TRF2 is conserved among many metazoan organisms, its role in various model organisms has been of considerable interest. Several studies found that inactivating TRF2 in nematode, fly, fish, and frog all resulted in lethality due to a block in embryogenesis. In contrast, germ cell-specific functions of TRF2 have also been reported for Drosophila and mice. In particular, while TRF2-null mice appear to display a modest non-Mendelian ratio of inheritance, the major defect manifests as a lack of spermiogenesis. Although these studies revealed that TRF2 provides nonredundant functions during development, these genetic studies were unable to link TRF2 to selective core-promoter recognition functions in vivo. For instance, direct TRF2 target genes responsible for these previously observed phenotypes have not been identified or characterized. The identification of histone H1 and ribosomal proteins as key gene products misregulated in TRF2-depleted Drosophila organs not only provides candidate TRF2 target genes responsible for the chromatin defects observed in TRF2-depleted Drosophila germ cells, but also underscores the potential role of TRF2 in other organisms. For example, TRF2-null mice display a major defect in chromocenter formation in spermatids. This suggests that, consistent with TRF2-mediated H1 regulation in Drosophila somatic cells, TRF2 may also target genes that are essential for chromatin structure in mammalian gonads. However, the precise molecular targets and mechanisms of TRF2 action may differ. Indeed, a recent report points to the involvement of a human DREF homolog in regulating transcription from a TATA-box-containing histone H1 promoter in human cells. In contrast, in Drosophila, it was found that the H1 gene is TATA-less and does not appear to be regulated by DREF (Isogai, 2007).
In addition, the finding that Drosophila TRF2 directs the expression of a large number of gene products critical for essential cell function such as growth (i.e., ribosomal subunits and histones) would be consistent with the lethality associated with the loss of TRF2 in most organisms. These findings also suggest that in mammals TRF2 may play an important role regulating essential cell functions in tissues other than testis. The biological context of TRF2 usage as an alternative core-promoter recognition factor may well be more universal than we had anticipated (Isogai, 2007).
Nucleosome-remodelling factors containing the ATPase ISWI, such as ACF, render DNA in chromatin accessible by promoting the sliding of histone octamers. Although the ATP-dependent repositioning of mononucleosomes is readily observable in vitro, it is unclear to which extent nucleosomes can be moved in physiological chromatin, where neighbouring nucleosomes, linker histones and the folding of the nucleosomal array restrict mobility. In this study arrays were assembled consisting of 12 nucleosomes or 12 chromatosomes (nucleosomes plus linker histone) from defined components and subjected to remodelling by Drosophila ACF or the ATPase CHD1. Both factors increased the access to DNA in nucleosome arrays. ACF, but not CHD1, catalysed profound movements of nucleosomes throughout the array, suggesting different remodelling mechanisms. Linker histones inhibited remodelling by CHD1. Surprisingly, ACF catalysed significant repositioning of entire chromatosomes in chromatin containing saturating levels of linker histone H1. H1 inhibited the ATP-dependent generation of DNA accessibility by only about 50%. This first demonstration of catalysed chromatosome movements suggests that the bulk of interphase euchromatin may be rendered dynamic by dedicated nucleosome-remodelling factors (Maier, 2008).
Due to the abundance of linker histones in interphase chromatin, H1-containing nucleosome arrays are probably the most common and physiological substrate for ATP-dependent chromatin remodelling factors. It is therefore important to understand whether and how these complexes can deal with the linker histone. So far, the literature mostly suggested that linker histones hinder chromatin remodelling. Residual remodelling activity has largely been attributed to incomplete loading of the substrate with linker histones. Attempts were made to rule out this experimental shortcoming by tightly controlling the stoichiometric incorporation of linker histones into chromatin arrays. Yet, ACF was able to induce the movement of entire chromatosome units throughout extended arrays. Importantly, the inability of CHD1 to remodel H1-containing chromatin confirms the inhibitory nature of the chromatosome array. These data are in accordance with previous findings in a crude, undefined system that nucleosome movements can occur within H1-containing chromatin, but they present the first direct demonstration of ATP-dependent chromatosome mobility in a defined chromatin array (Maier, 2008).
The results are surprising in light of the documented impediments of linker histones on nucleosome remodelling. First, H1 binding limits the amount of free linker DNA, which is known to determine the efficiency of ACF-dependent remodelling (Yang, 2006; Gangaraju, 2007). Second, H1 is likely to compete with ISWI-type remodellers for nucleosomal binding sites. In addition, H1 is believed to constrain the path of DNA entering and exiting the nucleosome and may therefore hinder DNA translocation. Finally, the increased compaction promoted by linker histones might restrict the access of remodelling factors towards the chromatin fibre. According to both currently favoured models for the structure of the 30-nm fibre, the linker DNA and hence all points of access for remodelling enzymes are located inside the chromatin fibre. The cation concentrations in these experiments promote the compaction of the nucleosomal array (Maier, 2008).
In spite of these possible constraints, a considerable ACF- and ATP-dependent repositioning of chromatosomes was observed. It is considered that H1 purified from Drosophila embryos might carry modifications, decreasing its affinity for chromatin. For example, the extensive phosphorylation of linker histone C-termini interferes with DNA binding and relieves its inhibitory impact on SWI/SNF-dependent chromatin remodelling. However, mass spectrometrical analysis of histone H1 purified from Drosophila embryos did not reveal extensive phosphorylation. It is therefore considered unlikely that phosphorylation impacted the outcome of these experiments (Maier, 2008).
The inhibitory effect of histone H1 on nucleosome remodelling was apparent when CHD1 was used as a remodelling enzyme. Notably, CHD1's activity on nucleosome arrays was equal to that of ACF, ruling out a defective activity of CHD1. Rather, ACF appears particularly suited for coping with linker histones. This is supported by the observation that ACF can assist the assembly of H1-containing chromatin arrays, whereas CHD1 can only promote assembly of H1-free chromatin. Recently, the ISWI-containing remodelling factor NURF has been suggested to be involved in modulating the association of H1 with chromosomes in vivo (Corona, 2007). The ability to slide chromatosomes may thus be a more widespread property of remodelling enzymes (Maier, 2008).
How might ACF achieve chromatosome repositioning? ACF may directly catalyse the eviction of H1 before nucleosome sliding, and a number of reports indicate that nucleosome-remodelling factors can, in principle, disrupt the DNA interactions of other proteins than core histones. Although it was not possible to detect free linker histones during remodelling, the analysis does not exclude that a fraction of H1 is transiently dislocated to secondary sites on the nucleosome array or an acceptor site on ACF. In vivo, linker histone displacement may be facilitated by cooperating histone chaperones. ACF and the histone chaperone NAP1 can act in concert towards the assembly of H1-containing chromatin, and it is thus conceivable that in cells ACF may cooperate with chaperones to catalyse the reverse reaction, which is the eviction of linker histones. However, since no chaperone was included in this experiment, alternative mechanisms have to be considered (Maier, 2008).
Chromatosome movements might already be facilitated if only the linker histone's globular domain was transiently detached from the nucleosome, while the C-terminal tail remained associated with the linker DNA. Such a scenario is reminiscent of documented changes on H1 interaction due to transcription, where selective crosslinking in Drosophila showed that the globular domain but not the C-terminal tail of linker histones was reversibly displaced from chromatin. In line with these considerations, the C-terminal tail contributes to H1 binding to DNA and determines its residence time on chromatin in living cells (Maier, 2008).
The analysis of chromatosome positions by primer extension revealed that in the arrays H1 protects DNA from nuclease digestion only on one side of the nucleosome, suggesting an asymmetrical binding of H1. This asymmetrical interaction, combined with the repetitive nature of the 601 array, endows the entire array with directionality. Although the precise topography of the ACF-nucleosome complex is not known at present, it has suggested on the basis of site-directed DNA affinity labelling that the related ISW2 complex interacts with linker DNA only on one side of the nucleosome (Kagalwala, 2004; Dang, 2006). It is thus speculated that ACF may interact with nucleosomal linker on the side that is not contacted by the globular domain of H1, in order to initiate the remodelling reaction. Propagation of a 'looped segment' of DNA around the histone octamer would then lead to movement of the histone octamer and concomitant displacement of the globular domain. The domain would then have to relocate and bind to the new nucleosome dyad and DNA entry point. A testable prediction of this hypothesis is that nucleosome sliding in presence of H1 would be unidirectional (Maier, 2008).
It is not knowm at this point whether ACF distributively targets individual nucleosomes within a nucleosome array or rather remodels neighbouring nucleosomes processively. In the latter case the fibre ends may provide points of entry. However, restriction enzyme accessibility assays did not reveal a gradient of increased accessibility towards the ends of the array, as might be expected from such a scenario. In contrast ACF is known to remain bound to its initial substrate during chromatin assembly, and it was observed earlier that nucleosomes within extended arrays were repositioned by Drosophila embryonic extract in apparent synchrony. Further experiments are required to clarify this issue (Maier, 2008).
This study provides the first evidence that ATP-dependent nucleosome-remodelling factors can mobilize entire chromatosomes, even if they reside in extensive arrays. Hence, the majority of euchromatin might be characterized by mobile nucleosomes and chromatosomes (Maier, 2008).
A cell free system of Drosophila preblastoderm embryos was devised for the efficient assembly of cloned DNA into chromatin. The chromatin assembly system utilizes endogenous core histones and assembly factors and yields long arrays of regularly spaced nucleosomes with repeat length of 180 bp. Chromatin assembled with the preblastoderm embryo extract is deficient in histone H1, because of the absence of H1 in early embryos. Exogenous H1 can be incorporated during nucleosome assembly in vitro. When chromatin is reconstituted in the presence of H1, an increased nucleosome repeat length is observed, from 180 bp to about 197 bp, identical to the in vivo repeat length for postblastoderm chromatin. Regular spacing of nucleosomes with or without H1 is sufficient to maximally repress transcription from hsp70 and fushi tarazu gene promoters. There is a modest increase in the level of repression that is dependent on exogenous histone H1. These results show that optimal assembly or regularly spaced nucleosome cores is sufficient to maximally repress transcription in vitro, even in the absence of histone H1 (Becker, 1992).
Chromatin structure must be flexible to allow the binding of regulatory proteins and to accommodate different
levels of gene activity. Chromatin assembled in a cell-free system derived from Drosophila embryos contains an
activity that hydrolyses ATP to render entire nucleosome arrays mobile (See ISWI). Nucleosome movements (most likely
their sliding) occurs even in the presence of the linker histone H1. Binding of more than one linker histone per nucleosome leads to a further compaction of chromatin or aggregation. This could explain the loss of accessability to endonuclease cleavage. The dynamic state of chromatin in the
presence of the ATP hydrolysing factor and ATP globally increases the accessibility of nucleosomal DNA to incoming proteins. This increase can even take place in the presence of H1, but only when H1 is limited to one histone unit per nucleosome. Such an unprecedented demonstration of energy-dependent nucleosome mobility identifies a new principle that is
likely to be fundamental to the mechanism of chromatin remodeling and the binding of regulatory proteins (Varga-Weisz, 1995).
Chromatin reconstituted in an extract taken from preblastoderm Drosophila embryos represses transcription by RNA
polymerase II. Transcriptional repression of immobilized chromatin is largely due to nucleosome
cores. When purified H1 is incorporated into chromatin (resulting in increased repeat lengths to 200-220 bp) the
contribution of H1 to transcriptional repression is negligible. If more H1 is added no regularly spaced
chromatin is obtained; only under these conditions is transcriptional inhibition by H1 apparent. It has been concluded that efficient repression of transcription by polymerase II in this system does not require the presence
of histone H1 (Sandaltzopoulos, 1994).
The 5'-untranslated region of the Drosophila gypsy retrotransposon contains an "insulator," which disrupts the interactions between distally located enhancers and proximal promoter elements. The insulator effect is dependent on the suppressor of Hairy-wing (su[Hw]) protein, which binds to reiterated sites within the 350 base pairs of the gypsy insulator, and additionally acts as a transcriptional activator of gypsy. This study shows that the 350-base pair su(Hw) binding site-containing gypsy insulator behaves as a matrix/scaffold attachment region (MAR/SAR), involved in interactions with the nuclear matrix. In vitro experiments using nuclear matrices from Drosophila, murine, and human cells demonstrate specific binding of the gypsy insulator, not observed with any other sequence within the retrotransposon. Moreover, it is shown that the gypsy insulator, like previously characterized MAR/SARs, specifically interacts with topoisomerase II and histone H1, i.e. with two essential components of the nuclear matrix. Experiments within cells in culture demonstrate differential effects of the gypsy MAR sequence on reporter genes, namely no effect under conditions of transient transfection and a repressing effect in stable transformants, as expected for a sequence involved in chromatin structure and organization (Nabirochkin, 1998).
The presence of a MAR/SAR within gypsy is not totally unexpected, since "boundary" elements are in general regions which contain not only enhancer and insulating elements, but also matrix attachment domains. The rather original feature of the gypsy sequence is that all three domains, which in general are sufficiently "dispersed" so as to allow isolation of "pure" enhancers, MAR/SAR, or insulators, are in the present case "gathered" within a single and relatively short (350 bp) sequence. This rather uncommon situation might in fact be relevant to the pressure for compactness within retroviral sequences, as it is known that retroviruses can only package a limited amount of genetic information. A consequence of compaction is that the gypsy insulator and its associated components are most probably interacting, in vivo, with elements of the nuclear matrix. Accordingly, proteins of the nuclear matrix might play a role in the insulation process, and conversely the su(Hw) protein (which is essential for insulation) might interact with proteins of the matrix. Such interactions could actually account for the data on gypsy insulation and fit with previously proposed models for the gypsy effects (Nabirochkin, 1998).
A first series of data strongly suggested that the gypsy insulator, like all previously characterized insulators, essentially prevents interactions between distal enhancer and promoter, without any direct repressing effect on the enhancer itself. This directional effect can most easily be accounted for by the "looping model" involving generation of structural domains isolated one from the other by attachment of boundary sequences (MAR/SAR) to the nuclear matrix. Alternatively, a series of data on gypsy insulation (essentially in mod(mdg4) mutants) discloses bidirectional repressing effects, which can be accounted for by a model involving heterochromatinization. The present data (showing that the gypsy insulator behaves as a MAR/SAR) are clearly in agreement with the structural looping model, but also support the heterochromatinization model. Indeed, the gypsy MAR/SAR DNA per se, in the absence of su(Hw) protein, is involved in histone H1 nucleation (as shown in this paper), and it has been demonstrated that histone H1 nucleation is associated with both DNA compaction and transcriptional silencing. Additionally, Laemmli and co-workers have found that histone H1 can be removed from MAR/SAR domains by distamycin and distamycin-like proteins (D-like proteins, such as the high mobility group proteins); this has led to the proposal that MAR/SARs can activate or repress transcription of adjacent genes depending on the nucleation/depletion of histone H1. The gypsy MAR/SAR could then be responsible for the repressing effect observed in the mod(mdg4) mutants, as well as in the present assay within heterologous cells (assuming further that appropriate D-like proteins are absent in those cells). Taking into account, in addition, that mutations in the mod(mdg4) or the su(Hw) genes modify position-effect variegation, it could be further hypothesized that the su(Hw)/mod(Mdg4) complex acts as the D-like proteins and modifies the nucleation processes to allow the switch from a repressing to an active state. Accordingly, a model in which the su(Hw) binding sites and the associated su(Hw)/mod(Mdg4) complex modulate the effects of the MAR/SAR DNA sequence could rather simply account for the biological effects of the gypsy insulator in both the wild type and su(Hw)/mod(mdg4) mutants. The proposed model would then reconcile the two previous models for gypsy insulation, i.e. the heterochromatinization and the looping models (Nabirochkin, 1998 and references).
Ubiquitination of histones has been linked to the complex processes that regulate the activation of eukaryotic transcription. However,
the cellular factors that interpose this histone modification during the processes of transcriptional activation are not well characterized.
A biochemical approach has identified the Drosophila coactivator TAFII250, the central subunit within the general transcription factor TFIID, as a histone-specific ubiquitin-activating/conjugating enzyme (ubac). TAFII250 mediates monoubiquitination of histone H1 in
vitro. Point mutations within the putative ubac domain of TAFII250 abolish H1-specific ubiquitination in vitro. In the Drosophila
embryo, inactivation of the TAFII250 ubac activity reduces the cellular level of monoubiquitinated histone H1 and the expression of
genes targeted by the maternal activator Dorsal. Thus, coactivator-mediated ubiquitination of proteins within the transactivation
pathway may contribute to the processes directing activation of eukaryotic transcription (Pham, 2000).
Polyubiquitination represents a mark on proteins that identifies them for degradation and requires the involvement of three enzymes: (1) ubiquitin-activating enzymes (E1), which mediate the adenosine triphosphate
(ATP)-dependent conjugation of E1 with ubiquitin via a
covalent thioester linkage; (2) ubiquitin-conjugating enzymes (E2),
which mediate the transfer of ubiquitin from E1 to E2, conjugate
ubiquitin via thioester bonds and, (3) together with
ubiquitin-protein ligase (E3), link ubiquitin to target proteins via
isopeptide bonds. Polyubiquitination requires
all three enzymes, whereas monoubiquitination of proteins requires E1
and E2 activities only. Unlike polyubiquitination, monoubiquitination of histones has been correlated with activation of gene expression. However, the functional connections between histone ubiquitination and activation of gene expression remain
unknown. Thus, as a first step toward understanding the role of histone
ubiquitination for transcriptional regulation, attempts were made to identify
enzymes that ubiquitinate histones in Drosophila embryonic
nuclear extract using an activity gel assay (Pham, 2000).
Nuclear extract was separated in SDS-polyacrylamide gels containing
histones. After electrophoresis (SDS-PAGE), gel-bound proteins were
subsequently denatured, renatured, and, to monitor enzymatic
activities, incubated with 32P-labeled ubiquitin. By using this assay, a protein was identified with a molecular mass of approximately 200 kD that
mediates ubiquitination of histones. The 200-kD activity coincides with TAFII250, suggesting
that TAFII250 may ubiquitinate histones (Pham, 2000).
TAFII250 most likely does not interact with E1, E2, or E3
enzymes. Since mono-ubiquitination requires at least E1 and E2 activities,
these results imply that TAFII250 may have intrinsic E1 and
E2 activities. The ubiquitin/H1 conjugates resisted reducing agents, suggesting that TAFII250
may mediate a covalent bond between ubiquitin and H1 by means of
isopeptide linkages. Since this enzymatic reaction is characteristic for
E2 enzymes, TAFII250 may have intrinsic E2 activity. The E1 enzyme requires ATP to conjugate with ubiquitin by means of
thioester bonds. Therefore, to explore whether TAFII250 has E1 activity, the capability
of TAFII250 for conjugating with ubiquitin by means of
thioester bonds was investigated. TAFII250 conjugated with
ubiquitin in an ATP-dependent manner in the absence, but not in the
presence, of reducing agents, suggesting that TAFII250 and ubiquitin form a covalent
bond by means of a thioester linkage. Thus, TAFII250 may
have both E1 and E2 activities and may therefore be a ubac (Pham, 2000).
To provide supporting evidence that TAFII250 mediates
ubiquitination of H1, solution assays were used. Reactions containing TAFII250,
32P-labeled ubiquitin, H1, and ATP mediate the formation
of a 39-kD protein that is recognized by antibodies to both ubiquitin
and H1. These results suggest that the 39-kD protein
represents a conjugate composed of one ubiquitin moiety (7 kD) and H1
(32 kD). By contrast, TAFII250 does not ubiquitinate other
histones, H2A/H2B dimers, H3/H4
tetramers, or core nucleosomes. Thus,
TAFII250 mediates monoubiquitination of H1 (Pham, 2000).
To determine the portion of TAFII250 that mediates
monoubiquitination of H1, TAFII250 mutants
truncated at the COOH-terminal were used. Membrane assays indicate that full-length
TAFII250 and 250deltaC850 (lacking the 850 amino acids
closest to the COOH-terminal), but not 250deltaC1300 (lacking the 1300 amino acids closest to the COOH-terminal) ubiquitinate H1. Thus, the H1-specific ubac activity
is likely to reside between amino acids 768 and 1218 (Pham, 2000).
Two Drosophila TAF250 alleles, TAF250XS-2232 and
TAF250S-625, have been described that
contain single-amino acid point mutations that reside within the
putative TAFII250 ubac domain. TAF250XS-2232 contains a valine-1072 to aspartic acid change, and TAF250S-625 an
arginine-1096 to proline change. To investigate the effect of these mutations on
TAFII250 ubac activity, the middle region of
TAFII250 containing amino acids 612 to 1140 (TAF250-M), TAFII250-M-V1072D (containing the V1072 to D mutation), and TAFII250-M-R1096P (containing the R1096 to P
mutation) were subjected to membrane assays. Although
TAF250-M ubiquitinates H1, the mutants do not. Wild-type and
mutant TAFII250-M proteins have histone acetyltransferase activity. The TAF250-M proteins (used for the membrane
assays) acetylate histones; this suggests that the lack of
ubac activity seen with TAF250-M-V1072D and
TAFII250-M-R1096P is most likely not due to a general
functional inactivity of the mutant proteins (Pham, 2000).
In Drosophila, TFIID mediates transcriptional
activation by the maternal activator Dorsal.
Dorsal activates the expression of the mesoderm-determining genes
twist (twi) and snail (sna), which are
transcribed in 20 and 18 of the ventral-most cells of cellularizing
embryos, respectively. To investigate the
functional relevance of TAFII250 ubac activity for
Dorsal-dependent transcriptional activation in vivo, in situ
hybridization was used to monitor twi and
sna expression in Drosophila embryos containing
reduced levels of Dorsal and expressing
TAFII250XS-2232 or
TAFII250S-625, which lack ubac activity in
vitro. Both twi and sna expression are severely
reduced in dl-sensitized,
TAF250XS-2232 embryos
and dl-sensitized, TAF250S-625 embryos,
but not in control embryos. Weak twi mRNA levels were detectable in 10 to 12 cells, and sna expression was restricted to 4 to 12 ventral-most cells and disrupted by gaps. Analyses of
cuticular preparations revealed that dl-sensitized TAF250XS-2232 mutants or dl-sensitized TAF250S-625 mutants, but not control embryos exhibit a dorsalized and twisted body pattern. These results indicate that Dorsal-dependent activation of transcription is impaired in embryos lacking TAFII250 ubac activity (Pham, 2000).
To investigate whether H1 may represent a target for
TAFII250 ubac activity in Drosophila, H1 was purified from nuclei prepared from 0- to 3-hour-old wild-type and
TAFII250 mutant embryos.
Western blot analyses indicate that antibodies to both H1 and ubiquitin
detect a monoubiquitin/H1 conjugate. This result
indicates that at least a fraction of H1 present in early
Drosophila embryos is monoubiquitinated. Moreover, Western
blot analyses indicate that compared with wild-type embryos, mutant
embryos that lack TAFII250 ubac activity contain a
significantly reduced level of monoubiquitinated H1. These
results suggest that TAFII250 ubac activities may
contribute to monoubiquitination of H1 in Drosophila (Pham, 2000).
How coactivators convert activation signals from activation domains of
transcription factors into enhanced levels of mRNA synthesis lies at
the heart of transcriptional regulation. These results suggest that one
coactivator, TAFII250, may use intrinsic ubiquitin-activating/conjugating activities to mediate activation of
transcription. Multiple-alignment analysis and comparison with protein
database sequences reveal that TAF250-M exhibits similarities to E1
and E2 enzymes. Thus, the result that
TAFII250 mediates monoubiquitination of H1 in vitro is in
agreement with other results suggesting that E1 and E2 activities are
sufficient to mediate monoubiquitination of proteins. As point mutations that abrogate TAFII250 ubac activity in vitro also reduce gene expression
in the Drosophila embryo, TAFII250 ubac activity may play an important role for the activation of gene expression in
Drosophila. Although the in vivo targets of
TAFII250 ubac activity remain unknown, the results
that H1 is monoubiquitinated in Drosophila and
that the level of monoubiquitinated H1 is significantly reduced in
embryos lacking TAFII250 ubac activity imply that H1 may
represent one in vivo target of TAFII250. Thus,
ubiquitination of H1 or other proteins within the transcription
machinery, or both, by TAFII250 may constitute an important
coactivator function of TAFII250 and, hence, may allow
TFIID to direct events during the processes of transcriptional
activation (Pham, 2000).
Steroid hormones fulfil important functions in animal development. In Drosophila, ecdysone triggers molting and metamorphosis through its effects on gene expression. Ecdysone works by binding to a nuclear receptor, EcR, which heterodimerizes with the retinoid X receptor homolog Ultraspiracle. Both partners are required for binding to ligand or DNA. Like most DNA-binding transcription factors, nuclear receptors activate or repress gene expression by recruiting co-regulators, some of which function as chromatin-modifying complexes. For example, p160 class coactivators associate with histone acetyltransferases and arginine histone methyltransferases. The Trithorax-related gene of Drosophila encodes the SET domain protein TRR. TRR is a histone methyltransferase capable of trimethylating lysine 4 of histone H3 (H3-K4). trr acts upstream of hedgehog (hh) in progression of the morphogenetic furrow, and is required for retinal differentiation. Mutations in trr interact in eye development with EcR, and EcR and TRR can be co-immunoprecipitated on ecdysone treatment. TRR, EcR and trimethylated H3-K4 are detected at the ecdysone-inducible promoters of hh and BR-C in cultured cells, and H3-K4 trimethylation at these promoters is decreased in embryos lacking a functional copy of trr. It is proposed that TRR functions as a coactivator of EcR by altering the chromatin structure at ecdysone-responsive promoters (Sedkov, 2003).
The products of trithorax group The Drosophila gene skittles encodes a putative PIP5KI, which is required for cell viability and germline and bristle development; sktl mutations affect the ovary, dorsal appendage, egg, and wing. Ash2 and Sktl bind directly to each other in vitro and in vivo and sktl mutations enhance the homeotic transformation phenotype of ash2 mutations. This study also shows that histone H1 hyperphosphorylation within euchromatin is dramatically increased on ash2 and sktl mutant polytene chromosomes. These results support a model in which PIP2 plays a role in maintaining transcriptionally active chromatin via histone H1 modification (Cheng, 2004 and references therein).
A result that shows functional significance of the physical association between Ash2 and Sktl is a similar dramatic increase in histone H1 hyperphosphorylation on both ash2 and sktl mutant chromosomes compared to wild-type chromosomes. Histone H1 is thought to be a general repressor of transcription by RNA polymerase II. The presence of histone H1 affects the ability of transcription factors to interact with DNA and is associated with transcription repression, while the removal of histone H1 is associated with transcriptional activation. Studies in mammals and Tetrahymena have found a correlation between transcriptional activation and increased histone H1 phosphorylation. Dephosphorylated histone H1 bound to chromatin over the mouse mammary tumor virus promoter is thought to restrict chromatin remodeling and transcription factor access. Phosphorylation of histone H1 has also been shown to regulate ATP-dependent chromatin-remodeling enzymes. The effect of phosphorylation is to create a region of negative charge, which may displace histone H1 from chromatin, allowing the binding of specific regulating factors. Alternatively, proteins that regulate transcription may recognize the phosphorylated residues (Cheng, 2004 and references therein).
However, histone H1 hyperphosphorylation has the opposite effect and is linked to high chromatin condensation, possibly by allowing the binding of accessory factors. During mitosis, histone H1 becomes hyperphosphorylated, which may facilitate the interaction with the DNA minor groove and factors involved in metaphase chromosome condensation. Therefore, increased histone H1 hyperphosphorylation as observed in ash2 and sktl mutants implies increased chromosome condensation and reduced transcription (Cheng, 2004).
Ash1 has been shown to be able to methylate K4 of histone H3 and ash1 mutant chromosomes show complete loss of histone H3 K4 methylation. This result suggests that Ash1 is required for all of the histone H3 K4 methylation that occurs in vivo. The S. cerevisiae SET1 complex, which contains two subunits that are thought to represent a bipartite functional homolog of Ash2, has also been shown to methylate K4 of histone H3. In Drosophila, if Ash2 was also in a complex that could methylate histone H3 K4, then it would be predicted that ash2 mutant chromosomes would show a decrease in histone H3 K4 methylation. Indeed, a decrease is seen in histone H3 K4 methylation on ash2 mutant chromosomes (Cheng, 2004 and references therein).
During the assembly of nucleosomes, histone acetylation regulates the binding of histone H1 and chromatin condensation. Displacement of histone H1 is required prior to acetylation of target genes and activation of transcription, because histone H1 inhibits histone H3 acetylation by hindering the access of histone acetyltransferases to the histone H3 tail. It has been predicted that chromatin-remodeling complexes would contain components that modify the interaction of histone H1 with chromatin. Ash2 and Sktl may represent such components. The results suggest that Ash2 and Sktl are direct binding partners that are associated in a complex. When the Ash2-Sktl complex binds to chromatin, a source of PIP2 (Sktl) is brought to the chromatin. PIP2 can bind to and displace histone H1 and/or be metabolized to IP3 and phosphorylated derivatives. The displacement of histone H1 would prevent its hyperphosphorylation and allow for chromatin decondensation, histone acetylation, and eventually, transcription activation. The presence of IP4 and IP5 would also stimulate transcription (Cheng, 2004).
Nuclear extracts from Drosophila Kc cells were used to characterize 3' end processing of Drosophila histone pre-mRNAs. Drosophila Stem-loop binding protein (SLBP) plays a critical role in recruiting the U7 snRNP (Dominski, 2003) to the pre-mRNA and is essential for processing all five Drosophila histone pre-mRNAs. The Drosophila processing machinery strongly prefers cleavage after a fourth nucleotide following the stem-loop and favors an adenosine over pyrimidines in this position. Increasing the distance between the stem-loop and the histone downstream element (HDE) does not result in a corresponding shift of the cleavage site, suggesting that in Drosophila processing the U7 snRNP does not function as a molecular ruler. Instead, SLBP directs the cleavage site close to the stem-loop. The upstream cleavage product generated in Drosophila nuclear extracts contains a 3' OH, and the downstream cleavage product is degraded by a nuclease dependent on the U7 snRNP, suggesting that the cleavage factor has been conserved between Drosophila and mammalian processing. A 2'O-methyl oligonucleotide complementary to the first 17 nt of the Drosophila U7 snRNA was not able to deplete the U7 snRNP from Drosophila nuclear extracts, suggesting that the 5' end of the Drosophila U7 snRNA is inaccessible. This oligonucleotide selectively inhibited processing of only two Drosophila pre-mRNAs and had no effect on processing of the other three pre-mRNAs. Together, these studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing share common features, there are also significant differences, likely reflecting divergence in the mechanism of 3' end processing between vertebrates and invertebrates (Dominski, 2005).
Metazoan replication-dependent histone pre-mRNAs do not contain introns, and the only processing reaction necessary to generate mature histone mRNAs is a single endonucleolytic cleavage of the mRNA precursors (pre-mRNAs) to form the 3' end. Studies on 3' end processing were initially carried out in Xenopus oocytes using synthetic pre-mRNAs and sea urchin histone genes and later were facilitated by the development of an in vitro system based on nuclear extracts from mammalian cells. Replication-dependent histone pre-mRNAs contain two cis elements required for 3' end processing: a highly conserved stem-loop structure consisting of a 6-bp stem and a 4-nt loop and a less conserved histone downstream element (HDE) located ~15 nt 3' of the stem-loop. Mammalian histone pre-mRNAs are cleaved between the two elements, 5 nucleotides downstream of the stem-loop. The stem-loop is recognized by the stem-loop binding protein (SLBP), also referred to as the hairpin binding protein (HBP). The HDE interacts with the U7 snRNP, which contains an ~60-nt U7 snRNA, and this interaction is primarily mediated by base-pairing between the HDE and the 5' end of U7 snRNA. In vitro studies in mammalian nuclear extracts suggest that SLBP stabilizes binding of the U7 snRNP to the pre-mRNA and is essential in processing of only those pre-mRNAs that do not form sufficiently stable duplexes with the U7 snRNA. This role of SLBP in mammalian processing is most likely mediated by ZFP100, a 100-kDa zinc finger protein associated with the U7 snRNP and interacting with the SLBP/stem-loop complex. In addition to bridging the two factors bound to their respective sequence elements, ZFP100 may also play other roles in 3' end processing, possibly including the recruitment of the cleavage factor (Dominski, 2005).
Purification of the U7 snRNP from mammalian cells resulted in identification of two novel Sm-like proteins: Lsm10 and Lsm11, which replace the D1 and D2 Sm proteins present in the spliceosomal snRNPs. Lsm11 interacts in vitro with ZFP100 and plays a key role in recognizing the unique sequence of the Sm binding site in U7 snRNA. Orthologs of Lsm10 and Lsm11 are also found in the Drosophila U7 snRNP, demonstrating that the unique structure of the U7 snRNP in vertebrates and invertebrates is conserved. A counterpart of ZFP100 has not been yet identified in the Drosophila genome, suggesting that ZFP100 is either weakly conserved between vertebrates and invertebrates or processing of histone pre-mRNAs in Drosophila does not require this protein (Dominski, 2005).
Nuclear extracts from Drosophila S-2 and Kc cultured cells and embryos are capable of 3' end processing of presynthesized Drosophila histone pre-mRNAs. Nuclear extracts from Kc cells are also capable of cotranscriptional processing of histone pre-mRNAs. Unlike the auxiliary role played by SLBP in mammalian in vitro processing, Drosophila SLBP is indispensable for processing of all Drosophila histone pre-mRNAs. This observation suggests that Drosophila SLBP plays a much more important role in recruiting the U7 snRNP to the pre-mRNA than it does in the mammalian processing. This study uses an in vitro system based on Drosophila nuclear extracts to characterize 3' end processing of Drosophila histone pre-mRNAs and to define differences and similarities in processing between this model invertebrate processing system and processing in mammalian nuclear extracts (Dominski, 2005).
These studies demonstrate that although Drosophila and mammalian histone pre-mRNA processing occur with similar chemistry and both require SLBP and the U7 snRNP, the two mechanisms differ significantly in the relative importance of these trans-acting factors and in the specification of the cleavage site (Dominski, 2005).
Drosophila nuclear extracts cleave histone pre-mRNAs after the fourth nucleotide following the stem-loop and prefer an adenosine preceding the cleavage site. Consistent with this, all natural Drosophila histone pre-mRNAs contain an adenosine in this position. If the fourth nucleotide is changed to a pyrimidine, cleavage is also efficient after an adenosine at the third position but not after an adenosine located 5 nt downstream of the stem-loop, i.e., at the site exclusively utilized during mammalian processing. Sea urchin histone mRNAs, the only other invertebrate histone mRNAs with the characterized 3' ends, terminate with an ACCA consensus sequence. Thus, cleavage after the fourth nucleotide following the stem-loop may be a general feature of 3' end processing of invertebrate histone pre-mRNAs. Both Drosophila and mammalian processing machineries are similar in their extreme resistance to EDTA, generation of a 3' hydroxyl group at the end of the upstream cleavage product, and degradation of the downstream cleavage product by a U7 snRNP dependent activity. These results suggest that both processing machineries utilize the same or a highly related cleavage factor in 3' end processing of histone pre-mRNAs (Dominski, 2005).
In mammalian processing, the site of cleavage is determined by the position of the HDE, and moving the HDE, and, hence, the U7 snRNP, away from the stem-loop by as few as 4 nt results in a corresponding shift of the cleavage site. This observation led to the hypothesis that U7 snRNP recruits the cleavage factor to the pre-mRNA and acts as a molecular ruler to specify the cleavage site. SLBP bound to the stem-loop facilitates binding of the U7 snRNP to the HDE but does not play a direct role in recruitment of the cleavage factor. Consistent with this model, removal of SLBP, or using a substrate that cannot bind SLBP, reduces processing activity but does not abolish it (Dominski, 2005).
In contrast to mammalian processing, processing of Drosophila histone pre-mRNA is absolutely dependent on SLBP. In addition, increasing the distance between the stem-loop and the HDE by 4 or 8 nt in Drosophila histone pre-mRNA moved the cleavage site only 1 nt upstream from its normal position and did not abolish processing at the normal site. Larger insertions between the stem-loop and the HDE resulted in low efficiency cleavage further away from the stem-loop, but cleavage at these sites was still dependent on SLBP. This is in direct contrast to mammalian histone processing, where cleavage at the distant sites is independent of SLBP. Thus, in Drosophila processing the U7 snRNP does not function as a molecular ruler, but instead SLBP plays the critical role in specifying the cleavage site (Dominski, 2005).
To explain the observed differences between processing in Drosophila and mammalian nuclear extracts, it is proposed that within the Drosophila processing complex SLBP tightly interacts with the U7 snRNP, and this interaction is essential for bringing the U7 snRNP to the pre-mRNA. The two factors remain associated even if their respective binding sites are separated by a larger distance, likely by looping out the inserted nucleotides. The mutant pre-mRNAs are preferentially cleaved close to the stem-loop, reflecting the critical role of SLBP in forming the processing complex, although the precise position of the cleavage site and efficiency of processing depends on the size of the insert. In mammalian processing, the region between the stem-loop and the HDE is either rigidified, thus precluding looping out the inserted nucleotides, as previously suggested, or the interaction between SLBP and the U7 snRNP is relatively weak and disrupted by larger insertions, so binding of the U7 snRNP to the pre-mRNA depends solely on the base-pairing interaction. It is likely that in Drosophila processing the cleavage factor is recruited to histone pre-mRNA by interaction with both the U7 snRNP and SLBP, and neither factor is competent to carry out this function individually (Dominski, 2005).
In mammalian nuclear extracts, processing of histone pre-mRNAs is efficiently inhibited by relatively short 2'O-methyl oligonucleotides complementary to the 5' end of the mammalian U7 snRNA. These oligonucleotides, including a 10-mer, are also very efficient in depleting the U7 snRNP from nuclear extracts and were successfully used to affinity purify U7 snRNP from mammalian cells, demonstrating that the 5' end of the mammalian U7 snRNA is readily accessible. In contrast, two relatively long oligonucleotides, alphaDa, complementary to the first 17 nt of the Drosophila U7 snRNA, and alphaDb, complementary to nt 4-23, were not effective in depleting the U7 snRNP from Drosophila nuclear extracts. These results suggest that the 5' end of U7 snRNA is not accessible in the Drosophila U7 snRNP (Dominski, 2005).
Surprisingly, the alphaDa 2'O-methyl oligonucleotide abolished processing of the dH3* and dH1* pre-mRNAs (hybrid pre-mRNAs consisting of the stem-loop and cleavage site from the mouse H2a-614 pre-mRNA) but did not significantly affect processing of the other three Drosophila histone pre-mRNAs. Three additional oligonucleotides complementary to the regions of the U7 snRNP located closer to the Sm binding site effectively blocked processing of all five histone pre-mRNAs. It is not understood why processing of only two Drosophila pre-mRNAs is affected by the alphaDa oligonucleotide and which features of the HDEs make processing of the Drosophila pre-mRNAs either sensitive or resistant to this oligonucleotide. Selective inhibition of processing by the alphaDa oligonucleotide depending on the type of pre-mRNA used in the reaction suggests that blocking of the U7 snRNA must occur during processing. One possibility is that the U7 snRNP is initially recruited to the pre-mRNA solely by SLBP bound to the pre-mRNA, and later this interaction is followed by formation of a duplex between the HDE and the U7 snRNA, as a result of unmasking of the 5' end of U7 snRNA. The alphaDa oligonucleotide might block binding of the U7 snRNA to the HDE in the hybrid dH1* and dH3* pre-mRNAs, but not in the other pre-mRNAs, during this later step, while the other oligonucleotides block binding to all the HDEs (Dominski, 2005).
Overall, thes studies indicate that the structure of the 5' end of the Drosophila U7 snRNA and the mechanism of its initial interactions with the HDE differ significantly from the recognition of the HDE in processing of mammalian histone pre-mRNAs (Dominski, 2005).
In vitro processing of all five Drosophila histone pre-mRNAs is absolutely dependent on SLBP. This study has demonstrated that SLBP is essential for recruitment of the U7 snRNP to the pre-mRNA. The necessity of SLBP for recruitment of the U7snRNP to the Drosophila pre-mRNAs suggests that either Drosophila HDEs are unable to form a strong duplex with the U7 snRNA or that the interaction of the U7 snRNP with the SLBP/pre-mRNA complex is necessary to promote base-pairing by making the 5' end of U7 snRNA accessible (Dominski, 2005).
Both the 5' end of the Drosophila U7 snRNA and Drosophila HDEs are AU rich, allowing a number of possible base-pair schemes for making a duplex between the two RNAs. It is hypothesized that the most likely alignment used during processing is the one that allows formation of the largest number of base pairs between the purine core of the HDE and the CUCUUU sequence in the U7 snRNA and not necessarily the alignment that allows formation of the overall most stable duplex. The CUCUUU sequence is highly conserved among all known U7 snRNAs and is involved in recognition of the purine core in sea urchin and mammalian histone pre-mRNAs. A 3-nt mutation within the purine core of the hybrid dH3* pre-mRNA abolishes processing, whereas a 6-nt mutation within the AU-rich region immediately downstream of the purine core only partially inhibits processing. These results support the interpretation that base-pairing between the U7 snRNA and the purine core is critical, whereas formation of additional base in other regions increases the efficiency of Drosophila processing. It is also possible that the base-pairing interaction is limited to the purine core and the CUCUUU sequence in the U7 snRNA, whereas the AU-rich sequences in the U7 snRNA and the HDE are brought together by protein-protein interactions (Dominski, 2005).
This study demonstrated that the HDE of the hybrid dH3* pre-mRNA can abolish processing of the full-length substrate, presumably by sequestering the U7 snRNP, only when present at very high concentrations. Interestingly, this weak interaction of Drosophila HDEs with the U7 snRNP is sufficient to recruit a 5'-3' exonuclease that specifically degrades the downstream cleavage product in a U7 dependent manner. Thus, the endonucleolytic cleavage must require much stronger binding of the U7 snRNP to the pre-mRNA, while degradation of the DCP by an exonuclease may require only loose association of the HDE with the U7 snRNP (Dominski, 2005).
The most notable difference between histone pre-mRNA processing in Drosophila and mammalian nuclear extracts is the absolute dependence of Drosophila processing on SLBP and the role of SLBP in specifying the cleavage site close to the stem-loop. The Drosophila U7 snRNP does not function as a molecular ruler in processing and this feature most likely reflects a critical role of SLBP in recruiting the cleavage factor as well as the U7 snRNP, to histone pre-mRNA. These data suggest that SLBP and the U7 snRNP may form a tight complex on the histone pre-mRNA, and this complex remains stable even in the presence of large insertions between the stem-loop and the HDE (Dominski, 2005).
The similarities in the chemistry of the cleavage reaction, including preference for an adenosine preceding the cleavage site and generation of the 3'OH group in the presence of EDTA, as well as degradation of the downstream cleavage product by a U7-dependent 5'-3' exonuclease suggest that the cleavage factor has been conserved between Drosophila and mammalian processing. It will be of interest to determine whether there are factors unique to only one of these two types of organisms emphasizing long evolutionary distance and the divergence between vertebrates and invertebrates (Dominski, 2005).
Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem–loop structure generated by an endonucleolytic cleavage involving the U7 snRNP, which interacts with histone pre-mRNAs through base-pairing between U7 snRNA and a purine-rich sequence in the pre-mRNA located downstream of the cleavage site. Null mutations of the single Drosophila U7 gene were generated and U7 snRNA was demonstrated to be required in vivo for processing all replication-associated histone pre-mRNAs. Mutation of U7 results in the production of poly A+ histone mRNA in both proliferating and endocycling cells because of read-through to cryptic polyadenylation sites found downstream of each Drosophila histone gene. A similar molecular phenotype also results from mutation of Slbp, which encodes the protein that binds the histone mRNA 3' stem–loop. U7 null mutants develop into sterile males and females, and these females display defects during oogenesis similar to germ line clones of Slbp null cells. In contrast to U7 mutants, Slbp null mutations cause lethality. This may reflect a later onset of the histone pre-mRNA processing defect in U7 mutants compared to Slbp mutants, due to maternal stores of U7 snRNA. A double mutant combination of a viable, hypomorphic Slbp allele and a viable U7 null allele is lethal, and these double mutants express polyadenylated histone mRNAs earlier in development than either single mutant. These data suggest that SLBP and U7 snRNP cooperate in the production of histone mRNA in vivo, and that disruption of histone pre-mRNA processing is detrimental to development (Godfrey, 2006).
Chromosome duplication during the cell cycle requires the production of histones during S phase to package newly replicated DNA into chromatin. Bulk histone production during S phase is achieved through the biosynthesis of replication-dependent histone mRNAs, which are cell-cycle regulated and accumulate only in S phase. In animal cells these histone mRNAs are unique: The 3' end terminates in a conserved 26-nt sequence that forms a stem–loop rather than in a poly A+ tail. Since histone genes lack introns, the only processing step required for mature histone mRNA production is endonucleolytic cleavage of the pre-mRNA to form the 3' end of the mRNA. Much of the cell-cycle regulation of histone mRNAs is post-transcriptional and is mediated by the 3' end of the mRNA. Thus, a complete understanding of cell-cycle-regulated histone mRNA production requires a full understanding of the factors required for histone pre-mRNA processing (Godfrey, 2006).
The processing of histone pre-mRNAs requires two cis elements and a number of trans-acting factors. The cis elements are the stem–loop at the 3' end of histone mRNA and a purine-rich region downstream of the cleavage site, termed the histone downstream element (HDE). A protein called stem–loop binding protein (SLBP) or hairpin binding protein (HBP) specifically binds the 3' end of histone mRNA. SLBP is required for histone pre-mRNA processing in vivo and accompanies the mRNA to the cytoplasm, where it promotes the translation of the histone mRNA. The HDE binds U7 snRNP by base-pairing with the 5' end of U7 snRNA. In mammals, SLBP, the U7 snRNP, and a U7 snRNP-associated zinc finger protein called ZFP100 cooperate to recruit an endonuclease complex that cleaves the pre-mRNA. Recent evidence indicates that CPSF73, a component of the complex that mediates AAUAAA-directed cleavage prior to polyadenylation, is the likely endonuclease. This revealed some unexpected overlap in the machinery carrying out histone pre-mRNA processing and canonical polyadenylation (Godfrey, 2006).
The U7 snRNA is a small RNA (55–70 nt) that, like the spliceosomal snRNAs, contains both a trimethyl guanosine cap and an Sm binding site, which is essential for its function. The Sm site in these snRNAs stably binds a complex of seven related proteins of the LSm/Sm family to form the core snRNP particle. Proteins of the LSm/Sm family share a common tertiary structure called the Sm fold that assembles into hexameric or heptameric rings capable of binding single-stranded RNA. The U snRNPs contain a heptameric Sm ring, with each of the seven individual subunits making a specific contact with a residue in the Sm binding site of the snRNA. The heptameric Sm ring of spliceosomal snRNPs contains the proteins SmB/B', SmD1, SmD2, SmD3, SmE, SmF, and SmG. In contrast, the U7 snRNP contains five of these Sm proteins (B/B1, D3, E, F, G) and two novel Sm proteins called LSm10 and LSm11 that replace SmD1 and SmD2 of the spliceosomal snRNPs. The Sm site found in U7 snRNAs is distinct from the Sm site in spliceosomal snRNAs and is responsible for incorporation of LSm10 and LSm11 into the U7 snRNP. In addition to the Sm fold that participates in ring formation, LSm11 contains an NH2 terminal extension that makes contacts with ZFP100 and possibly other components of the histone pre-mRNA processing machinery (Godfrey, 2006).
The role of U7 snRNP in histone pre-mRNA processing has been examined primarily in nuclear extract systems that support the processing of synthetic histone pre-mRNAs, and by monitoring the processing of histone pre-mRNAs injected into Xenopus ooctyes. Complementary mutations in U7 snRNA and the HDE provided early evidence that base-pairing between the 5' end of U7 and the HDE was an important part of U7 snRNP function. Furthermore, blocking the 5' end of the U7 snRNA with a complementary oligonucleotide specifically inhibits processing of synthetic histone pre-mRNAs in nuclear extracts. However, the contribution of U7 snRNA to endogenous histone mRNA biosynthesis and whether this contribution is important for animal development have not been examined. To explore these issues, U7 snRNA mutations in Drosophila were generated and characterized (Godfrey, 2006).
Drosophila SLBP, U7 snRNA, and U7 snRNP specific proteins Lsm10 and Lsm11, have all been identified, and steps have been taken to characterize them genetically. Mutations in the Drosophila Slbp gene block normal histone pre-mRNA processing during embryonic development and result in production of polyadenylated histone mRNAs as a consequence of read-through past the normal processing site. This occurs because each of the five Drosophila histone genes contains cryptic polyadenylation sites downstream of the HDE that are utilized in the absence of SLBP. Null mutations of Slbp cause lethality during larval and pupal stages, presumably because of the histone processing defects, although the precise cause of lethality is not known. Slbp mutant cells are capable of replicating chromatin, likely because the inappropriate polyadenylated mRNAs are translated. A hypomorphic Slbp mutant allele that produces reduced amounts of SLBP protein results in the production of both normal and poly A+ histone mRNAs during embryogenesis, but does not cause lethality. However, these viable mutant females lay eggs that contain reduced amounts of histone mRNA and protein and do not develop. Thus, SLBP is required during both zygotic development and oogenesis (Godfrey, 2006).
This study compared mutations in the U7 snRNA gene, and the resulting phenotypes were compared with those caused by mutation of Slbp. The results indicate that U7 snRNA is required for normal histone mRNA biosynthesis during Drosophila development and that, like Slbp mutations, loss of U7 snRNA results in the production of polyadenylated histone mRNAs. However, unlike Slbp null mutants, U7 null mutants are viable, but both males and females are sterile. This difference in terminal phenotype is most likely because the maternal supply of U7 snRNA delays the onset of the histone processing defect in U7 mutants relative to Slbp mutants, which do not have a significant maternal supply of SLBP protein. Both U7 and SLBP are required for normal histone mRNA biosynthesis in the female germ line, and mutation of either gene disrupts oogenesis. These data indicate that loss of SLBP and U7 cause similar molecular phenotypes in Drosophila and suggest that early expression of this molecular phenotype prevents normal development (Godfrey, 2006).
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