Temporal inhibition of chromatin looping and enhancer accessibility during neuronal remodeling

During development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation. The chromatin insulator-associated protein Alan Shepard (Shep) promotes Drosophila post-mitotic neuronal remodeling by repressing transcription of master developmental regulators, such as brain tumor (brat), specifically in maturing neurons. Since insulator proteins can promote looping, we hypothesized that Shep antagonizes brat promoter interaction with an as yet unidentified enhancer. Using chromatin conformation capture and reporter assays, we identified two enhancer regions that increase in looping frequency with the brat promoter specifically in pupal brains after Shep depletion. The brat promoters and enhancers function independently of Shep, ruling out direct repression of these elements. Moreover, ATAC-seq in isolated neurons demonstrates that Shep restricts chromatin accessibility of a key brat enhancer as well as other enhancers genome-wide in remodeling pupal but not larval neurons. These enhancers are enriched for chromatin targets of Shep and are located at Shep-inhibited genes, suggesting direct Shep inhibition of enhancer accessibility and gene expression during neuronal remodeling. Our results provide evidence for temporal regulation of chromatin looping and enhancer accessibility during neuronal maturation.

Insulators in Plants: Progress and Open Questions

The genomes of higher eukaryotes are partitioned into topologically associated domains or TADs, and insulators (also known as boundary elements) are the key elements responsible for their formation and maintenance. Insulators were first identified and extensively studied in Drosophila as well as mammalian genomes, and have also been described in yeast and plants. In addition, many insulator proteins are known in Drosophila, and some have been investigated in mammals. However, much less is known about this important class of non-coding DNA elements in plant genomes. In this review, we take a detailed look at known plant insulators across different species and provide an overview of potential determinants of plant insulator functions, including cis-elements and boundary proteins. We also discuss methods previously used in attempts to identify plant insulators, provide a perspective on their importance for research and biotechnology, and discuss areas of potential future research.

Temporal inhibition of chromatin looping and enhancer accessibility during neuronal remodeling

ABSTRACTDuring development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation. The chromatin insulator-associated protein Shep promotes Drosophila post-mitotic neuronal remodeling by repressing transcription of master developmental regulators, such as brain tumor (brat), specifically in maturing neurons. Since insulator proteins can promote looping, we hypothesized that Shep antagonizes brat promoter interaction with an as yet unidentified enhancer. Using chromatin conformation capture and reporter assays, we identified two novel enhancer regions that increase in looping frequency with the brat promoter specifically in pupal brains after Shep depletion. The brat promoters and enhancers function independently of Shep, ruling out direct repression of these elements. Moreover, ATAC-seq in isolated neurons demonstrated that Shep restricts chromatin accessibility of a key brat enhancer as well as other enhancers genome-wide in remodeling pupal but not larval neurons. These enhancers are enriched for chromatin targets of Shep and are located at Shep-inhibited genes, suggesting direct Shep inhibition of enhancer accessibility and gene expression during neuronal remodeling. Our results provide evidence for temporal regulation of chromatin looping and enhancer accessibility during neuronal maturation.

Genomic context sensitivity of insulator function

Compartmentalization of interactions between genomic regulatory elements and potential target genes is influenced by the binding of insulator proteins such as CTCF, which act as potent enhancer blockers when interposed between an enhancer and a promoter in a reporter assay. But only a minority of CTCF sites genome-wide function as bona fide insulators, depending on cellular and genomic context. To dissect the influence of genomic context on enhancer blocker activity, we integrated reporter constructs with promoter-only, promoter and enhancer, and enhancer blocker configurations at hundreds of thousands of genomic sites using the Sleeping Beauty transposase. Deconvolution of reporter activity by genomic position revealed strikingly different patterns of reporter function, including a compartment of enhancer blocker reporter integration sites with robust expression. The high density of integration sites permits quantitative delineation of characteristic genomic context sensitivity profiles, and their decomposition into sensitivity to both local and distant DNaseI hypersensitive sites. Furthermore, a single-cell expression approach permits direct linkage of reporters integrated into the same clonal lineage with differential endogenous gene expression, we observe that CTCF insulator activity does not completely abrogate reporter effects on endogenous gene expression. Collectively, our results lend new insight to genomic regulatory compartmentalization and its influence on the determinants of promoter-enhancer specificity.

A Phosphorylated Histone H2A Variant Displays Properties of Chromatin Insulator Proteins in Drosophila

Chromatin insulators are responsible for mediating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of topologically associating domains (TADs). Here, we demonstrate an interaction between proteins that associate with the gypsy insulator and the phosphorylated histone variant H2Av (γH2Av), a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome. Mutation of insulator components prevents stable H2Av phosphorylation in polytene chromatin. Phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2Av localization in insulator mutants. We also show that γH2Av is a component of insulator bodies, and that phosphatase activity is required for insulator body dissolution after recovery from osmotic stress. We further demonstrate a tight association between γH2Av and TAD boundaries. Together, our results indicate a novel mechanism linking insulator function with a histone H2A variant and with genome stability.

Structural and Functional Dissection of the 5′ Region of the Notch Gene in Drosophila melanogaster

Notch is a key factor of a signaling cascade which regulates cell differentiation in all multicellular organisms. Numerous investigations have been directed mainly at studying the mechanism of Notch protein action; however, very little is known about the regulation of activity of the gene itself. Here, we provide the results of targeted 5′-end editing of the Drosophila Notch gene in its native environment and genetic and cytological effects of these changes. Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system in combination with homologous recombination, we obtained a founder fly stock in which a 4-kb fragment, including the 5′ nontranscribed region, the first exon, and a part of the first intron of Notch, was replaced by an attachment Phage (attP) site. Then, fly lines carrying a set of six deletions within the 5′untranscribed region of the gene were obtained by ΦC31-mediated integration of transgenic constructs. Part of these deletions does not affect gene activity, but their combinations with transgenic construct in the first intron of the gene cause defects in the Notch target tissues. At the polytene chromosome level we defined a DNA segment (~250 bp) in the Notch5′-nontranscribed region which when deleted leads to disappearance of the 3C6/C7 interband and elimination of CTC-Factor (CTCF) and Chromator (CHRIZ) insulator proteins in this region.

3D genome organisation in Drosophila

Ever since Thomas Hunt Morgan’s discovery of the chromosomal basis of inheritance by using Drosophila melanogaster as a model organism, the fruit fly has remained an essential model system in studies of genome biology, including chromatin organisation. Very much as in vertebrates, in Drosophila, the genome is organised in territories, compartments and topologically associating domains (TADs). However, these domains might be formed through a slightly different mechanism than in vertebrates due to the presence of a large and potentially redundant set of insulator proteins and the minor role of dCTCF in TAD boundary formation. Here, we review the different levels of chromatin organisation in Drosophila and discuss mechanisms and factors that might be involved in TAD formation. The dynamics of TADs and enhancer–promoter interactions in the context of transcription are covered in the light of currently conflicting results. Finally, we illustrate the value of polymer modelling approaches to infer the principles governing the three-dimensional organisation of the Drosophila genome.

BEN-solo factors partition active chromatin to ensure proper gene activation in Drosophila

The Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in transcriptional repression and chromatin insulation. A fourth protein—Elba3—bridges Elba1 and Elba2 to form an ELBA complex. Here, we report comprehensive investigation of these proteins in Drosophila embryos. We assess common and distinct binding sites for Insv and ELBA and their genetic interdependencies. While Elba1 and Elba2 binding generally requires the ELBA complex, Elba3 can associate with chromatin independently of Elba1 and Elba2. We further demonstrate that ELBA collaborates with other insulators to regulate developmental patterning. Finally, we find that adjacent gene pairs separated by an ELBA bound sequence become less differentially expressed in ELBA mutants. Transgenic reporters confirm the insulating activity of ELBA- and Insv-bound sites. These findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators to partition transcription units.

Insulator proteins contribute to expression of gene loci repositioned into heterochromatin in the course of Drosophila evolution

Pericentric heterochromatin in Drosophila is generally composed of repetitive DNA forming a transcriptionally repressive environment. Nevertheless, dozens of genes were embedded into pericentric genome regions during evolution of Drosophilidae lineage and retained functional activity. However, factors that contribute to “immunity” of these gene loci to transcriptional silencing remain unknown. Here, we investigated molecular evolution of the essential Myb and Ranbp16 genes. These protein-coding genes reside in euchromatic loci of chromosome X in D. melanogaster and related species, while in other studied Drosophila species, including evolutionary distant ones, they are located in genomic regions highly enriched with the remnants of transposable elements (TEs), suggesting their heterochromatic nature and location. The promoter region of Myb exhibits a conserved structure throughout the Drosophila phylogeny and carries motifs for binding of chromatin remodeling factors, including insulator BEAF-32, regardless of eu- or heterochromatic surroundings. Importantly, BEAF-32 occupies not only the promoter region of Myb but is also found in the vicinity of transcriptional start sites (TSS) of Ranbp16 gene as well as in a wide range of genes located in the contrasting chromatin types in D. melanogaster and D. virilis, denoting the boundary of the nucleosome-free region available for RNA polymerase II recruitment and the surrounding heterochromatin. We also find that along with BEAF-32, insulators dCTCF and GAF are enriched at the TSS of heterochromatic genes in D. melanogaster. Thus, we propose that insulator proteins contribute to gene expression in the heterochromatic environment and, hence, facilitate the evolutionary repositioning of gene loci into heterochromatin.Author summaryHeterochromatin in Drosophila is generally associated with transcriptional silencing. Nevertheless, hundreds of essential genes have been identified in the pericentric heterochromatin of Drosophila melanogaster. Interestingly, genes embedded in pericentric heterochromatin of D. melanogaster may occupy different genomic loci, euchromatic or heterochromatic, due to repositioning in the course of evolution of Drosophila species. By surveying factors that contribute to the normal functioning of the relocated genes in distant Drosophila species, i.e. D. melanogaster and D. virilis, we identify certain insulator proteins (e.g.BEAF-32) that facilitate the expression of heterochromatic genes in spite of the repressive environment.