InteractiveFly: GeneBrief

Histone H1 variant BigH1: Biological Overview | References

Gene name - Histone H1 variant BigH1

Synonyms -

Cytological map position - 88C10-88C10

Function - histone

Keywords - germline linker histone that enhances transcriptional silencing - replaced by somatic dH1 in somatic cells - oogenesis and spermatogenesis - along with the translational regulator Bam forms a repressor loop essential for male germ stem cell differentiation

Symbol - BigH1

FlyBase ID: FBgn0038252

Genetic map position - chr3R:14,663,450-14,664,822

NCBI classification - linker histone H1 and H5 family

Cellular location - nuclear

NCBI links: EntrezGene, Nucleotide, Protein

BigH1 orthologs: Biolitmine
Recent literature
Climent-Canto, P., Carbonell, A., Tamirisa, S., Henn, L., Perez-Montero, S., Boros, I. M. and Azorin, F. (2021). The tumour suppressor brain tumour (Brat) regulates linker histone dBigH1 expression in the Drosophila female germline and the early embryo. Open Biol 11(5): 200408. PubMed ID: 33947246
Linker histones H1 are essential chromatin components that exist as multiple developmentally regulated variants. In metazoans, specific H1s are expressed during germline development in a tightly regulated manner. However, the mechanisms governing their stage-dependent expression are poorly understood. This question was addressed in Drosophila, which encodes for a single germline-specific dBigH1 linker histone. During female germline lineage differentiation, dBigH1 is expressed in germ stem cells and cystoblasts, becomes silenced during transit-amplifying (TA) cystocytes divisions to resume expression after proliferation stops and differentiation starts, when it progressively accumulates in the oocyte. This study finds that dBigH1 silencing during TA divisions is post-transcriptional and depends on the tumour suppressor Brain tumour (Brat), an essential RNA-binding protein that regulates mRNA translation and stability. Like other oocyte-specific variants, dBigH1 is maternally expressed during early embryogenesis until it is replaced by somatic dH1 at the maternal-to-zygotic transition (MZT). Brat also mediates dBigH1 silencing at MZT. Finally, the situation in testes is discussed, where Brat is not expressed, but dBigH1 is translationally silenced too.

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 (Pérez-Montero, 2013; Carbonell, 2017). 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 (Pérez-Montero, 2013). At this stage, dBigH1 is replaced by somatic dH1 in somatic cells, whereas it is retained in the primordial germ cells (PGC) (Pérez-Montero, 2013), 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).

Chromatin remodeling in Drosophila preblastodermic embryo extract

Chromatin is known to undergo extensive remodeling during nuclear reprogramming. However, the factors and mechanisms involved in this remodeling are still poorly understood and current experimental approaches to study it are not best suited for molecular and genetic analyses. This study reports on the use of Drosophila preblastodermic embryo extracts (DREX) in chromatin remodeling experiments. The results show that incubation of somatic nuclei in DREX induces changes in chromatin organization similar to those associated with nuclear reprogramming, such as rapid binding of the germline specific linker histone dBigH1 variant (Pérez-Montero, 2013) to somatic chromatin, heterochromatin reorganization, changes in the epigenetic state of chromatin, and nuclear lamin disassembly. These results raise the possibility of using the powerful tools of Drosophila genetics for the analysis of chromatin changes associated with this essential process (Satovic, 2018).

This study reports that incubation of somatic nuclei in DREX induces changes in chromatin organization similar to those associated with nuclear reprogramming. On one hand, rapid incorporation was observed of the Drosophila germline specific linker histone dBigH1 into the somatic nuclei. NT experiments performed in Xenopus and mammals showed that incorporation of the oocyte specific linker histone variants B4 and H1oo into the donor nuclei is an early event in nuclear reprogramming. B4 binding precedes loading of oocyte RNApol II and expression of a dominant negative B4 form significantly inhibits transcription of many reprogrammed genes. Along the same lines, expression of H1oo in mouse ESCs impairs differentiation although it does not improve iPSC formation. How oocyte specific H1s might contribute to nuclear reprogramming remains not well understood. Oocyte specific H1s are less positively charged than their somatic counterparts and, therefore, their interaction with DNA is weaker and condense chromatin less than somatic H1s, rendering it more accessible to chromatin modifiers, remodelers and transcription factors. In this regard, Xenopus B4 is more mobile than somatic H18 and B4-containing chromatin is more accessible to remodeling factors39. B4 binds pervasively across chromatin of the donor nuclei and, concomitantly, somatic H1s are released, suggesting competition of somatic H1s by the oocyte specific variants. However, this competition does not appear to play an important role in reprogramming since overexpression of somatic H1s does not interfere with B4 binding and subsequent activation of pluripotency genes. Moreover, in mouse fibroblasts, binding of H1oo is detected 10' after NT, while release of somatic H1s occurs later at 30' after NT20. Similarly, somatic H1s replacement can last hours in NT experiments with bovine cells. Finally, the results indicate that, upon incubation in DREX, dBigH1 binds along chromatin without affecting somatic dH1 occupancy. In fact, dH1 occupancy is significantly reduced only at short incubation times when dBigH1 binding is very low (Satovic, 2018).

The results also show that DREX induces changes in the epigenetic landscape of chromatin, which are in agreement with the global epigenetic remodeling of chromatin observed during reprogramming of somatic cells to iPSCs. In particular, increased global H3Ac was observed that is maintained throughout the incubation time course. Increased histone acetylation is observed in fully reprogrammed iPSCs40 and ESC chromatin is hyperacetylated compared to differentiated cells. It was also observed that H3K4me3 levels increased more intensively at promoters of developmentally regulated genes that are silent in S2 cells but highly expressed in early embryogenesis, suggesting their reactivation. Interestingly, pluripotency-related and developmentally regulated genes are known to acquire H3K4me3 at promoters during nuclear reprogramming. Finally, though not statistically significant, global levels of H3K4me3 and the chromatin bound promoter-proximal active RNApol IIoser form tend to increase at short incubation times. In this regard, NT experiments in Xenopus showed loading of oocyte basal transcription factors and RNApol II leading to genome-wide transcriptional reprogramming and selective activation of pluripotency genes. Notably, these results showed that increased histone acetylation induced by DREX does not require binding of dBigH1, suggesting that, at least in part, the epigenetic changes occurring during reprogramming do not depend only on the activities of the oocyte specific H1s (Satovic, 2018).

Incubation in DREX also induces profound changes in chromatin/nuclear organization. On one hand, at short incubation times, DREX induces heterochromatin reorganization since HP1a/H3K9me3 foci disassemble. A decrease in the number of HP1a foci has also been reported during reprogramming to iPSC. In this regard, chromatin of pluripotent cells is largely decondensed and heterochromatin is organized in larger and fewer domains that become smaller, more abundant and hypercondensed as cells differentiate. Interestingly, incubation in DREX did not decrease H3K9me2 occupancy at multiple heterochromatic elements, suggesting that DREX affects condensation but not the actual heterochromatin content of somatic nuclei. Oocyte specific H1s might be one of the factors contributing to heterochromatin decondensation since, in humans, H1oo is required for decondensation of sperm chromatin. At long incubation times, HP1a foci reform and extrude from nuclei. Interestingly, extrusion of heterochromatic sequences was also reported in somatic plant cells undergoing meiosis. Finally, it was also observed that DREX induces disassembly of nuclear lamin, a nuclear envelope component of differentiated cells that is absent in ESCs. Similar results were reported earlier using a Drosophila oocyte cell-free extract. Nuclear lamin disassembly is considered a marker of reprogrammed cells, since it is detected at the nuclear envelope in partial iPSCs, but not in fully reprogrammed iPSCs40. Interestingly, nuclear lamin disassembly strongly correlates with heterochromatin reorganization, which might account for the heterochromatin extrusion observed after long-term exposure to DREX (Satovic, 2018).

In summary, these results show that DREX induces several changes associates with gain of pluripotency, such as binding of the germline specific linker histone dBigH1, epigenetic remodeling, heterochromatin reorganization and nuclear lamin disassembly. However, it is highly unlikely that DREX induces full reprogramming of somatic nuclei. Nevertheless, the use of DREX offers the possibility of applying the powerful genetics techniques developed in Drosophila to the analysis of factors and mechanisms involved in chromatin remodeling during this essential process (Satovic, 2018).

The germline linker histone dBigH1 and the translational regulator Bam form a repressor loop essential for male germ stem cell differentiation

Drosophila spermatogenesis constitutes a paradigmatic system to study maintenance, proliferation, and differentiation of adult stem cell lineages. Each Drosophila testis contains 6-12 germ stem cells (GSCs) that divide asymmetrically to produce gonialblast cells that undergo four transit-amplifying (TA) spermatogonial divisions before entering spermatocyte differentiation. Mechanisms governing these crucial transitions are not fully understood. This study reports the essential role of the germline linker histone dBigH1 during early spermatogenesis. These results suggest that dBigH1 is a general silencing factor that represses Bam, a key regulator of spermatogonia proliferation that is silenced in spermatocytes. Reciprocally, Bam represses dBigH1 during TA divisions. This double-repressor mechanism switches dBigH1/Bam expression from off/on in spermatogonia to on/off in spermatocytes, regulating progression into spermatocyte differentiation. dBigH1 is also required for GSC maintenance and differentiation. These results show the critical importance of germline H1s for male GSC lineage differentiation, unveiling a regulatory interaction that couples transcriptional and translational repression (Carbonell, 2017).

Studies in Drosophila have provided important insights into the cellular pathways governing maintenance, proliferation, and differentiation of adult stem cell lineages, which is central to understanding normal tissue homeostasis and its alteration in disease. In particular, Drosophila spermatogenesis has become an ideal model system to study these questions. In the Drosophila testis, germ stem cells (GSCs) localize anterior, anchored to a niche of somatic cells (hub), and divide asymmetrically for self-renewal and to produce daughter progenitor gonialblast cells (GBs), which start the complex differentiation program that leads to the production of functional gametes. GBs are surrounded by 2 somatic cyst cells (Cs) and undergo four successive rounds of transit-amplifying (TA) mitoses with incomplete cytokinesis to produce a cyst of 16 sister spermatogonial cells that remain interconnected. Then, cysts differentiate to spermatocytes and undergo two meiotic divisions to produce 64 spermatids that develop to mature sperm cells (Carbonell, 2017).

bag-of-marbles (bam) is an important regulator of the first stages of spermatogenesis. Upon asymmetric division, daughter cells move away from the niche and escape Dpp/BMP-mediated repression. Bam expression increases during the first TA divisions, reaching a maximum at the 8-cell stage. Then, at the 16-cell stage, Bam expression decreases rapidly, TA proliferation stops, and differentiation into spermatocytes proceeds. How these crucial developmental transitions occurring during early male GSC lineage differentiation are regulated is not fully understood. Bam is a translational repressor that interacts with Bgcn (Benign gonial cell neoplasm) and Tut (tumorous testis) to repress Mei-P26 expression, establishing a regulatory feedback loop that governs spermatogonia proliferation. Bam also plays an important function in female oogenesis, in which it is repressed in GSCs by Dpp/BMP signaling and interacts with Bgcn to prevent translation of GSC maintenance factors (Carbonell, 2017).

This study report on the essential contribution of the Drosophila germline-specific linker histone H1 (dBigH1) to male GSC lineage development and differentiation. Linker H1s are intrinsic components of chromatin that interact with the nucleosome and regulate chromatin higher-order organization. In comparison to core histones, H1s are less well conserved, with most species containing several variants that play partially redundant functions. A conserved feature in metazoans is the presence of germline-specific variants that replace somatic H1s in germ cells (GCs) (Pérez-Montero, 2016). Vertebrates generally contain several male-specific variants (i.e., H1t, HILS1, and H1T2 in mice and humans) and one female-specific H1 (i.e., B4 in Xenopus and H1oo in mice and humans). In contrast, a single germline-specific linker histone dBigH1 exists in Drosophila, which is present in both the female and the male germline (Pérez-Montero, 2013). Female-specific H1s are generally retained during early embryogenesis until zygotic genome activation (ZGA) (Pérez-Montero, 2016). In this regard, in Drosophila, dBigH1 has been shown to maintain the zygotic genome silenced until ZGA is completed at cellularization, when dBigH1 is replaced by somatic dH1 (Pérez-Montero, 2013; Carbonell, 2017 and references therein).

Little is known about the functions that germline-specific H1s play in GSC lineage development and differentiation. In mammals, h1t2 mutant mice show several abnormalities during spermatogenesis and have reduced fertility. Similarly, hils1 expression is reduced in men suffering from reduced sperm motility. However, h1t mutants do not show detectable abnormality or fertility defects. In females, H1oo is required for maturation of germinal-vesicle stage oocytes. Finally, in Caenorhabditis elegans, depletion of H1.1/HIS-24, which is abundant in the germline, affects GCs proliferation and differentiation and reduces fertility. This study shows that in Drosophila, dBigH1 is essential for male GSC lineage differentiation. dBigH1 and Bam form a double-repressor loop that regulates progression into spermatocyte differentiation. It study also shows that dBigH1 acts as a general repressor in spermatocytes and that dBigH1 is required for male GSC maintenance. Altogether, these results unveil the essential contribution of germline-specific linker histone H1 variants to GSC lineage development and differentiation (Carbonell, 2017).

This study shows that dBigH1 is required to silence bam, which is a master regulator of spermatogonia proliferation and differentiation. During the first three TA divisions, Mei-P26 facilitates accumulation of Bam, which reaches a maximum at the 8-cell stage. Then, Bam levels decrease and spermatogonia stop proliferation and differentiate to spermatocytes (see dBigH1 and Bam Form a Double-Repressor Loop that Regulates Entrance into Spermatocyte Differentiation). Several mechanisms are known to contribute to Bam downregulation after the 8-cell stage. High Bam levels downregulate Mei-P26 translation, establishing a regulatory feedback loop (Insco, 2012). In addition, several microRNAs have been shown to downregulate Bam translation. The current results suggest a model by which, in addition to translational regulation, dBigH1-mediated transcriptional repression is required to silence bam during spermatocyte differentiation. In the absence of dBigH1, bam is not silenced; thus, entrance to the spermatocyte differentiation program is blocked and spermatogonial cells accumulate. This accumulation is not accompanied by increased spermatogonia proliferation; since dBigH1 is absent during the TA divisions and, therefore, its depletion is not affecting Bam accumulation to reach the threshold that dictates proliferation stop (Insco, 2009). The current results indicate that in addition to bam, dBigH1 represses expression of multiple other genes in spermatocytes, suggesting that like in early embryogenesis, dBigH1 acts as a general silencing factor in spermatocytes. Altogether, these observations support a model by which dBigH1 acts after spermatogonia cease proliferation to set up the specific gene expression program that governs spermatocyte differentiation (Carbonell, 2017).

The results also show that Bam, which is an important translational repressor, downregulates dBigH1 expression during TA divisions. dBigH1 expression in TA cells decreases parallel to the progressive accumulation of Bam, being detectable in all 2-cell cysts and in some 4-cell cysts. It is not known whether Bam directly interacts with dBigH1 mRNAs. However, dBigH1 mRNAs are likely present during TA divisions, as they are detected before spermatocyte differentiation (Vibranovski, 2009) and bam-GAL4-induced dBigH1 depletion in TA cells reduces dBigH1 content, blocking spermatocyte differentiation. Moreover, Bam represses dBigH1 expression specifically in TA spermatogonial cells when it is driven by the ubiquitously active vasa promoter. Altogether, these observations suggest that Bam expression during the TA divisions inhibits dBigH1 mRNA translation. Later, when Bam levels decrease, dBigH1 translation resumes, reinforcing Bam downregulation through transcriptional silencing. The results show that this dBigH1/Bam double-repressor loop is crucial to license spermatogonia into spermatocyte differentiation. The important contribution of mechanisms that regulate mRNA translation during spermatogenesis has been extensively studied. However, the actual role of transcription regulation in these processes is not well understood. From this point of view, this work unveils a functional interaction during the early stages of spermatogenesis that integrates both translational and transcriptional regulation (Carbonell, 2017).

These results also suggest that dBigH1 is required for GSC maintenance, as shown by the strong developmental defects observed in nos > bigH1RNAi testes in which dBigH1 depletion was induced in GSCs. These defects include the lack of testes in ∼10% of cases and the drastic loss of GCs in the rest of the affected testes. bam overexpression results in GSC loss. However, the contribution of dBigH1 to GSC maintenance is not likely reflecting a role in bam repression since, in knockdown nos > bigH1RNAi testes, no derepression of a bamP-GFP reporter was observed in vasa-positive hub-attached cells that showed no detectable dBigH1 expression. In this regard, it is known that bam is actively repressed in GSCs by the DNA binding proteins PMad/Medea that are downstream effectors of Dpp/BMP signals emanating from the somatic cells of the niche. Repression imposed by specific DNA binding proteins likely prevails over transcriptional silencing induced by general repressors such as dBigH1. dBigH1 expression is constrained to the primordial GSCs early in embryogenesis, being present in somatic cells as long as their transcriptional program is not turned on (Pérez-Montero, 2013). In this scenario, it is tempting to speculate that dBigH1 is required in GSCs to repress the somatic gene expression program throughout development. In this regard, its replacement by somatic dH1 during TA divisions is particularly intriguing. How this replacement takes place and what the consequences of its misregulation are remain to be determined (Carbonell, 2017).

The presence of germline-specific histone H1 is conserved in metazoans (Pérez-Montero, 2016). However, to date, detailed functional analysis of their contribution to germline development and differentiation was largely missing. From this point of view, this study unveils the fundamental functions that germline-specific linker histone H1 variants play in male GSC lineage differentiation, providing further understanding of the factors and mechanisms that regulate the dramatic developmental transitions associated with spermatogenesis (Carbonell, 2017).

BEN domain protein Elba2 can functionally substitute for linker histone H1 in Drosophila

Metazoan linker histones are essential for development and play crucial roles in organization of chromatin, modification of epigenetic states and regulation of genetic activity. Vertebrates express multiple linker histone H1 isoforms, which may function redundantly. In contrast, H1 isoforms are not present in Dipterans, including D. melanogaster, except for an embryo-specific, distantly related dBigH1. This study shows that Drosophila BEN domain protein Elba2, which is expressed in early embryos and has been hypothesized to have insulator-specific functions, can compensate for the loss of H1 in vivo. Although the Elba2 gene is not essential, its mutation causes a disruption of normal internucleosomal spacing of chromatin and reduced nuclear compaction in syncytial embryos. Elba2 protein is distributed ubiquitously in polytene chromosomes and strongly colocalizes with H1. In H1-depleted animals, ectopic expression of Elba2 rescues the increased lethality and ameliorates abnormalities of chromosome architecture and heterochromatin functions. Ectopic expression of BigH1 similarly complements the deficiency of H1 protein. Thus, in organisms that do not express redundant H1 isoforms, the structural and biological functions performed by canonical linker histones in later development, may be shared in early embryos by weakly homologous proteins, such as BigH1, or even unrelated, non-homologous proteins, such as Elba2 (Xu, 2016).

The embryonic linker histone H1 variant of Drosophila, dBigH1, regulates zygotic genome activation

Histone H1 is an essential chromatin component. Metazoans usually contain multiple stage-specific H1s. In particular, specific variants replace somatic H1s during early embryogenesis. In this regard, Drosophila was an exception because a single dH1 was identified that, starting at cellularization, is detected throughout development in somatic cells. This study has identified the embryonic H1 of Drosophila, dBigH1. dBigH1 is abundant before cellularization occurs, when somatic dH1 is absent and the zygotic genome is inactive. Upon cellularization, when the zygotic genome is progressively activated, dH1 replaces dBigH1 in the soma, but not in the primordial germ cells (PGCs) that have delayed zygotic genome activation (ZGA). In addition, a loss-of-function mutant shows premature ZGA in both the soma and PGCs. Mutant embryos die at cellularization, showing increased levels of active RNApol II and zygotic transcripts, along with DNA damage and mitotic defects. These results show an essential function of dBigH1 in ZGA regulation (Pérez-Montero, 2013).

This study has identified the early embryonic histone H1 variant of Drosophila, dBigH1. H1 variants that specifically replace somatic H1s during early embryogenesis have been reported in most metazoan species analyzed to date. Up to now, Drosophila has been a remarkable exception to this general rule, since a single dH1 variant was identified that, starting at cellularization, is detected throughout development in somatic cells. In this regard, HMGD has proposed to act as an early embryonic linker binding protein that plays a structural role similar to that of dH1. Subsequent work challenged this hypothesis, since null hmgD mutants are viable and show normal nuclear morphology and no detectable mitotic defects, in contrast to the strong mitotic defects and high early embryonic lethality of bigH1100 mutants. dBigH1 shows characteristic features of a histone H1: (1) it has a tripartite structure with significant homology to dH1, particularly at the central and C-terminal domains; (2) it localizes throughout chromatin; (3) it binds nucleosomes; and (4) it regulates nucleosomal spacing. Like other embryonic H1s, dBigH1 is very abundant during early embryogenesis, is replaced by dH1 at cellularization, and then is no longer detected except in germline cells. In adults, dBigH1 is abundant in ovaries, which is common to most other early embryonic H1s studied. In addition, dBigH1 is also detected in testes. The presence of specific H1s during male spermatogenesis has been studied in some detail only in mammals, in which several male-specific H1s, but no embryonic H1oo variant, have been identified. Whether the presence of embryonic H1s during spermatogenesis is a peculiarity of Drosophila or is conserved in other eukaryotes remains to be determined (Pérez-Montero, 2013).

dBigH1 is highly conserved in Drosophila, from D. grimshawi to D. simulans. An unusual feature of dBigH1 in comparison with somatic dH1 is its relative enrichment in acidic residues (17.8% versus 4.7%, respectively) in D. melanogaster. In part, this enrichment is due to the acquisition of a highly acidic domain at the N-terminal tail that occurred before the D. obscura and D. melanogaster groups separated >30 million years ago. In D. melanogaster, more than half of the residues in this extra domain are acidic, and the complete N-terminal tail shows an ~5-fold excess of acidic versus basic residues (37.9% versus 7.7%, respectively), which is in contrast to the net enrichment in basic residues of somatic dH1 The N-terminal tail of dBigH1 from ancient Drosophila species (i.e., D. willistoni), which lack the extra domain mentioned above, are also enriched in acidic residues, albeit to a lesser extent. In fact, embryonic H1s from other lineages are also more acidic than their somatic counterparts, indicating that increased content in acidic residues is a conserved feature of embryonic H1s. However, this enrichment can be detected at either the N-terminal tail (as in Drosophila [sea urchin CS-H1]) or the C-terminal tail (Xenopus B4, mammalian H1oo), or at both the N- and C-terminal tails (Zebrafish H1M). Notice that in parallel to the increased acidic content, embryonic H1s also show a decreased content in basic residues at both the N- and C-terminal tails. As a consequence, embryonic H1s are less positively charged than somatic H1s, which suggests a weaker interaction with DNA. As a matter of fact, several embryonic H1s have been shown to bind nucleosomes with lower affinity and condense chromatin to a lower extent than somatic H1s in vitro (Pérez-Montero, 2013).

The functional properties of embryonic H1s remain largely undetermined. This study has shown that in Drosophila, dBigH1 plays an essential role during early embryonic development in preventing premature ZGA in both the soma and the germline. The gene-expression program that governs embryogenesis is tightly regulated, with specific sets of genes being sequentially activated or repressed to induce specific developmental transitions. Thus, premature initiation of zygotic transcription would alter this finely tuned genetic program and have strongly deleterious effects, accounting for the observed high lethality of the bigH1100 mutation. This lethality is largely constrained to homozygous embryos that die at cellularization, suggesting that it mostly results from defective precellular zygotic dBigH1 expression. Although it has been generally accepted that the zygotic genome remains essentially silent before cellularization, evidence for precellular zygotic phenotypes has been reported for at least one gene, and precellular zygotic expression of multiple genes was recently reported in Drosophila. Zygotic transcription during cleavage stages has also been detected in echinoderms, amphibians, and zebrafish. Although the current results do not unambiguously establish the precise stage at which zygotic dBigH1 expression takes place, several observations support the hypothesis that it occurs during early blastoderm development. First, significant RNApol II binding was detected across the coding region in early 0-2 hpf embryos, which are mostly precellular. Furthermore, mutant bigH1100 embryos showing highly reduced dBigH1 content and mitotic defects are most abundant around syncytial blastoderm stage 4, indicating that their zygotic defect occurs at this stage. Finally, increased αdBigH1 staining was observed in stage 4 embryos at nuclear cycle 12 in comparison with nuclear cycle 11, albeit with relatively low frequency (~25% of WT embryos) (Pérez-Montero, 2013).

It was reported earlier that a deficiency uncovering dBigH1 does not show detectable defects prior to gastrulation (Müller, 1999). As a matter of fact, no genes on 3R appear to be required before cellularization, which is in contrast to the zygotic bigH1100 phenotype described in this study However, in the previous experiments, deficiency embryos were generated with the use of compound chromosomes and therefore carried a full diploid maternal dBigH1 dose, whereas in the current experiments the mutant bigH1100 embryos carried roughly half of the maternal dose because they were derived from heterozygous bigH1100 mothers. These observations suggest that the bigH1100 phenotype is also maternal, because the observed zygotic defects appear to depend on the heterozygous genotype of the mothers, and that zygotic dBigH1 expression in heterozygous bigH1100 embryos rescues the maternal defects. It was not possible to perform a classical germline clone analysis because dBigH1 is essential for gametogenesis in both females and males, as zygotically rescued homozygous bigH1100 flies are sterile and show gonads with strong developmental defects and incomplete gametogenesis. The results also show that partial rescue of the maternal dBigH1 contribution to ~80% of the WT content does not significantly increase the viability of homozygous bigH1100 embryos, suggesting that it is not sufficient to rescue the maternal defects. For a factor such as dBigH1, which is uniformly distributed across the genome and thus is rapidly used up during early embryogenesis, the maternal dose might be especially crucial to guarantee functional levels throughout embryogenesis. It must also be noted that zygotic dBigH1 expression appears to take place at late precellular stages, when the maternal contribution is rapidly declining, and just before replacement by dH1 occurs. In this regard, zygotic dBigH1 expression might be seen as a safeguard mechanism to ensure completion of cellularization. Most likely, the relatively modest rescue induced by ectopic zygotic dBigH1 expression reflects the short time window in which zygotic dBigH1 must be delivered to be functional. In this regard, the efficiency of zygotic rescue would strongly depend on the actual GAL4 levels available at this moment, so that changes in the maternal GAL4 dose would have a strong impact. This observation provides a reasonable explanation for the low efficiency observed when the rescue construct is provided maternally, since in this case, GAL4 is heavily used to activate the construct in the ovaries. Finally, it is uncertain whether a similar situation accounts for the observed PGC defects, since there is no reported evidence for zygotic expression in the PGC prior to cycle 14. Furthermore, this study observed PGC defects in heterozygous embryos, albeit with low frequency, suggesting a maternal effect. However, no major fertility defects were observed in heterozygous bigH1100 females or males, indicating that germline development was not seriously impaired (Pérez-Montero, 2013).

The mechanisms governing ZGA are not fully understood. In this regard, the results strongly suggest that in Drosophila, replacement of embryonic dBigH1 by somatic dH1 is an early ZGA event. In fact, the zygotic genome does not appear to become fully transcriptionally competent until somatic dH1 replaces dBigH1. This replacement is likely to be highly regulated since it occurs sequentially, first in euchromatin and later in heterochromatin, and appears to be regulated by phosphorylation, as phosphorylated dBigH1 is not detected at cellularization when dBigH1 is being replaced by dH1. Altogether, these observations suggest a model in which the presence of dBigH1 renders chromatin refractory to transcription, and replacement by dH1 licenses the zygotic genome for activation. The molecular mechanisms of dBigH1's contribution to ZGA regulation, and whether this contribution is conserved in other embryonic H1s, remain to be determined. However, it is tempting to speculate that increased acidic content, which is evolutionarily conserved, is a main functional determinant of embryonic H1s. It is possible that the presence of embryonic H1s modifies the electrostatic surface of chromatin, affecting the binding of key regulatory factors (Pérez-Montero, 2013).

In summary, these results identify dBigH1 as the early embryonic and germline-specific histone H1 variant of Drosophila, and unveil its essential role in the early embryo. This study constitutes the first functional characterization of the contribution of an embryonic H1 to development (Pérez-Montero, 2013).


Search PubMed for articles about Drosophila BigH1

Carbonell, A., Pérez-Montero, S., Climent-Canto, P., Reina, O. and Azorin, F. (2017). The germline linker histone dBigH1 and the translational regulator Bam form a repressor loop essential for male germ stem cell differentiation. Cell Rep 21(11): 3178-3189. PubMed ID: 29241545

Climent-Canto, P., Carbonell, A., Tatarski, M., Reina, O., Bujosa, P., Font-Mateu, J., Bernues, J., Beato, M. and Azorin, F. (2020). The embryonic linker histone dBigH1 alters the functional state of active chromatin. Nucleic Acids Res. PubMed ID: 32103264

Insco, M. L., Leon, A., Tam, C. H., McKearin, D. M. and Fuller, M. T. (2009). Accumulation of a differentiation regulator specifies transit amplifying division number in an adult stem cell lineage. Proc Natl Acad Sci U S A 106(52): 22311-22316. PubMed ID: 20018708

Insco, M. L., Bailey, A. S., Kim, J., Olivares, G. H., Wapinski, O. L., Tam, C. H. and Fuller, M. T. (2012). A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage. Cell Stem Cell 11(5): 689-700. PubMed ID: 23122292

Muller, H. A., Samanta, R. and Wieschaus, E. (1999). Wingless signaling in the Drosophila embryo: zygotic requirements and the role of the frizzled genes. Development 126(3): 577-586. PubMed ID: 9876186

Pérez-Montero, S., Carbonell, A., Moran, T., Vaquero, A. and Azorin, F. (2013). The embryonic linker histone H1 variant of Drosophila, dBigH1, regulates zygotic genome activation. Dev Cell 26(6): 578-590. PubMed ID: 24055651

Pérez-Montero, S., Carbonell, A. and Azorin, F. (2016). Germline-specific H1 variants: the "sexy" linker histones. Chromosoma 125(1): 1-13. PubMed ID: 25921218

Satovic, E., Font-Mateu, J., Carbonell, A., Beato, M. and Azorin, F. (2018). Chromatin remodeling in Drosophila preblastodermic embryo extract. Sci Rep 8(1): 10927. PubMed ID: 30026552

Vibranovski, M. D., Lopes, H. F., Karr, T. L. and Long, M. (2009). Stage-specific expression profiling of Drosophila spermatogenesis suggests that meiotic sex chromosome inactivation drives genomic relocation of testis-expressed genes. PLoS Genet 5(11): e1000731. PubMed ID: 19936020

Xu, N., Lu, X., Kavi, H., Emelyanov, A.V., Bernardo, T.J., Vershilova, E., Skoultchi, A.I. and Fyodorov, D.V. (2016). BEN domain protein Elba2 can functionally substitute for linker histone H1 in Drosophila in vivo. Sci Rep 6: 34354. PubMed ID: 27687115

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date revised: 20 June 2020

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