BIOLOGICAL OVERVIEW

TBP-related factor 2 (TRF2) selectively regulates the TATA-less Histone H1 gene promoter, while TBP/TFIID targets core histone transcription

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 anticipated (Isogai, 2007).

The chromodomain-containing NH(2)-terminus of Chromator interacts with histone H1 and is required for correct targeting to chromatin

The chromodomain protein, Chromator, can be divided into two main domains, a NH(2)-terminal domain (NTD) containing the chromodomain (ChD) and a COOH-terminal domain (CTD) containing a nuclear localization signal. During interphase Chromator is localized to chromosomes; however, during cell division Chromator redistributes to form a macromolecular spindle matrix complex together with other nuclear proteins that contribute to microtubule spindle dynamics and proper chromosome segregation during mitosis. It has previously been demonstrated that the CTD is sufficient for targeting Chromator to the spindle matrix. This study shows that the NTD domain of Chromator is required for proper localization to chromatin during interphase and that chromosome morphology defects observed in Chromator hypomorphic mutant backgrounds can be largely rescued by expression of this domain. Furthermore, this study shows that the ChD domain can interact with histone H1 and that this interaction is necessary for correct chromatin targeting. Nonetheless, that localization to chromatin still occurs in the absence of the ChD indicates that Chromator possesses a second mechanism for chromatin association and evidence is provided that this association is mediated by other sequences residing in the NTD. Taken together these findings suggest that Chromator's chromatin functions are largely governed by the NH(2)-terminal domain whereas functions related to mitosis are mediated mainly by COOH-terminal sequences (Yao, 2012).

The chromodomain protein, Chromator, has multiple functions depending on the developmental context. During interphase Chromator is localized to interband regions of Drosophila polytene chromosomes and has been demonstrated to interact with other chromosomal proteins such as the zinc-finger protein Z4 and the histone H3S10 kinase JIL-1 and to contribute to the maintenance of polytene chromosome morphology. However, during cell division Chromator redistributes to form a macro molecular spindle matrix complex together with at least three other nuclear derived proteins Skeletor, Megator, and EAST. It has recently been proposed that this structure may take the form of a hydrogel-like matrix with viscoelastic properties that contribute to microtubule spindle dynamics and proper chromosome segregation during mitosis. Evidence that Chromator may participate in spindle matrix function has been provided by mutational analysis with two loss-of-function alleles, Chro⁷¹ and Chro⁶¹². The analysis showed that neuroblasts from Chro⁷¹/Chro⁶¹² brain squash preparations have severe microtubule spindle and chromosome segregation defects that were associated with a developmental small brain phenotype. Furthermore, time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggested that the chromosome segregation defects were the results of incomplete alignment of chromosomes at the metaphase plate, possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase (Yao, 2012).

Chromator can be divided into two main domains, an NH₂-terminal domain (NTD) containing the chromodomain (ChD) and a COOH-terminal domain (CTD) containing a nuclear localization signal. Studies have shown that the CTD of Chromator was sufficient for localization to spindles and that expression of this domain alone could partially rescue mutant spindle defects. However, the function of the NTD and whether it plays a role in targeting Chromator to chromatin was not determined. This study provides evidence that the NTD of Chromator is responsible for correct targeting to chromatin, that it interacts with histone H1, and that the chromodomain is required for these interactions (Yao, 2012).

This study shows that the NTD domain of Chromator is required for proper localization to chromatin and that chromosome morphology defects observed in Chromator mutant backgrounds can be largely rescued by expression of this domain. Evidence is provided that the ChD domain can interact with histone H1 suggesting that this interaction is necessary for the correct chromatin targeting. Nonetheless, that localization to chromatin still occurs in the absence of the ChD indicates that Chromator possesses a second mechanism for chromatin association, and evidence is provided that this association is mediated by other sequences residing in the NTD. Such an association could in principle be mediated by other molecular interaction partners of Chromator that also localize to chromatin such as JIL-1 or Z4. However, studies in S2 cells with RNAi mediated Chromator depletion and in JIL-1^z2 homozygous null mutant backgrounds demonstrated that neither protein was dependent on the other for its chromatin localization. The interaction of Chromator with Z4 was identified in co-immunoprecipitation experiments and the two proteins colocalize extensively at interband polytene regions. Recently, evidence was provided that Chromator and Z4 may directly interact and that localization of Z4 to chromatin depends on Chromator, but not vice versa. Another candidate for mediating chromatin localization is Skeletor. The interaction between Chromator and Skeletor was first detected in a yeast two-hybrid screen and subsequently confirmed by pull-down assays. Immunocytochemical labeling of Drosophila embryos, S2 cells, and polytene chromosomes demonstrated that the two proteins show extensive co-localization during the cell cycle although their distributions are not identical. During interphase Chromator is localized on polytene chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. During mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure with Chromator additionally localizing to centrosomes that are devoid of Skeletor-antibody labeling. Thus, the extensive co-localization of the two proteins is compatible with a direct physical interaction; however, at present it is not known whether such an interaction occurs throughout the cell cycle or is present only at certain stages, with additional proteins mediating complex assembly at other stages. Regardless, it is likely that Chromator together with Skeletor functions in at least two different molecular complexes, one associated with the spindle matrix during mitosis and one associated with nuclear and chromatin structure during interphase. Furthermore, taken together these findings suggest that Chromator's chromatin functions are largely governed by the NH₂-terminal domain whereas functions related to mitosis are mediated by COOH-terminal sequences. The molecular mechanisms of how the two distinct chromatin binding affinities residing within the NH₂-terminal domain of Chromator interact to confer proper localization to interbands remains to be elucidated (Yao, 2012).

An important feature of the Chromator protein is the presence of a chromodomain, the only conserved motif found in database searches . Structure determination of the prototype chromodomain has revealed a small, three-stranded antiparallel β-sheet supported by an α-helix that runs across the sheet. Classic chromodomains contain three conserved aromatic amino acids that confer binding affinity for methylated histone H3. However, several chromodomains have been identified that vary at some of these structurally important positions but that still conform well to the overall folding of the prototype chromodomain. One example of this is the chromo-shadow domain also found in HP1a that is a protein-protein interaction domain that allows HP1a to homodimerize via its α-helices. In addition, various chromodomains have been demonstrated to bind to a wide variety of proteins including transcription corepressors, remodeling ATPases, lamin B receptor, and chromatin assembly factors. Thus, relatively small sequence variations in the otherwise conserved structural scaffold of chromodomains can confer considerable variation in molecular interactions. Evidence is provided by modeling that the chromodomain of Chromator is likely to adopt the canonical chromodomain tertiary configuration very similar to the chromodomain of HP1a. However, due to amino acid substitutions at two of the three conserved aromatic amino acid positions it is not likely to bind to methylated histone H3. Rather this study provides evidence by overlay and pull down assays that it binds to the linker histone H1. A candidate region for providing such a binding fold or surface is the additional α-helical stretch found in the chromodomain of Chromator just prior to the main α-helix of the chromodomain structure. In future experiments it will be of interest to further determine the structural basis for the interaction of Chromator with histone H1 and specifically how the chromodomain contributes to Chromator's role in nucleosome and chromatin organization (Yao, 2012).

Regulatory functions and chromatin loading dynamics of linker histone H1 during endoreplication in Drosophila

Eukaryotic DNA replicates asynchronously, with discrete genomic loci replicating during different stages of S phase. Drosophila larval tissues undergo endoreplication without cell division, and the latest replicating regions occasionally fail to complete endoreplication, resulting in underreplicated domains of polytene chromosomes. This study shows that linker histone H1 is required for the underreplication (UR) phenomenon in Drosophila salivary glands. H1 directly interacts with the Suppressor of UR (SUUR) protein and is required for SUUR binding to chromatin in vivo. These observations implicate H1 as a critical factor in the formation of underreplicated regions and an upstream effector of SUUR. It was also demonstrated that the localization of H1 in chromatin changes profoundly during the endocycle. At the onset of endocycle S (endo-S) phase, H1 is heavily and specifically loaded into late replicating genomic regions and is then redistributed during the course of endoreplication. The data suggest that cell cycle-dependent chromosome occupancy of H1 is governed by several independent processes. In addition to the ubiquitous replication-related disassembly and reassembly of chromatin, H1 is deposited into chromatin through a novel pathway that is replication-independent, rapid, and locus-specific. This cell cycle-directed dynamic localization of H1 in chromatin may play an important role in the regulation of DNA replication timing (Andreyeva, 2017).

This study demonstrated that virtually all major sites of UR throughout the Drosophila genome exhibit a substantial increase in salivary gland DNA copy number upon depletion of the linker histone H1, thus implicating H1 in the regulation of endoreplication. In control knockdown salivary glands, 46 underreplicated domains were identified. While these regions are in general agreement with previous efforts to map underreplicated domains by less sensitive microarray analyses, fewer underreplicated sites were identified than a recent report that used high-throughput sequencing of salivary gland DNA. Notably, the underreplicated domains that the current analyses failed to detect represent sites with the weakest degree of UR. One possible source of variation is the distinct technical approach that was used compared with Yarosh (2014), as simultaneous sequencing of a nonpolytenized (embryonic) genome as a means to normalize the reads from underrepresented sequences in polytenized tissues (Yarosh, 2014) likely provides additional sensitivity. Another potential explanation could lie in the relative sequencing depth of the respective assays (approximately fourfold lower in the current study), considered crucial for the analyses of next-generation sequencing data. However, this explanation is less likely, as subsampling of the current reads to much lower depths yielded no appreciable difference in the number and location of identified underreplicated sites or the change in copy number upon H1 knockdown (Andreyeva, 2017).

On average, a moderate knockdown of H1 led to an ~50% copy number gain at the center of underreplicated domains in intercalary heterochromatin (IH; large dense bands scattered in euchromatin comprising clusters of repressed genes. The copy number is not restored to the same degree as that in a SuUR genetic mutant. The difference is likely attributable to the incomplete depletion of H1. In fact, in an independent biological validation experiment that resulted in an ~95% depletion of H1, an almost complete restoration of copy number was observed. The observation of an almost complete reversal of UR in cells depleted of H1 (but still wild type for SuUR) strongly suggests an epistatic mechanism of action in which both H1 and SUUR act together in the same biochemical pathway (Andreyeva, 2017).

This study found that H1 and SUUR are also involved in UR of PH. For instance, both the mapped pericentric regions and TE sequences, which are highly abundant in pericentric regions, exhibit an increase of DNA copy number upon H1 knockdown. The SuURES mutation also results in a robust loss of UR at PH, as measured by changes in DNA copy number at TEs. The abrogation of H1 expression gives rise to a somewhat weaker effect on the UR of PH than that of IH, which is consistent with an almost complete elimination of SUUR protein from polytene chromosome arms in salivary glands depleted of H1 by RNAi but the persistence of residual SUUR at their PH. The role of H1 in maintaining the underreplicated state of PH may be relevant to its important regulatory functions in constitutive heterochromatin, where it recruits Su(var)3-9, facilitates H3K9 methylation, and maintains TEs in a transcriptionally repressed state. Recently, it was proposed that TE repression in ovarian somatic cells involves an H3K9 methylation-independent process through recruitment of H1 by Piwi-piRNA complexes, resulting in reduced chromatin accessibility. The current results also implicate UR of TE sequences in polytenized cells as yet another putative mechanism that contributes to regulation of their expression. Interestingly, it was shown previously that double mutants encompassing both the Su(var)3-9 and SuUR mutant alleles exhibit a synthetically increased predominance of novel band-interband structures at PH compared with the mutation of SuUR alone. While the evidence suggests a relationship between UR and transcriptionally repressive epigenetic states, such as H3K9 methylation, the nature of this relationship remains largely speculative (Andreyeva, 2017).

This study demonstrated that SUUR protein physically interacts with H1 in both a complex mixture of whole-cell extracts that contain endogenous native H1 and recombinant purified H1 polypeptides. Furthermore, the particular structural domains of the two proteins were delimited that are required for the interaction. SUUR protein contains several sequence features that have been implicated in regulation of UR and binding to specific proteins. Although SUUR possesses a putative bromodomain, it contains no identifiable DNA-binding domain, so the mechanism that allows SUUR to exhibit a preference for specific genomic underreplicated loci is unknown. The positively charged central region is both necessary and sufficient to interact with heterochromatin protein 1a (HP1a), which suggests a possible involvement of HP1a in tethering SUUR to H3K9me2/3-rich PH. However, the specific localization of SUUR to underreplicated IH, which is not enriched for H3K9me2/3, remains enigmatic. This study now demonstrates that the central region of SUUR is also sufficient for binding directly to H1 in vitro. Considering that the central region of SUUR is essential for the faithful localization of the protein to chromatin in vivo, including underreplicated IH, it seems likely that H1 directly mediates the tethering of SUUR to chromatin in underreplicated regions (Andreyeva, 2017).

The tripartite structure of H1 provides multiple binding interfaces for interacting proteins and thus allows H1 to mediate several biochemically separable functions in vivo. For instance, the globular domain and proximal 25% of the CTD are required for H1 loading into chromatin, while the proximal 75% of the CTD is needed for normal polytene morphology, H3K9 methylation, and physical interactions with Su(var)3-9. This study discovered a previously unknown function for the distal 25% of the H1 CTD, which is shown to be essential for binding to SUUR. Deletion of this region of H1 results in a near-complete loss of the interaction with SUUR. Thus, in addition to its critical functions in heterochromatin structure and activity, the CTD of H1 is likely also important in facilitating UR (Andreyeva, 2017).

One of the most striking findings in this study is the observation that the genomic occupancy of H1 undergoes profound changes during the endoreplication cycle. It also remains largely mutually exclusive with that of DNA polymerase clamp loader PCNA, which is consistent with the observed depletion of H1 in nascent chromatin compared with mature chromatin (Andreyeva, 2017).

H1 is heavily loaded into late replicating loci at the onset of replication (when these loci are silent for replication). Combined, the current observations indicate that the chromosome distribution of H1 during the endocycle is governed by at least three independent processes. Two of them [replication-dependent (RD) eviction of H1 and RD deposition of H1 after the passage of replication fork] are related to the well-recognized obligatory processes of chromatin disassembly and reassembly during replication. The third pathway, which directs early deposition of H1 into late replicating loci, has not been described previously. This process is (1) replication-independent (RI); (2) locus-specific, with a strong preference for late replicating sites; and (3) apparently more rapid than the RD deposition of H1, since very high levels of H1 occupancy are observed in all nuclei immediately after the initiation of endo-S. It is possible that the RI pathway of H1 loading into chromatin is mediated by a selective recruitment of H1 based on epigenetic core histone modification-dependent mechanisms. For instance, mammalian H1.2 was reported to recognize H3K27me3, and this modification is very abundant in IH (Sher et al. 2012) (Andreyeva, 2017).

Also, the RI mechanism for deposition of H1 probably does not involve de novo nucleosome assembly, as H1 is known to exhibit a mutually exclusive distribution with RI core histone variants, and there is no known nuclear process during early S phase that requires core histone turnover. In the future, it will be interesting to further confirm that RI nucleosome assembly does not take place during early replication in salivary gland polytene chromosomes. Finally, the locus-specific RI deposition of H1 in early endo-S chromatin may be conserved in the normal S phase of diploid tissues, and it will require independent experimentation with sorted mitotically dividing cells to confirm this possibility (Andreyeva, 2017).

This study also provides cytological evidence that the functions of H1 and SUUR are biochemically linked. Specifically, it was demonstrated that SUUR localizes to a subset of H1-positive bands and requires H1 for its precise distribution in polytene chromosomes, nuclear localization, and stability in salivary gland cells. These observations implicate H1 as an upstream effector of SUUR functions in vivo and an essential component of the biological pathway that maintains loci of reduced ploidy in polytenized cells. Importantly, this finding adds to a growing list of biochemical partners of H1 that mediate their chromatin-directed functions in an H1-dependent fashion (Andreyeva, 2017).

Interestingly, even a moderate depletion of H1 (to ~30% of normal) results in a complete removal of SUUR from chromosome arms. Thus, H1-dependent localization of SUUR requires high concentrations of the linker histone in chromatin. This conclusion is also consistent with SUUR colocalization with polytene loci that are the most strongly stained for H1. In contrast, elimination of the H3K9me2 mark from polytene spreads requires very extensive depletion of H1, whereas the moderate depletion of H1 does not strongly affect H3K9 dimethylation in the chromocenter or polytene arms. Therefore, the robust effect of even moderate H1 depletion on SUUR localization in chromatin is unlikely to be mediated indirectly through disorganization of heterochromatin structure (Andreyeva, 2017).

Unexpectedly, the cell cycle-dependent temporal pattern of H1 localization is not identical to that of SUUR. In contrast to H1, SUUR protein (1) is only weakly present in IH during early endo-S phase, (2) achieves the maximal occupancy at IH loci only in the late endo-S, and (3) colocalizes with PCNA at certain sites. The observations made in this study and in previous works can be summarized in the following model for H1-mediated regulation of SUUR association with chromatin. The initiation of the deposition of SUUR in chromosomes is strongly dependent on H1. More specifically, SUUR is preferentially localized to chromatin domains that are highly enriched for H1. For instance, the tremendously elevated concentration of H1 in IH of early endo-S cells promotes and nucleates the initiation of deposition of SUUR into these regions. However, the pattern of SUUR occupancy at these sites does not occur temporally in parallel with that of H1. Initially, the exceptionally high abundance of H1 in late replicating loci during early endo-S is not paralleled by a simultaneous comparable increase of SUUR occupancy. Rather, loading of SUUR into these sites lags significantly behind H1 occupancy. Thus, the rate of SUUR localization to H1-rich IH appears to be much slower than that of the RI deposition of H1 into these loci. After the initial recruitment, further loading of SUUR does not require H1, and SUUR continues (in a slower fashion) to accumulate at IH throughout the endo-S phase even when H1-enriched domains dissipate in the course of DNA endoreplication. The additional loading of SUUR in chromatin is likely facilitated by its self-association through dimerization of the N terminus and physical interactions with the replication fork, as proposed previously. In this fashion, SUUR achieves its maximal concentration in IH loci by the late endo-S (Andreyeva, 2017).

This study has demonstrated that H1 has a pivotal function in the establishment of UR of specific IH loci in polytenized salivary gland cells. The findings that H1 interacts directly with SUUR in vitro and is required for SUUR localization to late replicating IH in polytene chromosomes in vivo strongly suggest that the H1-mediated recruitment of SUUR promotes UR by obstructing replication fork progression in its cognate underreplicated loci but does not affect replication origin firing. However, the remarkable temporal pattern of H1 distribution in endoreplicating polytene chromosomes suggests that it may also play a direct SUUR-independent role in regulation of endoreplication. This is especially plausible considering that the temporal distribution patterns of SUUR and H1 are dissimilar (Andreyeva, 2017).

In contrast to the role of SUUR in slowing down the replication fork progression during late endo-S phase, H1 (acting in the absence of SUUR during early endo-S) may function to repress the initiation of endoreplication, as proposed in several studies. DNA-seq analyses also suggest this mechanism. Compared with the relatively smooth, flat profiles of DNA copy numbers in SuURES mutant salivary glands, the profiles in H1-depleted cells exhibit a jagged, uneven appearance, indicative of aberrant local initiation of replication. Unfortunately, the experimental system (cytological analyzes of salivary glands) cannot be used to further confirm this idea. First, an extensive depletion of H1 results in the loss of polytene morphology; second, since the staging of endo-S progression is based on PCNA staining, a spurious activation of ectopic replication origins would result in an incorrect calling of the stage. To further complicate these analyses, polytenized cells are not amenable to other methods of cell cycle staging, such as fluorescence-activated cell sorting (FACS). In the future, it will be important to examine the role of H1 in regulation of DNA replication timing in sorted Drosophila diploid cells (Andreyeva, 2017).

Dynamic placement of the linker histone H1 associated with nucleosome arrangement and gene transcription in early Drosophila embryonic development

The linker histone H1 is critical to maintenance of higher-order chromatin structures and to gene expression regulation. However, H1 dynamics and its functions in embryonic development remain unresolved. This study has profiled gene expression, nucleosome positions, and H1 locations in early Drosophila embryos. The results show that H1 binding is positively correlated with the stability of beads-on-a-string nucleosome organization likely through stabilizing nucleosome positioning and maintaining nucleosome spacing. Strikingly, nucleosomes with H1 placement deviating to the left or the right relative to the dyad shift to the left or the right, respectively, during early Drosophila embryonic development. H1 occupancy on genic nucleosomes is inversely correlated with nucleosome distance to the transcription start sites. This inverse correlation reduces as gene transcription levels decrease. Additionally, H1 occupancy is lower at the 5' border of genic nucleosomes than that at the 3' border. This asymmetrical pattern of H1 occupancy on genic nucleosomes diminishes as gene transcription levels decrease. These findings shed new lights into how H1 placement dynamics correlates with nucleosome positioning and gene transcription during early Drosophila embryonic development (Hu, 2018).

This study determined the placement pattern of the linker histone H1 and its impact on nucleosome positioning and gene transcription during early Drosophila embryonic development. The results show that nucleosomes with H1 placement deviating to the left relative to the dyad in 3-4 h AEL embryos shift to the left when embryos develop to 14-15 h AEL. Similarly, nucleosomes with H1 placement deviating to the right relative to the dyad shift to the right when embryos develop to 14-15 h AEL. Additionally, H1 occupancy on the genic nucleosomes is negatively correlated with the nucleosome distance to TSS. This negative correlation reduces as transcription levels decrease. Intriguingly, H1 occupancy is lower at the 5' border of the genic nucleosomes than the 3' border. This uneven pattern of H1 occupancy on the genic nucleosomes diminishes as transcription levels decrease. These findings greatly improve understanding of the impact of histone H1 on nucleosome positioning dynamics and gene activity during early Drosophila embryonic development (Hu, 2018).

The location of the linker histone H1 in the chromatosome has remained controversial. Some studies suggested that the globular domain bound to the DNA located at the dyad and interacted with both linker DNAs. Some studies showed that the globular domain bound to the DNA located at the dyad but interacted with only one linker DNAs. This study clearly demonstrates that H1 tends to position at the border of the nucleosome. This finding favors the model that the globular domain interacts with both linker DNAs. Moreover, H1 locates at the border of the nucleosome with a much higher frequency than at the dyad. This consists with the previous finding that human H1.4 linker histones bind to the nucleosome off the dyad. However, this is also partially due to the fact that the C-terminal interacts with linker DNA. Of note, both H1 and nucleosome position is dynamic. The aforementioned models of H1 binding were based on the structural studies using NMR and cryo-EM that represent the static structures in vitro. For example, the globular domain favors binding to the more rigid linker DNA on the most open chromatin conformations while the flexible linker DNA is selected for binding in a compact chromatin structure. Although the epigenomics study using high-throughput sequencing failed to identify the structural domains and specific residues for the interactions between H1 linker histones and nucleosomal DNA, the results revealed the patterns of in vivo H1 binding within a chromatosome (Hu, 2018).

This study shows that the left-skewed or right-skewed placement of H1 on nucleosomes is predictive for nucleosome shift during early Drosophila embryonic development. The results agree with the previous finding that H1 binding to the nucleosomal DNA has a strong inhibitory effect on nucleosome mobility. When H1 binding deviates from the left border of the nucleosome, it creates space between H1 and nucleosome core particle. Consequently, nucleosomes shift to the left and H1 binding becomes symmetric to the dyad as Drosophila embryos develop next stage. Similarly, nucleosomes shift to the right when H1 binding deviates from the right border of the nucleosome. Thus the impact of H1 binding on nucleosome positioning alters chromatin structure. ATP-dependent chromatin remodeling enzymes are one of the important factors regulating nucleosome positioning. Knockdown of the chromatin remodeling complex Brahma extensively altered nucleosome positioning and led to the arrest of early Drosophila embryonic development. Which chromatin remodeling complex, chromatin chaperone, and other factors drive skew binding of H1 in the first place and further interfere with the eventual shifting of the asymmetric H1-bound nucleosomes? More studies are needed to address these questions (Hu, 2018).

In addition to the structural role in chromatin, H1 also plays an important role in regulation of gene transcription. It has long been thought that H1 was a repressor of transcription. This view consists with the structural role in chromatin that the placement of H1 can inhibit nucleosome mobility and stabilize higher-order chromatin structure. Consistently, this study shows that H1 occupancy is lower in highly-expressed genes than the lowly-expressed genes. Thus, the asymmetric distribution of H1 on the two borders of the nucleosome, low at the 5' border and high the 3' border, exists in the active genes not repressed genes. Of note, H1 appears to be not a repressor of global transcriptional activity in mammals. For example, with the involvement of DNA methylation, both increase and decrease in gene expression were observed in the H1 depleted mouse embryonic stem cells. Deletion of H1 also down-regulated active euchromatic genes located in Drosophila. Collectively, dynamics of H1 positioning regulates gene transcription through altering chromatin structure and with the involvement of DNA methylation in some cases (Hu, 2018).

The embryonic linker histone dBigH1 alters the functional state of active chromatin

Linker histones H1 are principal chromatin components, whose contribution to the epigenetic regulation of chromatin structure and function is not fully understood. In metazoa, specific linker histones are expressed in the germline, with female-specific H1s being normally retained in the early-embryo. Embryonic H1s are present while the zygotic genome is transcriptionally silent and they are replaced by somatic variants upon activation, suggesting a contribution to transcriptional silencing. This study directly address this question by ectopically expressing dBigH1 in Drosophila S2 cells, which lack dBigH1. dBigH1 was shown to bind across chromatin, replaces somatic histone H1 and reduces nucleosome repeat length (NRL). Concomitantly, dBigH1 expression down-regulates gene expression by impairing RNApol II binding and histone acetylation. These effects depend on the acidic N-terminal ED-domain of dBigH1 since a truncated form lacking this domain binds across chromatin and replaces dH1 like full-length dBigH1, but it does not affect NRL either transcription. In vitro reconstitution experiments using Drosophila preblastodermic embryo extracts corroborate these results. Altogether these results suggest that the negatively charged N-terminal tail of dBigH1 alters the functional state of active chromatin compromising transcription (Climent-Canto, 2020).

Linker histones H1 constitute an evolutionarily conserved family of chromosomal proteins that play an important structural role in regulating chromatin compaction and higher order chromatin organization. In metazoan species, histones H1 usually exist as multiple variants, some of which are specifically expressed in the germline. For instance, four of the eleven mice/human H1 isoforms are germline specific, of which three are expressed in males (H1T, HILS1 and H1T2) and one in females (H1oo). Female-specific variants usually accumulate in the oocyte and are retained during early embryogenesis. In comparison to most metazoa, H1 complexity in Drosophila is much reduced since it contains a single somatic dH1 variant, which is ubiquitously expressed throughout development, and a single germline specific variant dBigH1, which is expressed in both the female and male germlines, and it is retained in the early embryo. Embryonic H1s persist as long as the zygotic genome remains transcriptionally silent, being replaced by somatic variants when transcription begins during zygotic genome activation (ZGA). In Drosophila, dBigH1 is present during early embryogenesis until ZGA onset at cellularization. At this stage, dBigH1 is replaced by somatic dH1 in somatic cells, whereas it is retained in the primordial germ cells (PGC), which remain transcriptionally silent (Climent-Canto, 2020).

These observations suggest that dBigH1, and embryonic H1s in general, are general transcriptional regulators that contribute to silencing. Linker histones H1 have been usually associated with transcription repression, but somatic H1s are readily detected across expressed genes. In this regard, it has been reported that somatic H1s can even enhance the synergism between transcription factors. In contrast, in the presence of dBigH1, chromatin appears to be transcriptionally silent, suggesting that dBigH1 enhances transcriptional silencing. This study analyzed the mechanisms of action of dBigH1. For this purpose, ectopic expression experiments were performed in Drosophila S2 cells, which lack dBigH1. These experiments confirm the contribution of dBigH1 to transcriptional silencing, identifying the acidic N-terminal ED-domain as responsible for its negative effect on transcription (Climent-Canto, 2020).

The mechanism of dBigH1 action in transcription regulation was addressed using ectopic dBigH1 expression experiments in S2 cells. Though weakly, the genomic distribution of ectopically expressed dBigH1 positively correlates with those observed in embryos and testes, where dBigH1 is naturally expressed, suggesting that the mechanisms governing dBigH1 deposition might be partially conserved in S2 cells. The results suggest that binding of dBigH1 negatively affects transcription. Upon dBigH1 expression, more than two-thirds of the differentially expressed genes were down-regulated. This effect was probably underestimated since, though only in one replicate, was a global decrease in gene expression observed that, considering the methodology used for normalization, could hamper identification of differentially down-regulated genes. This down-regulation occurred at the transcriptional level, as down-regulated genes showed reduced RNApol II content. Conversely, RNApol II content of up-regulated genes was not increased upon dBigH1 expression, suggesting that the observed up-regulation was not transcriptional. Moreover, in vitro experiments showed that dBigH1 inhibited transcription of a chromatin template. Consistent with the negative effect on transcription, dBigH1 expression specifically decreased H3K36me3 levels at CDS of down-regulated genes (Climent-Canto, 2020).

The results show that dBigH1 replaces dH1. In these experiments, dBigH1 binding to chromatin was likely taking place in the absence of DNA replication, as dBigH1 induction was sustained for 24h and, during this time, cell density did not increase noticeably. Whether dBigH1 deposition involves active dH1 replacement remains to be determined. In vitro, incubation of purified nuclei with Drosophila embryo extract results in binding of dBigH1 to chromatin without dH1 displacement, suggesting that the replacement observed in S2 cells responds to an active process. Along the same lines, it was observed that dBigH1 was preferentially deposited at regions enriched in dH1. Replacement of somatic H1s by embryonic H1s has been reported in nuclear transfer experiments and NAP-1 has been shown to be involved in both B4/H1M deposition and somatic H1s removal in Xenopus. Further work is required to determine the mechanisms regulating dBigH1 deposition (Climent-Canto, 2020).

The acidic ED-domain of dBigH1 is required to inhibit transcription since expression of the truncated dBigH1ΔED form, which also replaced somatic dH1, did not down-regulate gene expression either affected RNApol II loading or H3Kac levels. The presence of the negatively charged acidic ED-domain in dBigH1 is peculiar as histones are highly positively charged. It is possible that, due to the negative charge of the ED-domain, the structural organization of chromatin is compromised in the presence of dBigH1. Actually, the overall NRL changed upon dBigH1 expression, but not when dBigH1ΔED was expressed. Interestingly, although the ED-domain of dBigH1 is not conserved outside of the Drosophila genus, embryonic H1s are generally more acidic than somatic ones. In this regard, it was shown that both the Xenopus B4/H1M and the mammalian H1oo embryonic linker histones alter chromatin organization and dynamics. An altered chromatin organization would perturb access to chromatin and/or functioning of chromatin remodelers/modifiers and transcription factors that, ultimately, would affect RNApol II loading and transcription. In fact, regardless of the actual transcriptional outcome, dBigH1 expression globally affected H3Kac. In contrast, it was reported earlier that incubation of purified nuclei with Drosophila embryo extract, which also results in dBigH1 binding, increased H3Kac levels. However, it is important to note that the increase in H3Kac levels observed in this case was independent of dBigH1 binding (Climent-Canto, 2020).

It might be argued that the down-regulation observed upon dBigH1 expression is a consequence of increased global linker histones content. However, similar or even higher levels of expression of the truncated dBigH1ΔED and dBigH1ΔNTD forms did not affect transcription. Moreover, binding of dBigH1 is compensated by removal of dH1, thus total linker histones content is not greatly increased (Climent-Canto, 2020).

dBigH1 binding affected expression of a relatively small subset of genes. This may reflect the fact that in the experimental setup used in this study, dBigH1 accounted for only 20-25% of total linker histones. Thus, from this point of view, affected genes appear to correspond to a subset of genes more sensitive to dBigH1 levels. In this regard, it was observed that down-regulated genes had strong RNApol II pausing, which tended to decrease upon dBigH1 expression. In addition, though dBigH1 binding reduced H3Kac globally, only the down-regulated genes were transcriptionally affected. Interestingly, impairing RNApol II pausing generally down-regulates gene expression, while reduced H3Kac levels preferentially affects expression of highly paused genes. These observations suggest that the higher sensitivity to dBigH1 expression of down-regulated genes is likely due to the way their transcription is regulated (Climent-Canto, 2020).

In summary, this study has presented evidence supporting that the acidic N-terminal tail of the embryonic dBigH1 linker histone of Drosophila compromises transcription by altering the functional epigenetic state of active chromatin. Other embryonic H1s might share similar properties since they are generally more acidic than their somatic counterparts (Climent-Canto, 2020).

Earlier Histone H1 Studies

In eukaryotic cells, the combination of DNA with numerous proteins known as chromatin organizes what would otherwise be a tangled DNA helix. In the absence of linker histones, chromatin forms a 10nm thick nucleosome-containing chromatin fiber, the first order of DNA - protein packing. The linker histone H1, serves to stabilize a higher order 30 nm diameter chromatin fiber that is fundamental to the structural organization of chromosomes. The linker histone binds to each nucleosome, and by self affinity, links these nucleosomes together resulting in the 30 nm chromatin fiber. Modulation of linker histone binding is thought to be an additionally important element in the ordering of chromatin structure accompaning activation and inactivation of gene transcription.

The first order of chromatin compaction consists of 146 bp of DNA wound in two superhelical turns around a core histone octamer consisting of two molecules each of histones H2A, H2B, H3 and H4. The core histones, constituting the nucleosome, interact with DNA to form the 10 nm thick chromatin fiber. A single Histone H1 polypeptide interacts with an additional 20 bp of DNA as it enters and leaves the nucleosomal core.

Just where does linker histones bind to the nucleosomal core? The traditional model for H1 binding to nucleosomes cores posits that the globular domain of the linker histone binds to the outward facing DNA, at the site where DNA enters and leaves the core nucleosome structure. In this model, linker histones span the entering and exiting DNA, holding the DNA in place. Linker histones exposed on the surface of nucleosomes are then free to self associate, resulting in the higher order 30 nm chromatin fiber (Thomas 1992 and citations).

More recently it has become clear that linker histone might actually nestle inside the coils of the DNA that wrap around core histones, and that linker histone is not found symmetrically associated with entering and exiting DNA but is displaced by approximately 60 nucleotides from the center (dyad axis) of the nucleosome bound DNA (Pruss, 1996 and Hayes, 1996). Crystal structure analysis has shown that each linker histone consists of a globular 'winged-helix" central domain flanked by basic NH2- and COOH-terminal tail domains (Ramakrishnan, 1993). If linker histone-DNA interactions were analogous to those of the winged helix transcription factor HNF-3-DNA - that is, if linker histone were to bind within the major groove of DNA - then the globular domain would not be centered at the nucleosomal dyad (the earlier hypothesis), because at that place the major groove of the nucleosomal DNA faces the histone octamer, and only the minor groove faces out and is available for additional interaction.

The newer idea of an asymmetic nucleosome described above might impart a directionality to the folding of the chromatin fiber, consistent with a polar head-to-tail arrangement of linker histone molecules. The orientation of the winged-helix domain would favor interaction of the basic COOH-terminal tail of the linker histone with linker DNA (the DNA between nucleosomes), facilitating chromatin compaction. The binding of the winged-helix domain in the major groove would account for sequence selectivity of nucleosome position and the restriction of nucleosome mobility that is dependent on linker histones. Removal of histone H1 as seen on transcriptional activation of inducible promoters would remove a major positional signal for chromatin organization; the resulting mobility of histone octamers with respect to DNA sequence could greatly facilitate transcriptional activation (Pruss, 1996 and references).

Histone H1 is absent from Drosophila embryos early in development, but appears during midblastula transition, at a time when zygotic messenger RNA synthesis becomes activated. The high mobility group protein HMG-D, present at high levels prior to midblastula transition declines in prevalence relative to H1 concentration. It is thought that this change in relative protein concentrations is an important factor in activation of zygotic transcription. More information about this important interaction is found at the HMG-D site.

GENE STRUCTURE

The plasmid cDm500 consists of a 4.8-kb sequence of genes coding for five histone genes, H1, H3, H4, H2a, and H2b repeated in tandem 1.8 times. The five genes are consecutively oriented on alternate strands, and thus each successive gene is transcribed in the alternate direction. Three genes (H3, H2A and H1) are therefore transcribed from one DNA strand, and two (H4 and H2B) from the other strand. The reassociation kinetics of this repeat unit indicate that its sequence is repeated approximately 100 times per haploid genome. Virtually all copies of the DNA sequence are located in the region 39DE of salivary gland polytene chromosomes, a region that appears to span most of the 12 chromomeres associated with 39DE. In several species of sea urchin these five genes are likewise tandemly repeated, but all the genes are transcribed in the same direction. The finding that both sea urchin and the fly contain all five genes arranged in such a way, leads to the belief that the five histone genes were linked in species whose descendents subsequently diverged to give rise to Protosomia and Deuterostomia (Lifton, 1977).

Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure

Mutant alleles of Drosophila were generated in which expression of the linker histone H1 could be be down-regulated over a wide range by RNAi. When the H1 protein level is reduced to ~20% of the level in wild-type larvae, lethality occured in the late larval - pupal stages of development. This study shows that H1 has an important function in gene regulation within or near heterochromatin. It is a strong dominant suppressor of position effect variegation (PEV). Similar to other suppressors of PEV, H1 is simultaneously involved in both the repression of euchromatic genes brought to the vicinity of pericentric heterochromatin and the activation of heterochromatic genes that depend on their pericentric localization for maximal transcriptional activity. Studies of H1-depleted salivary gland polytene chromosomes show that H1 participates in several fundamental aspects of chromosome structure and function. First, H1 is required for heterochromatin structural integrity and the deposition or maintenance of major pericentric heterochromatin-associated histone marks, including H3K9Me2 and H4K20Me2. Second, H1 also plays an unexpected role in the alignment of endoreplicated sister chromatids. Finally, H1 is essential for organization of pericentric regions of all polytene chromosomes into a single chromocenter. Thus, linker histone H1 is essential in Drosophila and plays a fundamental role in the architecture and activity of chromosomes in vivo (Lu, 2009).

This work provides evidence that maintaining the level of histone H1 expression is essential for proper Drosophila development. In vivo transcription of an H1-specific dsRNA 'hairpin' was used to induce post-transcriptional gene silencing in Drosophila. Lethality caused by abrogation of histone H1 synthesis is temperature-dependent. In this system, the transcription of the H1-specific hairpin RNA is activated ubiquitously by the yeast transactivator protein GAL4, which is known to exert stronger effects at elevated temperatures. Indeed, the depletion of H1 protein and penetrance of the RNAi-induced lethality in transgenic strains both directly correlated with the temperature . Thus, the temperature dependence of GAL4 transcriptional activity allows temporal control over the post-transcriptional silencing of H1; that is, by transferring developing animals from the permissive (18^oC) to the restrictive (29^oC) temperatures, or vice versa, one can target the RNAi effect to a specific developmental time period. For instance, it was found that activating the synthesis of the H1-specific RNAi during late stages of Drosophila development (in pupae and adults) did not cause an appreciable effect on viability, in contrast to H1 abrogation in embryos and larvae. Thus, there may be a less stringent requirement for maintaining H1 expression after metamorphosis. Alternatively, the endogenous H1 protein that accumulates in larvae prior to metamorphosis may be sufficient for proper cell function throughout the rest of the life cycle in Drosophila (Lu, 2009).

Previous studies with single and compound H1 subtype-specific knockout mice also revealed a direct correlation between the levels of H1 expression and survival. Mice lacking only one or two H1 subtypes, but containing a normal H1 to nucleosome ratio, survive and appear normal. On the other hand, mice lacking five H1 alleles, with a reduction from 20% to up to 50% in the H1-to-nucleosome ratios in different tissues, were small and born at a significantly lower rate than the single and double H1 knockout mice. Embryos lacking six alleles (three H1 subtypes) and containing approximately half of the normal H1 levels developed multiple abnormalities and died in midgestation, an indication that a minimum threshold level of H1 protein is required for normal mammalian embryonic development. These data in Drosophila parallel these findings, since at subpermissive temperatures (26°C or lower), intermediate reduction of H1 expression to ~70% of the wild-type larval level resulted in partial survival of affected animals. Thus, in contrast to simpler eukaryotes, in which the linker histone is not essential, metazoans require maintenance of a certain level of H1 expression for normal development (Lu, 2009).

Pericentric heterochromatin has been implicated in gene silencing that occurs when euchromatic genes are placed adjacent to heterochromatin by chromosome rearrangement or transposition—a phenomenon that was initially described in Drosophila as PEV. Through genetic screening, many important chromatin regulators have been identified, which, when mutated, act as modifiers (suppressors or enhancers) of PEV. Thus, PEV in Drosophila represents a valuable assay for identification and molecular study of evolutionarily conserved functions controlling epigenetic programming in eukaryotes. This study observed that the linker histone H1 stimulates silencing in pericentric heterochromatin. Although it was not feasible to make a classical mutant of the H1 genes, dose reduction of H1 by ~15% resulted in PEV suppression. In that respect, H1 resembles other dominant suppressors of PEV, such as Su(var)2-5, which encodes HP1. Dose reduction of HP1 in Su(var)2-5 heterozygotes results in strong PEV suppression. The data indicate that H1 is an essential structural component of pericentric heterochromatin, or it is necessary for recruitment of another such essential biochemical component(s) to heterochromatin. In fact, it was found that the level of H1 does affect the localization of two major markers of pericentric heterochromatin, HP1 and H3K9Me₂ (Lu, 2009).

HP1 is an abundant nonhistone chromosomal protein first discovered in Drosophila because of its association with heterochromatin. HP1 is conserved in many eukaryotes, including fission yeast, insects, and mammals; involved in gene silencing; and consistently associated with pericentric heterochromatin and telomeres. In Drosophila polytene chromosomes, HP1 is diagnostic of heterochromatin, and the vast majority of HP1 protein concentrates at the chromocenter. Indirect immunofluorescence staining of polytene chromosomes indicates that histone H1 is abundant in pericentric heterochromatin. Furthermore, the chromocenter is severely disrupted in polytene chromosomes of salivary gland cells with depleted H1, and H1 abrogation also results in a delocalization of HP1. The dispersion of the chromocenter is not produced by mechanical stress during squashing, since it is similarly observed in whole-mount salivary gland cells. Thus, H1 plays important roles in the establishment and/or maintenance of the structure as well as in the biochemical composition of proximal heterochromatin in Drosophila larvae. It remains to be seen whether H1 is directly required for faithful deposition/recruitment of HP1 to its cognate loci in pericentric heterochromatin, or mislocalization of HP1 in chromosomes of H1-depleted cells is a secondary effect mediated by disruption of other nuclear processes that are regulated by the abundance of H1 (e.g., transcription). The former explanation is certainly possible since there are several reports that HP1 interacts directly with H1 (Lu, 2009).

Methylation of histone H3 Lys 9 (H3K9) has a well-established role in heterochromatin formation in metazoans, and H3K9Me₃ (H3K9Me₂ in Drosophila) is highly enriched in condensed heterochromatin. The chromodomain of HP1 specifically recognizes methylated H3K9, which facilitates its recruitment and leads to an overlapping distribution of HP1 and the H3K9 methylation mark in the genome. Upon H1 abrogation, however, very little or no H3K9Me₂ is detected in the loci where HP1 remains present. It is concluded that in polytene chromosomes of H1-depleted larvae, HP1 is deposited by a mechanism that does not require histone H3 dimethylation. The persistence of HP1 in proximal heterochromatin in the absence of dimethylated H3K9 is consistent with reports indicating that HP1 can bind nonspecifically to nucleosome core particles and even to naked DNA. It is also consistent with findings that used a tethering system to recruit HP1 to euchromatic sites: these showed that HP1-mediated silencing can operate in a Su(var)3-9-independent manner. The current findings strengthen the view that, whereas HP1 may normally cooperate with Su(var)3-9 and K9-methylated H3 in heterochromatin formation and gene silencing at pericentric chromosome sites, it can be deposited in these regions independently of these other components, and even without the presence of H1 (Lu, 2009).

The Su(var)3-9-null mutants, although also lacking an appreciable level of H3K9Me₂ signal in immunofluorescence-stained polytene chromosomes, do not exhibit the same spectrum of phenotypes as H1-depleted animals. For instance, the single polytene chromocenter is not disrupted in Su(var)3-9-null mutants. Thus, the observed phenotypes and defects in chromatin structure upon abrogation of H1 cannot be explained exclusively by the loss of H3K9 dimethylation, and H1 is therefore predicted to play a separate and unique role in the establishment and/or maintenance of pericentric heterochromatin. In the future, it will be interesting to see whether in addition to the reversal of heterochromatic silencing, similar to other suppressors of variegation, H1 depletion also affects other properties of heterochromatin, such as the reduced rates of meiotic recombination normally observed in these regions (Lu, 2009).

It is an intriguing observation that H3K9Me₂ is not detectable in chromatin of H1-depleted salivary glands by indirect immunofluorescence, although total protein levels in cell lysates are elevated rather than reduced. Thus, H1 may be required for H3K9Me₂ deposition in chromatin. Alternatively, if histone H3 Lys 9 is dimethylated by Su(var)3-9 predominantly in the context of a nucleosome, H1 depletion may result in specific expulsion of the K9-dimethylated form of H3 from pericentric regions and potentially other H3K9Me₂-enriched loci. The presence of other repressive, heterochromatin-specific histone marks, such as H4K20Me₂, H3K9Me₁, and H3K9Me₃, was examined in polytene chromosomes of H1 knockdown larvae by IF microscopy. It was discovered that they were all largely absent in pericentric heterochromatin. In contrast, there was no substantial effect on the active H3K4Me₂ mark, which remained widely distributed in polytene chromosomes. Thus, H1 appears to be required for global maintenance of repressive marks in heterochromatin, rather than stimulation of particular programs/enzymes that affect specific histone modification states. This function of H1 might be linked to its role in the transcriptional activity of heterochromatin. Indeed, studies of heterochromatic gene expression in H1-depleted larvae showed that low levels of H1 cause altered transcriptional activity in heterochromatin. Further studies of the dynamics of formation and maintenance of H3K9Me₂ and other repressive marks in H1-depleted chromatin may lead to a better understanding of this relationship (Lu, 2009).

H1 depletion has a dramatic effect on the distribution of H3K9Me₂-containing nucleosomes in the genome. It is possible that H1 is similarly involved in maintenance of other repressive histone marks in Drosophila. However, it is unlikely that H1 is involved in Polycomb silencing, since no homeiotic phenotypes were observed in adult escapers that survive partial H1 depletion (at 26^oC and below) (Lu, 2009).

Previous work with H1-depleted mouse ES cells, as well as studies in other species, suggested that H1 may participate in both transcriptional activation as well as repression in vivo. Likewise, studies with H1-depleted Drosophila larvae support dual roles for H1 in transcriptional regulation. Similar to other suppressors of PEV, H1 stimulates silencing of genes that are brought into juxtaposition with heterochromatin. In contrast, certain Drosophila genes that are embedded in heterochromatin (e.g., concertina, light, and rolled) are dependent on their genomic localization for proper transcriptional regulation, as their expression is reduced when their genomic loci are rearranged to lie next to a euchromatic breakpoint or when heterochromatin component genes are mutated. By qRT-PCR assay, it was demonstrated that concertina, light, and rolled are repressed in third instar larval salivary glands upon reduction of H1 levels. Thus, H1 is also required for activation of heterochromatic genes within the context of pericentric heterochromatin (Lu, 2009).

It has been proposed that heterochromatin-associated proteins function to support normal transcription of heterochromatic genes when those genes are at their normal chromosomal sites and that position effects result when these genes are deprived of such essential proteins by displacement away from heterochromatin 'compartments.' Similarly, H1 may contribute to the formation of a particular chromatin structure that interferes with activation of euchromatic genes but to which heterochromatic genes have become adapted. The loss of H1 would deplete the nucleus of this particular chromatin conformation, releasing silenced genes from repression while simultaneously depriving the resident heterochromatin genes of their functional context. Interestingly, mutations of rolled, similar to H1 depletion, lead to late larval or early pupal lethality and defective imaginal disc formation. It remains to be seen whether one of the effects contributing to the lethality of H1-depleted animals is down-regulation of specific heterochromatic genes (Lu, 2009).

As a control, a limited analysis of possible effects of H1 abrogation was performed on expression of several euchromatic genes. So far, no euchromatic in vivo transcriptional target for H1 has been found in Drosophila larvae. However, this lack of apparent effect can be explained by the limited sample size (four genes) and the choice of targets. Only abundant, ubiquitous genes, were assayed, whose transcription units in the wild-type animals (without H1 abrogation) may be positioned within chromatin that already contains little or no H1. In the future, it will be important to extend this analysis to tissue-specific, tightly regulated genes and to perform this experiment in an unbiased, genome-wide (microarray) format (Lu, 2009).

Although the Drosophila polytene chromosome has served as a model to study chromatin structure, remarkably little is known about its spatial organization or the molecular mechanisms that maintain the alignment of sister chromatids. Previous studies suggested that interchromatid cohesion is generated and maintained in the banded regions. H1 is widely distributed in euchromatic arms of polytene chromosomes; however, it localizes predominantly to bands of compacted chromatin. H1 depletion disrupts the normal band-interband structure of polytene chromosomes. Thus, H1 functions to establish or maintain the parallel alignment of band chromosome fibrils. When depleted by RNAi, residual H1 protein is not distributed uniformly in polytene chromosomes. Remarkably, the residual H1 maxima correlate with the persistent band-interband structure over short fragments of the H1-depleted polytene chromosomes. This result emphasizes the requirement for H1 in polytene chromatid alignment/adhesion. Similarly, the dissociation of the normal single chromocenter in polytene chromosomes into several foci of HP1 localization in the H1 knockdown larvae may also be related to the loss of adhesion (Lu, 2009).

Linker histone H1 is an abundant protein component of chromatin. It binds to DNA outside the core particle region, and its function in internucleosomal interactions and chromatin condensation is widely accepted. It is possible that internucleosomal interactions directly mediated by H1 can occur in trans between two distinct chromatin fibrils and, thus, play a role in adhesion of sister chromatids in polytene chromosomes. In that case, genomic regions of intrinsically higher H1 density (bands) would then cluster ('align') in polytene chromosomes. This direct mechanism is consistent with the partial conservation of the polytene chromosome banding structure of H1-depleted salivary gland cells in regions that contain elevated levels of residual H1. However, a possibility that H1 activity in chromatid alignment is mediated through interactions with other molecules important for chromatin structure maintenance, such as H3S10 kinase JIL-1, cannot be excluded (Lu, 2009).

Although JIL-1 hypomorphic or null alleles exhibit a defect in polytene chromosome alignment comparable with that observed in H1 knockdown alleles, other functions of these proteins are remarkably dissimilar. Unlike H1, JIL-1 localizes to gene-active interbands and counteracts the function of Su(var)3-9. JIL-1 is also an enhancer of PEV. Furthermore, in JIL-1 alleles, polytene chromosome arms are highly condensed and interband regions are missing, with the male X chromosome affected the most severely. None of these phenotypes are observed in H1 knockdown animals. On the contrary, H1-depleted polytene chromosomes are rather extended, probably due to the dispersal of normally compacted band regions. However, both H1 and JIL-1 appear to contribute to polytene fibril alignment. It is possible that the polytene chromosome structure is established through interplay between antagonistic effects mediated by several effectors, such as H1 and JIL-1 (or its substrates). In the future, it will be interesting to elucidate fine details of these putative functional interactions between H1 and JIL-1 (Lu, 2009).

Although H1 is clearly required for chromatid alignment in endoreplicating cells, it is likely dispensable or less critical for sister chromatid alignment in G2-M of proliferating cells. Mutations that affect Drosophila genes coding for the Rad21 subunit of cohesin, CAP-G subunit of condensin, and Orc2 and Orc5 subunits of the origin recognition complex have been shown previously to affect sister chromatid alignment and segregation in vivo. Mutations in these genes result in massive missegregation of chromosomes during mitosis, which was not observed in H1-depleted animals. In contrast, these mutations do not cause any abnormalities in polytene chromosome structure. Thus, adhesion of replicating chromatin in dividing and endoreplicating cells in Drosophila is likely to be maintained through distinct mechanisms (Lu, 2009).

In conclusion, this study demonstrated that the linker histone H1 is essential for normal development in Drosophila and required for proper chromosome structure and function. Specifically, H1 is involved in the establishment of repressive pericentric heterochromatin and deposition/maintenance of the several histone modification marks that are localized in proximal heterochromatin. Furthermore, reduced H1 expression results in defective polytene chromosome structure with dissociation of the chromocenter and an almost complete loss of the banding pattern in the chromosome arms. Thus, linker histone H1 plays an essential role in the architecture and activity of metazoan chromosomes (Lu, 2009).

PROTEIN STRUCTURE

Amino Acids - 255 (Murphy, 1986)

Structural Domains

By searching the current protein sequence databases using sequences from human and chicken histones H1/H5, H2A, H2B, H3 and H4, a new database has been constructed consisting of aligned histone protein sequences with statistically significant sequence similarity to the search sequence. A nucleotide sequence database of the corresponding coding regions for these proteins has also been assembled. The region of each of the core histones containing the histone fold motif is identified in the protein alignments. The database contains >1300 protein and nucleotide sequences. All sequences and alignments in this database are available through the World Wide Web at Histone fold motif This should be checked online (Baxevanis, 1996).

date revised: 12 December 2022

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