The mutation restores female fertility to with a null mutation.

Compensatory mutations offer clues to deciphering the role of a particular protein in cellular processes. Here we investigate an unknown compensatory mutation, present in the fly line, that provides sufficient rescue of the defective ovary phenotype caused by null alleles to allow maintenance of fly stocks lacking the chromatin domain insulator proteins Boundary Element-Associated Factors BEAF-32A and BEAF-32B. We call this mutation .

The Drosophila BEAF insulator protein interacts with the polybromo subunit of the PBAP chromatin remodeling complex.

The Drosophila Boundary Element-Associated Factor of 32 kDa (BEAF) binds in promoter regions of a few thousand mostly housekeeping genes. BEAF is implicated in both chromatin domain boundary activity and promoter function, although molecular mechanisms remain elusive. Here, we show that BEAF physically interacts with the polybromo subunit (Pbro) of PBAP, a SWI/SNF-class chromatin remodeling complex.

Overlapping but Distinct Sequences Play Roles in the Insulator and Promoter Activities of the BEAF-Dependent scs' Insulator.

Chromatin domain insulators are thought to help partition the genome into genetic units called topologically associating domains (TADs). In , TADs are often separated by inter-TAD regions containing active housekeeping genes and associated insulator binding proteins. This raises the question of whether insulator binding proteins are involved primarily in chromosomal TAD architecture or gene activation, or if these two activities are linked.

Promoter-Proximal Chromatin Domain Insulator Protein BEAF Mediates Local and Long-Range Communication with a Transcription Factor and Directly Activates a Housekeeping Promoter in .

BEAF (Boundary Element-Associated Factor) was originally identified as a chromatin domain insulator-binding protein, suggesting a role in gene regulation through chromatin organization and dynamics. Genome-wide mapping found that BEAF usually binds near transcription start sites, often of housekeeping genes, suggesting a role in promoter function. This would be a nontraditional role for an insulator-binding protein.

Using a phiC31 "Disintegrase" to make new attP sites in the Drosophila genome at locations showing chromosomal position effects.

An engineered phiC31 "Disintegrase" able to make an attP site in Drosophila out of an attR-attL pair is described. This was used to generate attP sites at genomic locations where a mini-white (mini-w) transgene was subject to chromosomal position effects (CPE). The first step was random genomic integration of a P-element-based transposon with an insulated mini-w transgene.

4C-seq characterization of Drosophila BEAF binding regions provides evidence for highly variable long-distance interactions between active chromatin.

Chromatin organization is crucial for nuclear functions such as gene regulation, DNA replication and DNA repair. Insulator binding proteins, such as the Drosophila Boundary Element-Associated Factor (BEAF), are involved in chromatin organization. To further understand the role of BEAF, we detected cis- and trans-interaction partners of four BEAF binding regions (viewpoints) using 4C (circular chromosome conformation capture) and analyzed their association with different genomic features.

Characterization of the Drosophila BEAF-32A and BEAF-32B Insulator Proteins.

Data implicate the Drosophila 32 kDa Boundary Element-Associated Factors BEAF-32A and BEAF-32B in both chromatin domain insulator element function and promoter function. They might also function as an epigenetic memory by remaining bound to mitotic chromosomes. Both proteins are made from the same gene.

Do the BEAF insulator proteins regulate genes involved in cell polarity and neoplastic growth?

It was reported that a chromosome with the BEAF(NP6377) (NP6377) allele leads to a loss of cell polarity and neoplastic growth in Drosophila melanogaster when homozygous (Gurudatta et al., 2012). We had previously generated the BEAF(AB-KO) (AB-KO) allele by homologous recombination and did not note these phenotypes (Roy et al.

Genome-wide studies of the multi-zinc finger Drosophila Suppressor of Hairy-wing protein in the ovary.

The Drosophila Suppressor of Hairy-wing [Su(Hw)] protein is a globally expressed, multi-zinc finger (ZnF) DNA-binding protein. Su(Hw) forms a classic insulator when bound to the gypsy retrotransposon and is essential for female germline development. These functions are genetically separable, as exemplified by Su(Hw)(f) that carries a defective ZnF10, causing a loss of insulator but not germline function.

Lack of the Drosophila BEAF insulator proteins alters regulation of genes in the Antennapedia complex.

In a screen based on a rough eye phenotype caused by a dominant negative form of the BEAF-32A and BEAF-32B insulator proteins, we previously identified 17 proteins that genetically interact with BEAF. Eleven of these are developmental transcription factors, seven of which are encoded by the Antennapedia complex (ANT-C). While investigating potential reasons for the genetic interactions, we obtained evidence that BEAF plays a role in the regulation of genes in the ANT-C.

Targeted gene replacement by homologous recombination in Drosophila stimulates production of second-site mutations.

Gene replacement by homologous recombination is a powerful technique for generating mutations in Drosophila. while using this technique for the BEAF gene, we encountered non-targeted lethal mutations on the target chromosome that complicated the analysis of the BEAF mutations until they were discovered and removed by meiotic recombination. Subsequent experiments indicated that the gene-targeting method leads to a modest but significant three-fold increase in the rate of production of non-targeted lethal mutations.

Genome-wide mapping of boundary element-associated factor (BEAF) binding sites in Drosophila melanogaster links BEAF to transcription.

Insulator elements play a role in gene regulation that is potentially linked to nuclear organization. Boundary element-associated factors (BEAFs) 32A and 32B associate with hundreds of sites on Drosophila polytene chromosomes. We hybridized DNA isolated by chromatin immunoprecipitation to genome tiling microarrays to construct a genome-wide map of BEAF binding locations.

BEAF regulates cell-cycle genes through the controlled deposition of H3K9 methylation marks into its conserved dual-core binding sites.

Chromatin insulators/boundary elements share the ability to insulate a transgene from its chromosomal context by blocking promiscuous enhancer-promoter interactions and heterochromatin spreading. Several insulating factors target different DNA consensus sequences, defining distinct subfamilies of insulators. Whether each of these families and factors might possess unique cellular functions is of particular interest.

Characterization of BEAF mutations isolated by homologous recombination in Drosophila.

The Drosophila BEAF-32A and BEAF-32B proteins bind to the scs' insulator and to hundreds of other sites on Drosophila chromosomes. These two proteins are encoded by the same gene. We used ends-in homologous recombination to generate the null BEAF(AB-KO) allele and also isolated the BEAF(A-KO) allele that eliminates production of only the BEAF-32A protein.

A genetic screen supports a broad role for the Drosophila insulator proteins BEAF-32A and BEAF-32B in maintaining patterns of gene expression.

Chromatin domain insulators are thought to insulate adjacent genes, including their regulatory elements, from each other by organizing chromatin into functionally independent domains. Thus insulators should play a global role in gene regulation by keeping regulatory domains separated. However, this has never been demonstrated.

The Drosophila boundary element-associated factors BEAF-32A and BEAF-32B affect chromatin structure.

Binding sites for the Drosophila boundary element-associated factors BEAF-32A and -32B are required for the insulator activity of the scs' insulator. BEAF binds to hundreds of sites on polytene chromosomes, indicating that BEAF-utilizing insulators are an important class in Drosophila. To gain insight into the role of BEAF in flies, we designed a transgene encoding a dominant-negative form of BEAF under GAL4 UAS control.

Studies of the role of the Drosophila scs and scs' insulators in defining boundaries of a chromosome puff.

Insulators are DNA elements that establish independent transcriptional domains within eukaryotic genomes. The Drosophila scs and scs' insulators localize near the borders of a structural domain in the polytene chromosomes, known as a puff, produced by transcription of the 87A heat shock protein (hsp) genes. It has been suggested that scs and scs' are boundary elements that delimit this decondensed chromatin domain, reflecting the mechanism by which these sequences act to constrain regulatory interactions.

Histone methyltransferase activity of a Drosophila Polycomb group repressor complex.

Polycomb group (PcG) proteins maintain transcriptional repression during development, likely by creating repressive chromatin states. The Extra Sex Combs (ESC) and Enhancer of Zeste [E(Z)] proteins are partners in an essential PcG complex, but its full composition and biochemical activities are not known. A SET domain in E(Z) suggests this complex might methylate histones.

Identification of a multicopy chromatin boundary element at the borders of silenced chromosomal domains.

The insulating properties required to delimit higher-order chromosomal domains have been shown to be shared by a variety of chromatin boundary elements (BEs). Boundary elements have been described in several species, from yeast to human, and we have previously reported the existence of a class of chromatin BEs in Drosophila melanogaster whose insulating activity requires the DNA-binding protein BEAF (boundary element-associated factor). Here we focus on the characterization of a moderately repeated 1.