Histone H1

DEVELOPMENTAL BIOLOGY

Embryonic

The first appearance of H1 takes place during cycles 7 and 8. It is during cycle 7 that the size of the nuclei begins to decrease. By cycles 10-12 a sufficient amount of H1 has accumulated to allow the reorganization of chromatin to a transcriptionally active state. Subsequently, increased zygotic transcription elevates H1 levels even further. This exponential increase, together with the increased number of nuclei, rapidly depletes the relative levels of HMG-D protein. It has been suggested that the chromatin generated in the presence of HMG-D is transcriptionally silent, and that transcription begins only when H1 levels have reached a particular threshold value and overcome the HMG-D effects, around nuclear cycle 10 (Ner, 1994).

The amount of histone H1 relative to core histones (See Histone H4) has been determined in three Drosophila species (D. melanogaster, D. texana and D. virilis) in chromatin from several tissues differing in chromatin structure and genetic activity. Low levels of H1 are found in relatively undifferentiated, early embryos as well as in a line of cultured cells. In late embryos the content of H1 is highest in D. virilis, which possesses larger amounts of and a partially more compacted constitutive heterochromatin than the two other species. Polytene chromatin from larval salivary glands shows increased levels of H1 compared with diploid chromatin; the degree of phosphorylation of this histone is relatively low. The degree of phosphorylation of H2A is found to be drastically reduced in polytene as compared with diploid embryonic chromatin, which parallels the extensive underreplication of constitutive heterochromatin. In diploid chromatin, a qualitative correlation is also observed between the relative amounts of heterochromatin and the levels of H2A phosphorylation. These findings suggest a connection between H2A phosphorylation and heavy compaction of interphase chromatin (Holmgren, 1985).

HMG-D is an abundant chromosomal protein associated with condensed chromatin during the first nuclear cleavage cycles of the developing Drosophila embryo. It previously suggested that HMG-D might substitute for the linker histone H1 in the preblastoderm embryo and that this substitution might result in the characteristic less compacted chromatin. The association of HMG-D with chromatin has been studied using a cell-free system for chromatin reconstitution derived from Drosophila embryos. Association of HMG-D with chromatin, like that of histone H1, increases the nucleosome spacing indicative of binding to the linker DNA between nucleosomes. HMG-D interacts with DNA during the early phases of nucleosome assembly but is gradually displaced as chromatin matures. By contrast, purified chromatin can be loaded with stoichiometric amounts of HMG-D, and this can be displaced upon addition of histone H1. A direct physical interaction between HMG-D and histone H1 was observed in a Far Western analysis. The competitive nature of this interaction is reminiscent of the apparent replacement of HMG-D by H1 during mid-blastula transition. These data are consistent with the hypothesis that HMG-D functions as a specialized linker protein prior to appearance of histone H1 (Ner, 2001).

Histone H1 and HMGB1 proteins could influence chromatin structure in a similar manner by binding to linker DNA sequence. Histone H1 associates with linker DNA sequences and organizes nucleosomal arrays into higher order chromatin structures, such as the 30-nm chromatin fiber. However, little is known about how HMGB1 interacts with the nucleosome and about the consequences in structure and function. H1 and HMGB1 share important features; both protect linker DNA sequences from nuclease digestion, and both bind four-way junctions. Consistent with the idea that interaction of HMGB1 might replace histone H1, in the very early stages of Drosophila embryogenesis histone H1 is absent, but the high mobility group protein D (HMG-D) is present in vast excess. Based on the similarities between HMG-D and H1, a role for HMG-D as a linker protein compatible with and perhaps required for the fast condensation-decondensation cycles associated with the very rapid nuclear division cycles found in preblastoderm embryos has been suggested. An analogous role has been proposed for the Xenopus HMGB1 and B4 proteins; both proteins have been demonstrated to bind di-nucleosomal DNA (Ner, 2001).

The fact that recombinant HMG-D increases the nucleosome repeat length (NRL) in a cell-free chromatin assembly system strongly supports this hypothesis. The NRL is strongly dependent on the ionic environment such that polycations are particularly effective in increasing the average separation between adjacent nucleosomes. In accordance with these findings the data implicate the polycationic basic region (residues 85-99; net charge, +10) of HMG-D in this function. However, the HMG-D-dependent increase in NRL is mediated both by the full-length protein and by HMG-D¹⁰⁰. These forms differ substantially in net charge +7 (for HMG-D) and +17 (for HMG-D¹⁰⁰), suggesting that the chromatin DNA can compete effectively with the polyanionic acidic tail of HMG-D. Histone H1 and the HMGN1 and HMGN2 proteins (formerly HMG-14 and HMG-17) are the only other proteins reported to cause such a change, in the case of H1 presumably by binding to the linker DNA. The binding of H1 to the linker sequence appears to differ from that of HMG-D. Increasing concentrations of histone H1 added to the assembly reaction will continue to increase the NRL to well over 220 bp before the regular nucleosomal array is lost. HMG-D, on the other hand, increases the NRL to only ~180 bp. This may reflect the stoichiometry of binding to the linker sequence. Di-nucleosomal DNA reconstituted by dialysis has been shown to be able to bind two molecules of H1 but only a single molecule of HMGB1. Although the exact nature of the binding remains unknown, HMG-D binds ~14 bp of DNA, and consequently 1-2 molecules of HMG-D could potentially occupy the linker space (Ner, 2001).

A tight correlation between nucleosome spacing and the folding of the nucleosomal fiber into a 30-nm fiber has been observed, which led to the suggestion that different NRLs would correspond to particular fiber geometries and, therefore, compaction states. Accordingly, increased nucleosome spacing is indicative of more compacted chromatin. The observation that HMG-D does not increase the NRL beyond 185 bp as H1 may indicate that HMG-D-containing chromatin is folded but is less compacted (Ner, 2001).

Like other HMG domain proteins such as LEF-1 and SRY, HMG-D can introduce sharp bends or kinks into DNA. The current estimates of the magnitude of the DNA kinks induced by HMG-D range from 100-120° for the full-length protein to 60° to >90° for HMG-D¹⁰⁰. These values are substantially greater than the average curvature of DNA wrapped around the histone octamer and indicate that HMG-D bound DNA is not smoothly curved. In the context of linker DNA, such a state would be consistent with both the lack of UV-induced thymine dimer formation in the linker and also, with evidence from electric dichroism studies, that the trajectory of linker DNA differs from that of DNA bound to the core histones. Of particular relevance are the observations that, in the presence of histone H1 derivatives containing a major proportion of the basic C-terminal domain, the linker DNA enters and leaves a single chromatosome as a straight rod approximately perpendicular to the superhelical axis. A similar structure has been observed in chromatin fibers. This organization implies that the DNA must bend sharply as it enters and leaves the chromatosome. A possible role for HMG-D would be to induce such sharp bends by kinking the DNA and thereby promoting a higher level of chromatin folding (Ner, 2001).

Evidence has been provided for an interaction of HMGB1 with the nucleosome and it has been suggested that it might replace histone H1 in the nucleosome. Evidence has been provided for interactions between histone H1 and HMGB1. The results are consistent with these observations. (1) In a Far Western analysis, H1 is the predominant protein identified when labeled HMG-D was used as a probe. (2) Using chromatin assembled on DNA attached to paramagnetic beads and preloaded with HMG-D protein, HMG-D is displaced upon titration of histone H1. It is noted that the full-length HMG-D and HMG-D¹⁰⁰ both interact with H1 in a Far Western analysis. The alanine-lysine-rich region (amino acids 84-100, AKKRAKPAKKVAKKSKK) is very similar to a region found in histone H1. Far Western analysis suggests that this region, or possibly the region immediately preceding glycine-rich linker, is interacting with H1. In HMG-D this sequence contains a serine residue that is phosphorylated by casein kinase II.

Although it is possible to argue for a structural role for HMG-D and HMGB1 in early embryonic chromatin, in vitro observations show that in the absence of H1 HMG-D, although initially present at high levels, is displaced to below 1 molecule/10-20 nucleosomes as the reaction proceeds and the chromatin matures. This would argue against a purely structural role for HMG-D and suggest that the protein may fulfill a different role. One possibility is that HMG-D functions as a chaperone molecule and preconfigures the DNA to facilitate the chromatin assembly process. HMG-D could participate to bend the DNA at the exit and entry points to the nucleosome, and this bend is then stabilized by histone H1. Under such a scenario, as chromatin assembly proceeds and the core histones are recruited, HMG-D molecules are displaced. The linker sequences would be the only locations where the protein would persist for longer duration. However, this too would be displaced on the addition of other chromatin-associated proteins (transcription factors, assembly factors). Such a mechanism would be very similar to that proposed for the recruitment of transcription factors. Similarly the displacement and competition with histone H1 can be envisaged as part of a process in which the DNA is kinked by HMG-D, and then the binding of the linker histone stabilizes this kink (Ner, 2001).

Preblastoderm embryonic chromatin clearly differs profoundly from post-blastoderm chromatin. Early syncytial nuclei are much larger and contain chromatin that is less compacted than later nuclei. In the early embryo HMG-D is highly abundant, although not all molecules are necessarily available for DNA binding. It is deposited in the egg by the mother but thereafter is maintained at an approximately constant level per embryo. Consequently, with each nuclear division the average number of HMG-D molecules per nucleus falls, although during nuclear cycles 7-14 the amount of H1 rapidly increases. Only during cycle 7 does the size of the nuclei begin to decrease. By cycles 10-12 a sufficient amount of histone H1 has accumulated to allow the reorganization of chromatin to a transcriptionally active state. Subsequently, increased zygotic transcription elevates histone H1 levels further. This exponential increase of histone H1 together with the increasing number of nuclei rapidly deplete HMG-D protein to levels that cannot have global effects on chromatin structure. What could be the physiological significance of different linker proteins? HMG-D- or H1-containing chromatin may differ profoundly in the degree or mode of compaction. The looser structure formed in the absence of H1 could facilitate the rapid condensation and decondensation required during the very short early cleavage cycles (Ner, 2001).

The switch from HMG-D- to H1-containing chromatin correlates with the acquisition of global transcriptional competence. Similar observations have been described in the Xenopus system in which B4, an H1 variant, and HMGB1 disappear during mid-blastula transition, again correlating with a change in the accessibility of embryonic chromatin to class III transcriptional machinery. The cell-free system employed in this study may facilitate the detailed analysis of this major switch in genome function during embryonic development (Ner, 2001).

Adult

A genomic fragment was cloned from a DNA library constructed from a Drosophila enhancer trap line in which reporter gene expression was observed at the anterior-most tip of the ovaries and testes. This genomic clone was identified as the L-repeat of the Drosophila melanogaster histone gene cluster. Northern blotting and in situ hybridization to RNA in tissues with individual cDNAs and PCR-generated probes for each histone confirm that gene expression is greatest at the anterior portion of each ovariole, in the germarium, and is also elevated in a few individual nurse cells and somatic follicle cells within the egg chamber during early developmental stages. Histone H1 and each of the core histones have a similar expression pattern that is correlated to cell division. Maternal stores of histone transcripts are also transported to the mature oocyte from the nurse cells at a later stage of oogenesis (stage 10), when virtually all the nurse cells contain high levels of histone transcripts. The results are consistent with expression of the somatic histone gene cluster during oogenesis as a coordinate unit. There does not seem to be a reduced level of somatic type H1 in the germ-line, as is observed in some other species. The relationship between the P[lacZ] expression pattern in the germarium and the overall expression of the histone cluster suggests there are specific regulatory elements for germ-line expression (Walker, 1998).

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).

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date revised: 15 December 2011

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