Phosphorylated histone variant γH2Av is associated with chromatin insulators in Drosophila.

Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of Topologically Associating Domains (TADs). Here, we demonstrate an association between gypsy insulator proteins and the phosphorylated histone variant H2Av (γH2Av), normally a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome, in polytene chromosomes and in diploid cells in which Chromatin IP data shows it is enriched at TAD boundaries. Mutation of insulator components su(Hw) and Cp190 results in a significant reduction in γH2Av levels in chromatin and phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2Av localization in insulator mutants. We also show that γH2Av, but not H2Av, is a component of insulator bodies, which are protein condensates that form during osmotic stress. Phosphatase activity is required for insulator body dissolution after stress recovery. Together, our results implicate the H2A variant with a novel mechanism of insulator function and boundary formation.

Mutations in the insulator protein Suppressor of Hairy wing induce genome instability.

Insulator proteins orchestrate the three-dimensional organization of the genome. Insulators function by facilitating communications between regulatory sequences and gene promoters, allowing accurate gene transcription regulation during embryo development and cell differentiation. However, the role of insulator proteins beyond genome organization and transcription regulation remains unclear. Suppressor of Hairy wing [Su(Hw)] is a Drosophila insulator protein that plays an important function in female oogenesis. Here we find that su(Hw) has an unsuspected role in genome stability during cell differentiation. We show that su(Hw) mutant developing egg chambers have poorly formed microtubule organization centers (MTOCs) in the germarium and display mislocalization of the anterior/posterior axis specification factor gurken in later oogenesis stages. Additionally, eggshells from partially rescued su(Hw) mutant female germline exhibit dorsoventral patterning defects. These phenotypes are very similar to phenotypes found in the important class of spindle mutants or in piRNA pathway mutants in Drosophila, in which defects generally result from the failure of germ cells to repair DNA damage. Similarities between mutations in su(Hw) and spindle and piRNA mutants are further supported by an excess of DNA damage in nurse cells, and because Gurken localization defects are partially rescued by mutations in the ATR (mei-41) and Chk1 (grapes) DNA damage response genes. Finally, we also show that su(Hw) mutants produce an elevated number of chromosome breaks in dividing neuroblasts from larval brains. Together, these findings suggest that Su(Hw) is necessary for the maintenance of genome integrity during Drosophila development, in both germline and dividing somatic cells.

The insulator protein Suppressor of Hairy wing is required for proper ring canal development during oogenesis in Drosophila.

Chromatin insulators orchestrate gene transcription during embryo development and cell differentiation by stabilizing interactions between distant genomic sites. Mutations in genes encoding insulator proteins are generally lethal, making in vivo functional analyses of insulator proteins difficult. In Drosophila, however, mutations in the gene encoding the Suppressor of Hairy wing insulator protein [Su(Hw)] are viable and female sterile, providing an opportunity to study insulator function during oocyte development. Whereas previous reports suggest that the function of Su(Hw) in oogenesis is independent of its insulator activity, many aspects of the role of Su(Hw) in Drosophila oogenesis remain unexplored. Here we show that mutations in su(Hw) result in smaller ring canal lumens and smaller outer ring diameters, which likely obstruct molecular and vesicle passage from nurse cells to the oocyte. Fluorescence microscopy reveals that lack of Su(Hw) leads to excess accumulation of Kelch (Kel) and Filament-actin (F-actin) proteins in the ring canal structures of developing egg chambers. Furthermore, we found that misexpression of the Src oncogene at 64B (Src64B) may cause ring canal development defects as microarray analysis and real-time RT-PCR revealed there is a three fold decrease in Src64B expression in su(Hw) mutant ovaries. Restoration of Src64B expression in su(Hw) mutant female germ cells rescued the ring phenotype but did not restore fertility. We conclude that loss of su(Hw) affects expression of many oogenesis related genes and down-regulates Src64B, resulting in ring canal defects potentially contributing to obstruction of molecular flow and an eventual failure of egg chamber organization.

Expanding the roles of chromatin insulators in nuclear architecture, chromatin organization and genome function.

Of the numerous classes of elements involved in modulating eukaryotic chromosome structure and function, chromatin insulators arguably remain the most poorly understood in their contribution to these processes in vivo. Indeed, our view of chromatin insulators has evolved dramatically since their chromatin boundary and enhancer blocking properties were elucidated roughly a quarter of a century ago as a result of recent genome-wide, high-throughput methods better suited to probing the role of these elements in their native genomic contexts. The overall theme that has emerged from these studies is that chromatin insulators function as general facilitators of higher-order chromatin loop structures that exert both physical and functional constraints on the genome. In this review, we summarize the result of recent work that supports this idea as well as a number of other studies linking these elements to a diverse array of nuclear processes, suggesting that chromatin insulators exert master control over genome organization and behavior.

<i>Drosophila</i> insulator proteins exhibit in vivo liquid-liquid phase separation properties.

Mounting evidence implicates liquid-liquid phase separation (LLPS), the condensation of biomolecules into liquid-like droplets in the formation and dissolution of membraneless intracellular organelles (MLOs). Cells use MLOs or condensates for various biological processes, including emergency signaling and spatiotemporal control over steady-state biochemical reactions and heterochromatin formation. Insulator proteins are architectural elements involved in establishing independent domains of transcriptional activity within eukaryotic genomes. In <i>Drosophila</i>, insulator proteins form nuclear foci known as insulator bodies in response to osmotic stress. However, the mechanism through which insulator proteins assemble into bodies is yet to be investigated. Here, we identify signatures of LLPS by insulator bodies, including high disorder tendency in insulator proteins, scaffold-client-dependent assembly, extensive fusion behavior, sphericity, and sensitivity to 1,6-hexanediol. We also show that the cohesin subunit Rad21 is a component of insulator bodies, adding to the known insulator protein constituents and γH2Av. Our data suggest a concerted role of cohesin and insulator proteins in insulator body formation and under physiological conditions. We propose a mechanism whereby these architectural proteins modulate 3D genome organization through LLPS.

A Drosophila insulator interacting protein suppresses enhancer-blocking function and modulates replication timing.

Insulators play important roles in genome structure and function in eukaryotes. Interactions between a DNA binding insulator protein and its interacting partner proteins define the properties of each insulator site. The different roles of insulator protein partners in the Drosophila genome and how they confer functional specificity remain poorly understood. The Suppressor of Hairy wing [Su(Hw)] insulator is targeted to the nuclear lamina, preferentially localizes at euchromatin/heterochromatin boundaries, and is associated with the gypsy retrotransposon. Insulator activity relies on the ability of the Su(Hw) protein to bind the DNA at specific sites and interact with Mod(mdg4)67.2 and CP190 partner proteins. HP1 and insulator partner protein 1 (HIPP1) is a partner of Su(Hw), but how HIPP1 contributes to the function of Su(Hw) insulator complexes is unclear. Here, we demonstrate that HIPP1 colocalizes with the Su(Hw) insulator complex in polytene chromatin and in stress-induced insulator bodies. We find that the overexpression of either HIPP1 or Su(Hw) or mutation of the HIPP1 crotonase-like domain (CLD) causes defects in cell proliferation by limiting the progression of DNA replication. We also show that HIPP1 overexpression suppresses the Su(Hw) insulator enhancer-blocking function, while mutation of the HIPP1 CLD does not affect Su(Hw) enhancer blocking. These findings demonstrate a functional relationship between HIPP1 and the Su(Hw) insulator complex and suggest that the CLD, while not involved in enhancer blocking, influences cell cycle progression.

Chromatin insulators specifically associate with different levels of higher-order chromatin organization in Drosophila.

Chromatin insulators are required for proper temporal and spatial expression of genes in metazoans. Here, we have analyzed the distribution of insulator proteins on the 56F-58A region of chromosome 2R in Drosophila polytene chromosomes to assess the role of chromatin insulators in shaping genome architecture. Data show that the suppressor of Hairy-wing protein [Su(Hw)] is found in three structures differentially associated with insulator proteins: bands, interbands, and multi-gene domains of coexpressed genes. Results show that bands are generally formed by condensation of chromatin that belongs to genes containing one or more Su(Hw) binding sites, whereas, in interbands, Su(Hw) sites appear associated with open chromatin. In addition, clusters of coexpressed genes in this region form bands characterized by the lack of CP190 and BEAF-32 insulator proteins. This pattern correlates with the distribution of specific chromatin marks and is conserved in nurse cells, suggesting that this organization may not be limited to one cell type but represents the basic organization of interphasic chromosomes.

Changes in chromatin structure correlate with transcriptional activity of nucleolar rDNA in polytene chromosomes.

Ribosomal DNA genes (rDNA) are found in tandem arrays of hundreds of repeated genes, but only a fraction of these genes are actively transcribed. The regulatory mechanism controlling the transition between active and inactive rDNA in higher eukaryotes is vital for cell survival. Here, we show that the nucleolus from Drosophila salivary gland cells contains two levels of chromatin organization reflecting differences in transcriptional activity: Decondensed chromatin is highly occupied with TATA-box-binding protein (TBP), phosphorylated H3S10, and acetylated H3K14, suggesting that rDNA in decondensed nucleolar areas is actively transcribed. Condensed chromatin lacks TBP, phosphorylated H3S10, or acetylated H3K14 and is enriched in the rDNA retrotransposons R1 and R2. The data show that R1 and R2 retrotransposons are not actively transcribed in salivary glands and may lead to the epigenetic silencing of flanking rDNA genes and that the silencing mechanisms of these sequences might be partially independent of heterochromatin formation by methylation of histone H3 at lysine 9 and binding of heterochromatin protein 1.

The phylogenetic distribution of non-CTCF insulator proteins is limited to insects and reveals that BEAF-32 is Drosophila lineage specific.

Chromatin insulators are DNA sequences found in eukaryotes that may organize genomes into chromatin domains by blocking enhancer-promoter interactions and preventing heterochromatin spreading. Considering that insulators play important roles in organizing higher order chromatin structure and modulating gene expression, very little is known about their phylogenetic distribution. To date, six insulators and their associated proteins have been characterized, including Su(Hw), Zw5, CTCF, GAF, Mod(mdg4), and BEAF-32. However, all insulator proteins, with the exception of CTCF, which has also been identified in vertebrates and worms, have been exclusively described in Drosophila melanogaster. In this work, we have performed database searches utilizing each D. melanogaster insulator protein as a query to find orthologs in other organisms, revealing that except for CTCF all known insulator proteins are restricted to insects. In particular, the boundary element-associated factor of 32 kDa (BEAF-32), which binds to thousands of sites throughout the genome, was only found in the Drosophila lineage. Accordingly, we also found a significant bias of BEAF-32 binding sites in relation to transcription start sites (TSSs) in D. melanogaster but not in Anopheles gambiae, Apis mellifera, or Tribolium castaneum. These data suggest that DNA binding proteins such as BEAF-32 may have a dramatic impact in the genome of single evolutionary lineages. A more thorough evaluation of the phylogenetic distribution of insulator proteins will allow for a better understanding of whether the mechanism by which these proteins exert their function is conserved across phyla and their impact in genome evolution.

Insulator and Ovo proteins determine the frequency and specificity of insertion of the gypsy retrotransposon in Drosophila melanogaster.

The gypsy retrovirus of Drosophila is quite unique among retroviruses in that it shows a strong preference for integration into specific sites in the genome. In particular, gypsy integrates with a frequency of > 10% into the regulatory region of the ovo gene. We have used in vivo transgenic assays to dissect the role of Ovo proteins and the gypsy insulator during the process of gypsy site-specific integration. Here we show that DNA containing binding sites for the Ovo protein is required to promote site-specific gypsy integration into the regulatory region of the ovo gene. Using a synthetic sequence, we find that Ovo binding sites alone are also sufficient to promote gypsy site-specific integration into transgenes. These results indicate that Ovo proteins can determine the specificity of gypsy insertion. In addition, we find that interactions between a gypsy provirus and the gypsy preintegration complex may also participate in the process leading to the selection of gypsy integration sites. Finally, the results suggest that the relative orientation of two integrated gypsy sequences has an important role in the enhancer-blocking activity of the gypsy insulator.

The Fragile X Protein and Genome Function.

The fragile X syndrome (FXS) arises from loss of expression or function of the FMR1 gene and is one of the most common monogenic forms of intellectual disability and autism. During the past two decades of FXS research, the fragile X mental retardation protein (FMRP) has been primarily characterized as a cytoplasmic RNA binding protein that facilitates transport of select RNA substrates through neural projections and regulation of translation within synaptic compartments, with the protein products of such mRNAs then modulating cognitive functions. However, the presence of a small fraction of FMRP in the nucleus has long been recognized. Accordingly, recent studies have uncovered several mechanisms or pathways by which FMRP influences nuclear gene expression and genome function. Some of these pathways appear to be independent of the classical role for FMRP as a regulator of translation and point to novel functions, including the possibility that FMRP directly participates in the DNA damage response and in the maintenance of genome stability. In this review, we highlight these advances and discuss how these new findings could contribute to our understanding of FMRP in brain development and function, the neural pathology of fragile X syndrome, and perhaps impact of future therapeutic considerations.

Trans-splicing as a novel mechanism to explain interallelic complementation in Drosophila.

Two mutant alleles of the same gene, each located in one of the two homologous chromosomes, may in some instances restore the wild-type function of the gene. This is the case with certain combinations of mutant alleles in the mod(mdg4) gene. This gene encodes several different proteins, including Mod(mdg4)2.2, a component of the gypsy insulator. This protein is encoded by two separate transcription units that can be combined in a trans-splicing reaction to form the mature Mod(mdg4)2.2-encoding RNA. Molecular characterization of complementing alleles shows that they affect the two different transcription units. Flies homozygous for each allele are missing the Mod(mdg4)2.2 protein, whereas wild-type trans-heterozygotes are able to synthesize almost normal levels of the Mod(mdg4)2.2 product. This protein is functional as judged by its ability to form a functional insulator complex. The results suggest that the interallelic complementation in the mod(mdg4) gene is a consequence of trans-splicing between two different mutant transcripts. A conclusion from this observation is that the trans-splicing reaction that takes place between transcripts produced on two different mutant chromosomes ensures wild-type levels of functional protein.

The Drosophila gypsy insulator supports transvection in the presence of the vestigial enhancer.

Though operationally defined as cis-regulatory elements, enhancers can also communicate with promoters on a separate homolog in trans, a mechanism that has been suggested to account for the ability of certain alleles of the same gene to complement one another in a process otherwise known as transvection. This homolog-pairing dependent process is facilitated in Drosophila by chromatin-associated pairing proteins, many of which remain unknown and their mechanism of action uncharacterized. Here we have tested the role of the gypsy chromatin insulator in facilitating pairing and communication between enhancers and promoters in trans using a transgenic eGFP reporter system engineered to allow for targeted deletions in the vestigial Boundary Enhancer (vgBE) and the hsp70 minimal promoter, along with one or two flanking gypsy elements. We found a modest 2.5-3x increase in eGFP reporter levels from homozygotes carrying an intact copy of the reporter on each homolog compared to unpaired hemizygotes, although this behavior was independent of gypsy. However, detectable levels of GFP protein along the DV wing boundary in trans-heterozygotes lacking a single enhancer and promoter was only observed in the presence of two flanking gypsy elements. Our results demonstrate that gypsy can stimulate enhancer-promoter communication in trans throughout the genome in a context-dependent manner, likely through modulation of local chromatin dynamics once pairing has been established by other elements and highlights chromatin structure as the master regulator of this phenomenon.

Chromatin insulator bodies are nuclear structures that form in response to osmotic stress and cell death.

Chromatin insulators assist in the formation of higher-order chromatin structures by mediating long-range contacts between distant genomic sites. It has been suggested that insulators accomplish this task by forming dense nuclear foci termed insulator bodies that result from the coalescence of multiple protein-bound insulators. However, these structures remain poorly understood, particularly the mechanisms triggering body formation and their role in nuclear function. In this paper, we show that insulator proteins undergo a dramatic and dynamic spatial reorganization into insulator bodies during osmostress and cell death in a high osmolarity glycerol-p38 mitogen-activated protein kinase-independent manner, leading to a large reduction in DNA-bound insulator proteins that rapidly repopulate chromatin as the bodies disassemble upon return to isotonicity. These bodies occupy distinct nuclear territories and contain a defined structural arrangement of insulator proteins. Our findings suggest insulator bodies are novel nuclear stress foci that can be used as a proxy to monitor the chromatin-bound state of insulator proteins and provide new insights into the effects of osmostress on nuclear and genome organization.

Setting the boundaries of chromatin domains and nuclear organization.

The nuclear architecture of the interphase nucleus is established by laying down an intricate three-dimensional framework of higher-order chromatin structure. This arrangement is essential for the integration of complex biological processes such as DNA replication, RNA processing, and transcription. Boundary or insulator elements are emerging as key players in the establishment and maintenance of this organization.

Extensive exon reshuffling over evolutionary time coupled to trans-splicing in Drosophila.

The relative position of exons in genes can be altered only after large structural mutations. These mutations are frequently deleterious, impairing transcription, splicing, RNA stability, or protein function, as well as imposing strong inflexibility to protein evolution. Alternative cis- or trans-splicing may overcome the need for genomic structural stability, allowing genes to encode new proteins without the need to maintain a specific exon order. Trans-splicing in the Drosophila melanogaster modifier of mdg4 (mod[mdg4]) gene is the best documented example in which this process plays a major role in the maturation of mRNAs. Comparison of the genomic organization of this locus among several insect species suggests that the divergence between the lineages of the mosquito Anopheles gambiae and D. melanogaster involved an extensive exon rearrangement, requiring >11 breakpoints within the mod(mdg4) gene. The massive reorganization of the locus also included the deletion or addition of a new function as well as exon duplications. Whereas both DNA strands are sense strands in the Drosophila gene, the coding region in mosquito lays in a single strand, suggesting that trans-splicing may have originated in the Drosophila lineage and might have been the triggering factor for such a dramatic reorganization.

Phosphorylation of histone H3 during transcriptional activation depends on promoter structure.

Covalent modifications of histone N-terminal tails are required for the proper assembly and activation of the general transcription factors at promoters. Here, we analyze histone acetylation and phosphorylation in Drosophila transgenes activated by the yeast Gal4 transcriptional activator in the context of different promoters. We show that, independent of the promoter, transcription does not correlate with acetylation of either H3-Lys 14 or H4-Lys 8. Histone H3 associated with the DNA of Gal4-induced transcribing transgenes driven by the Drosophila Hsp70 promoter is hyperphosphorylated at Ser 10 during transcription. Surprisingly, histone H3 at Gal4-induced transgenes driven by the P element Transposase promoter is not hyperphosphorylated. The data suggest that transcription occurs without acetylated H4 and H3 in both transgenes in Drosophila polytene chromosomes. Instead, phosphorylation of H3 is linked to transcription and can be modulated by the structure of the promoter.