Linker histone H1 regulates homeostasis of heterochromatin-associated cRNAs.

Chromatin-associated RNAs (cRNAs) are a poorly characterized fraction of cellular RNAs that co-purify with chromatin. Their full complexity and the mechanisms regulating their packaging and chromatin association remain poorly understood. Here, we address these questions in Drosophila. We find that cRNAs constitute a heterogeneous group of RNA species that is abundant in heterochromatic transcripts. We show that heterochromatic cRNAs interact with the heterogeneous nuclear ribonucleoproteins (hnRNP) hrp36/hrp48 and that depletion of linker histone dH1 impairs this interaction. dH1 depletion induces the accumulation of RNA::DNA hybrids (R-loops) in heterochromatin and, as a consequence, increases retention of heterochromatic cRNAs. These effects correlate with increased RNA polymerase II (RNAPII) occupancy at heterochromatin. Notably, impairing cRNA assembly by depletion of hrp36/hrp48 mimics heterochromatic R-loop accumulation induced by dH1 depletion. We also show that dH1 depletion alters nucleosome organization, increasing accessibility of heterochromatin. Altogether, these perturbations facilitate annealing of cRNAs to the DNA template, enhancing R-loop formation and cRNA retention at heterochromatin.

Lysine 27 dimethylation of Drosophila linker histone dH1 contributes to heterochromatin organization independently of H3K9 methylation.

Post-translational modifications (PTMs) of core histones are important epigenetic determinants that correlate with functional chromatin states. However, despite multiple linker histone H1s PTMs have been identified, little is known about their genomic distribution and contribution to the epigenetic regulation of chromatin. Here, we address this question in Drosophila that encodes a single somatic linker histone, dH1. We previously reported that dH1 is dimethylated at K27 (dH1K27me2). Here, we show that dH1K27me2 is a major PTM of Drosophila heterochromatin. At mitosis, dH1K27me2 accumulates at pericentromeric heterochromatin, while, in interphase, it is also detected at intercalary heterochromatin. ChIPseq experiments show that >98% of dH1K27me2 enriched regions map to heterochromatic repetitive DNA elements, including transposable elements, simple DNA repeats and satellite DNAs. Moreover, expression of a mutated dH1K27A form, which impairs dH1K27me2, alters heterochromatin organization, upregulates expression of heterochromatic transposable elements and results in the accumulation of RNA:DNA hybrids (R-loops) in heterochromatin, without affecting H3K9 methylation and HP1a binding. The pattern of dH1K27me2 is H3K9 methylation independent, as it is equally detected in flies carrying a H3K9R mutation, and is not affected by depletion of Su(var)3-9, HP1a or Su(var)4-20. Altogether these results suggest that dH1K27me2 contributes to heterochromatin organization independently of H3K9 methylation.

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. Here we directly address this question by ectopically expressing dBigH1 in Drosophila S2 cells, which lack dBigH1. We show that dBigH1 binds across chromatin, replaces somatic dH1 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.

Linker histone H1 prevents R-loop accumulation and genome instability in heterochromatin.

Linker histone H1 is an important structural component of chromatin that stabilizes the nucleosome and compacts the nucleofilament into higher-order structures. The biology of histone H1 remains, however, poorly understood. Here we show that Drosophila histone H1 (dH1) prevents genome instability as indicated by the increased γH2Av (H2AvS137P) content and the high incidence of DNA breaks and sister-chromatid exchanges observed in dH1-depleted cells. Increased γH2Av occurs preferentially at heterochromatic elements, which are upregulated upon dH1 depletion, and is due to the abnormal accumulation of DNA:RNA hybrids (R-loops). R-loops accumulation is readily detectable in G1-phase, whereas γH2Av increases mainly during DNA replication. These defects induce JNK-mediated apoptosis and are specific of dH1 depletion since they are not observed when heterochromatin silencing is relieved by HP1a depletion. Altogether, our results suggest that histone H1 prevents R-loops-induced DNA damage in heterochromatin and unveil its essential contribution to maintenance of genome stability.While structural importance of linker histone H1 in packaging eukaryotic genome into chromatin is well known, its biological function remains poorly understood. Here the authors reveal that Drosophila linker histone H1 prevents DNA:RNA hybrids accumulation and genome instability in heterochromatin.

Histone H1: Lessons from Drosophila.

Eukaryotic genomes are structured in the form of chromatin with the help of a set of five small basic proteins, the histones. Four of them are highly conserved through evolution, form the basic unit of the chromatin, the nucleosome, and have been intensively studied and are well characterized. The fifth histone, histone H1, adds to this basic structure through its interaction at the entry/exit site of DNA in the nucleosome and makes an essential contribution to the higher order folding of the chromatin fiber. Histone H1 is the less conserved histone and the less known of them. Though for long time considered as a general repressor of gene expression, recent studies in Drosophila have rejected this view and have contributed to uncover important functions on genome stability and development. Here we present some of the most recent data obtained in the Drosophila model system and discuss how the lessons learnt in these studies compare and could be applied to all other eukaryotes.

The Drosophila histone demethylase dKDM5/LID regulates hematopoietic development.

dKDM5/LID regulates transcription of essential developmental genes and, thus, is required for different developmental processes. Here, we report the essential contribution of dKDM5/LID to hematopoiesis in Drosophila. Our results show that dKDM5/LID is abundant in hemocytes and that its depletion induces over-proliferation and differentiation defects of larval hemocytes and disrupts organization of the actin cytoskeleton. We also show that dKDM5/LID regulates expression of key factors of hematopoietic development. In particular, dKDM5/LID depletion up-regulates expression of several transcription factors involved in hemocytes proliferation and differentiation as well as of several small-GTPases that link signaling effectors to actin cytoskeleton formation and dynamics.

New insights for Drosophila GAGA factor in larvae.

GAGA factor plays important roles during Drosophila embryogenesis and its maternal contribution is essential for early development. Here, the role of GAGA factor was studied in 3rd instar larvae using depletion and overexpression conditions in wing disc and transcriptome analysis. We found that genes changing expression were different to those previously described using GAGA mutants in embryos. No apparent phenotypes on GAGA depletion could usually be observed at larval stages in imaginal discs but a strong effect on salivary gland polytene chromosomes was observed. In the adult, GAGA depletion produced many defects like abnormal cell proliferation in the wing, impaired dorsal closure and resulted in homeotic transformation of abdominal segment A5. Unexpectedly, no effects on Ultrabithorax expression were observed. Short overexpression of GAGA factor in 3rd instar larvae also resulted in activation of a set of genes not previously described to be under GAGA regulation, and in lethality at pupa. Our results suggest a little contribution of GAGA factor on gene transcription in wing discs and a change of the genes regulated in comparison with embryo. GAGA factor activity thus correlates with the global changes in gene expression that take place at the embryo-to-larva and, later, at the larva-to-pupa transitions.

GAGA factor repression of transcription is a rare event but the negative regulation of Trl is conserved in Drosophila species.

GAGA is a highly conserved Drosophila transcription factor encoded by the Trithorax-like (Trl) gene. While GAGA usually activates transcription, it represses its own promoter. Here we show that GAGA-mediated repression of Trl is conserved between two distant Drosophila species. A detailed promoter study showed that GAGA repressive activity can't be attributed to any discrete element in the Trl promoter. Genome-wide analysis of the transcriptome in S2 cells indicated that repression of Trl is very likely unique, being GAGA factor a transactivator for all the other promoters. Taken together, our results suggest a new mechanism to explain GAGA-mediated repression that involves a dose-dependent change in the architecture of the Trl promoter.

Combined bottom-up and top-down mass spectrometry analyses of the pattern of post-translational modifications of Drosophila melanogaster linker histone H1.

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.

Drosophila melanogaster linker histone dH1 is required for transposon silencing and to preserve genome integrity.

Histone H1 is an intrinsic component of chromatin, whose important contribution to chromatin structure is well-established in vitro. Little is known, however, about its functional roles in vivo. Here, we have addressed this question in Drosophila, a model system offering many advantages since it contains a single dH1 variant. For this purpose, RNAi was used to efficiently deplete dH1 in flies. Expression-profiling shows that dH1 depletion affects expression of a relatively small number of genes in a regional manner. Furthermore, depletion up-regulates inactive genes, preferentially those located in heterochromatin, while active euchromatic genes are down-regulated, suggesting that the contribution of dH1 to transcription regulation is mainly structural, organizing chromatin for proper gene-expression regulation. Up-regulated genes are remarkably enriched in transposons. In particular, R1/R2 retrotransposons, which specifically integrate in the rDNA locus, are strongly up-regulated. Actually, depletion increases expression of transposon-inserted rDNA copies, resulting in synthesis of aberrant rRNAs and enlarged nucleolus. Concomitantly, dH1-depleted cells accumulate extra-chromosomal rDNA, show increased γH2Av content, stop proliferation and activate apoptosis, indicating that depletion causes genome instability and affects proliferation. Finally, the contributions to maintenance of genome integrity and cell proliferation appear conserved in human hH1s, as their expression rescues proliferation of dH1-depleted cells.

The structure of a transcription activation subcomplex reveals how σ(70) is recruited to PhoB promoters.

PhoB is a two-component response regulator that activates transcription by interacting with the σ(70) subunit of the E. coli RNA polymerase in promoters in which the -35 σ(70)-recognition element is replaced by the pho box. The crystal structure of a transcription initiation subcomplex that includes the σ(4) domain of σ(70) fused with the RNA polymerase ß subunit flap tip helix, the PhoB effector domain and the pho box DNA reveals how σ(4) recognizes the upstream pho box repeat. As with the -35 element, σ(4) achieves this recognition through the N-terminal portion of its DNA recognition helix, but contact with the DNA major groove is less extensive. Unexpectedly, the same recognition helix contacts the transactivation loop and helices α2 and α3 of PhoB. This result shows a simple and elegant mechanism for polymerase recruitment to pho box promoters in which the lost -35 element contacts are compensated by new ones with the activator. In addition, σ(4) is reoriented, thereby suggesting a remodelling mechanism for transcription initiation.

Acetylation of GAGA factor modulates its interaction with DNA.

GAGA is a Drosophila transcription factor that shows a high degree of post-translational modification. Here, we show that GAGA factor is acetylated in vivo. Lysine residues K325 and K373 on basic regions BR1 and BR3 of the DNA binding domain, respectively, are shown to be acetylated by PCAF. While BR1 is strictly required to stabilize DNA binding, BR3 is dispensable. However, acetylation of both lysine residues, either alone or in combination, weakens the binding to DNA. Despite the high degree of conservation of K325 and K373 in flies, their mutation to glutamine does not affect DNA binding. Molecular dynamics simulations, using acetylated K325 and a K325Q mutant of GAGA DNA binding domain in complex with DNA, are fully consistent with these results and provide a thermodynamic explanation for this observation. We propose that while K325 and K373 are not essential for DNA binding they have been largely conserved for regulatory purposes, thus highlighting a key regulatory system for GAGA factor in flies.

New aminoacyl-tRNA synthetase-like protein in insecta with an essential mitochondrial function.

Aminoacyl-tRNA synthetases (ARS) are modular enzymes that aminoacylate transfer RNAs (tRNA) for their use by the ribosome during protein synthesis. ARS are essential and universal components of the genetic code that were almost completely established before the appearance of the last common ancestor of all living species. This long evolutionary history explains the growing number of functions being discovered for ARS, and for ARS homologues, beyond their canonical role in gene translation. Here we present a previously uncharacterized paralogue of seryl-tRNA synthetase named SLIMP (seryl-tRNA synthetase-like insect mitochondrial protein). SLIMP is the result of a duplication of a mitochondrial seryl-tRNA synthetase (SRS) gene that took place in early metazoans and was fixed in Insecta. Here we show that SLIMP is localized in the mitochondria, where it carries out an essential function that is unrelated to the aminoacylation of tRNA. The knockdown of SLIMP by RNA interference (RNAi) causes a decrease in respiration capacity and an increase in mitochondrial mass in the form of aberrant mitochondria.

Activation properties of GAGA transcription factor.

GAGA is a Drosophila transcription factor that has been involved in many nuclear activities. We present evidence that GAGA factor enhances transcription by stabilizing pre-initiation complex (PIC) and promoting reinitiation. Formation of PIC prior to GAGA addition prevents activation suggesting that GAGA is required early in the formation of activated complexes. GAGA stimulation of transcription can be attributed in part to a stabilization of PIC. All these properties depend on the GAGA C-terminal glutamine-rich domain and, in addition to other roles and together with previous data, support a role of GAGA as a transcription factor.

General, negative feedback mechanism for regulation of Trithorax-like gene expression in vivo: new roles for GAGA factor in flies.

Expression of every gene is first regulated at the transcriptional level. While some genes show acute and discrete periods of expression others show a rather steady expression level throughout development. An example of the latter is Trithorax-like (Trl) a member of the Trithorax group that encodes GAGA factor in Drosophila. Among other functions, GAGA factor has been described to stimulate transcription of several genes, including some homeotic genes. Here we show that GAGA factor is continuously down-regulating the expression of its own promoter using a negative feedback mechanism in vivo. Like its expression, repression by GAGA factor is ubiquitous, prevents its accumulation, and takes place throughout development. Experimental alteration of GAGA factor dosage results in several unexpected phenotypes, not related to alteration of homeotic gene expression, but rather to functions that take place later during development and affect different morphogenetic processes. The results suggest that GAGA factor is essential during development, even after homeotic gene expression is established, and indicate the existence of an upper limit for GAGA factor dosage that should not be exceeded.

The GAGA protein of Drosophila is phosphorylated by CK2.

The GAGA factor of Drosophila is a sequence-specific DNA-binding protein that contributes to multiple processes from the regulation of gene expression to the structural organisation of heterochromatin and chromatin remodelling. GAGA is known to interact with various other proteins (tramtrack, pipsqueak, batman and dSAP18) and protein complexes (PRC1, NURF and FACT). GAGA functions are likely regulated at the level of post-translational modifications. Little is known, however, about its actual pattern of modification. It was proposed that GAGA can be O-glycosylated. Here, we report that GAGA519 isoform is a phosphoprotein that is phosphorylated by CK2 at the region of the DNA-binding domain. Our results indicate that phosphorylation occurs at S388 and, to a lesser extent, at S378. These two residues are located in a region of the DNA-binding domain that makes no direct contact with DNA, being dispensable for sequence-specific recognition. Phosphorylation at these sites does not abolish DNA binding but reduces the affinity of the interaction. These results are discussed in the context of the various functions and interactions that GAGA supports.

Repression by TTK69 of GAGA-mediated activation occurs in the absence of TTK69 binding to DNA and solely requires the contribution of the POZ/BTB domain of TTK69.

tramtrack 69 (TTK69) is known to repress GAGA-mediated activation of the eve promoter in S2 cells. Here, we show that repression by TTK69 occurs in the absence of bona fide TTK69-binding sites on the template, indicating that it does not require the binding of TTK69 to DNA. Consistent with this interpretation, the POZ/BTB domain of TTK69, which does not bind DNA, is sufficient for repression. Moreover, a fusion protein in which the POZ/BTB domain of GAGA is replaced by that of TTK69 is not capable of activating the eve promoter but efficiently represses GAGA-dependent activation. Repression involves GAGA-TTK69 interaction because TTK69 is not capable of repressing basal transcription. Most probably, GAGA-TTK69 interaction occurs at the promoter because GAGA.TTK69 complexes are fully competent in binding DNA in vitro. Our results also show that repression by TTK69 of GAGA-dependent activation of the eve promoter is not mediated by any of the co-repressors known to interact with TTK69 (dMi2 or C-terminal binding protein) or by trichostatin A-sensitive histone deacetylases. Altogether, these observations strongly suggest that the binding of TTK69 prevents the interaction of GAGA with the transcription machinery and, therefore, compromises its activation potential.

The Drosophila transcription factor tramtrack (TTK) interacts with Trithorax-like (GAGA) and represses GAGA-mediated activation.

In this study, we report the interaction of the Drosophila transcription factors Trithorax-like (GAGA) and tramtrack (TTK). This interaction is documented both in vitro, through GST pull-down assays, as well as in vivo, in yeast and Schneider S2 cells. GAGA and TTK share in common the presence of an N-terminal POZ/BTB domain that was found to be necessary and sufficient for GAGA-TTK interaction. Structural models that could account for this interaction are discussed. GAGA is known to activate the expression of many genes in Drosophila. On the other hand, TTK was proposed to act as a maternally provided repressor of several pair-rule genes, such as even-skipped (eve). As with many Drosophila genes, eve contains at its promoter region binding sites for GAGA and TTK. Here, in transient expression experiments, we showed that GAGA activates transcription from the eve stripe 2 promoter element and that TTK inhibits this GAGA-dependent activation. Repression by TTK of the eve promoter requires its activation by GAGA and depends on the presence of the POZ/BTB domains of TTK and GAGA. These results indicate that GAGA-TTK interaction contributes to the regulation of gene expression in Drosophila.

GAGA factor down-regulates its own promoter.

GAGA factor is involved in many nuclear transactions, notably in transcription as an activator in Drosophila. The genomic region corresponding to the Trl promoter has been obtained, and a minimal version of a fully active Trl promoter has been defined using transient transfection assays in S2 cells. DNase I footprinting analysis has shown that this region contains multiple GAGA binding sites, suggesting a potential regulatory role of GAGA on its own promoter. The study shows that GAGA down-regulates Trl expression. The repression does not depend on the GAGA isoform, but binding to DNA is absolutely required. A fragment of the Trl promoter can mediate repression to a heterologous promoter only upon GAGA overexpression in transiently transfected S2 cells. Chromatin immunoprecipitation analysis of S2 cells confirmed that GAGA factors are bound to the Trl promoter over a region of 1.4 kbp. Using a double-stranded RNA interference approach, we show that endogenous GAGA factors limit Trl expression in S2 cells. Our results open the possibility of observing similar GAGA repressive effects on other promoters.

HMGB1 interacts with many apparently unrelated proteins by recognizing short amino acid sequences.

The chromatin high mobility group protein 1 (HMGB1) is a very abundant and conserved protein that is structured into two HMG box domains plus a highly acidic C-terminal domain. From the ability to bind DNA nonspecifically and to interact with various proteins, several functions in DNA-related processes have been assigned to HMGB1. Nevertheless, its functional role remains the subject of controversy. Using a phage display approach we have shown that HMGB1 can recognize several peptide motifs. A computer search of the protein data bases found peptide homologies with proteins already known to interact with HMGB1, like p53, and have allowed us to identify new potential candidates. Among them, transcriptional activators like the heterogeneous nuclear ribonucleoprotein K (hnRNP K), repressors like methyl-CpG binding protein 2 (MeCP2), and co-repressors like the retinoblastoma susceptibility protein (pRb) and Groucho-related gene proteins 1 (Grg1) and 5 (Grg5) can be found. A detailed analysis of the interaction of Grg1 with HMGB1 confirmed that the binding region contained the sequence homologous to one of the peptides identified. Our results have led us to propose that HMGB1 may play a central role in the stabilization and/or assembly of several multifunctional complexes through protein-protein interactions.