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.

<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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.