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

Abstract

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 (Pbro) subunit of PBAP, a SWI/SNF-class chromatin remodeling complex. BEAF also shows genetic interactions with Pbro and other PBAP subunits. We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing (PRO-seq) and micrococcal nuclease sequencing (MNase-seq) after RNAi-mediated knockdown in cultured S2 cells. Our results are consistent with the interaction playing a subtle role in gene activation. Fewer than 5% of BEAF-associated genes were significantly affected after BEAF knockdown. Most were down-regulated, accompanied by fill-in of the promoter nucleosome depleted region (NDR) and a slight upstream shift of the +1 nucleosome. Pbro knockdown caused down-regulation of several hundred genes, and showed a correlation with BEAF knockdown but a better correlation with promoter-proximal GAGA factor binding. MNase-seq supports that BEAF binds near housekeeping gene promoters while Pbro is more important at regulated genes. Yet there is a similar general but slight reduction of promoter-proximal pausing by RNA polymerase II and increase in NDR nucleosome occupancy after knockdown of either protein. We discuss the possibility of redundant factors keeping BEAF-associated promoters active and masking the role of interactions between BEAF and the Pbro subunit of PBAP in S2 cells. We identify FACT and NURF as candidate redundant factors.

TheTofumutation restores female fertility toDrosophilawith a nullBEAFmutation

“…Our lab previously performed a co-immunoprecipitation of BEAF from Drosophila embryos and by tandem mass spectrometry, identified around 100 co-immunoprecipitated factors with more than 1 peptide identified, a Benjamini-Hochberg corrected p-value less than 0.10, and a ratio of experimental to control IP greater than 1.0 (McKowen et al, 2022). One of these was rib (3 peptides, 1.8-fold enriched, 0.0390 p-value), making the SNP in the upstream PRE a candidate for Tofu .…”

“…ChIP-seq data from embryonic salivary glands identified 494 Rib-associated genes in the developing glands (Loganathan et al, 2016). We previously generated a list of 3241 BEAF-associated genes from S2 cell ChIP-seq data (Liang et al, 2014; McKowen et al, 2022), and found overlap with 30% (150) of the Rib-associated genes. A motif related to the BEAF-32B DNA binding consensus motif, CGATA (Hart et al, 1997; Zhao et al, 1995), represented around 15% of the occurrences of the top 10 motifs found in the Rib ChIP-seq peaks associated with the 494 genes (Loganathan et al, 2016).…”

New Drosophila promoter-associated architectural protein Mzfp1 interacts with CP190 and is required for housekeeping gene expression and insulator activity

“…To study the colocalization of Mzfp1 with CP190 and well-characterized architectural C2H2 proteins in euchromatic regions (Figure 2D ), we included previous data for M1BP ( 39 ), Su(Hw) ( 104 ), Pita, ZIPIC ( 32 ), dCTCF ( 87 ), BEAF-32 ( 119 , 120 ), and CP190 ( 64 ). All tested architectural C2H2 proteins have been shown to interact with CP190 ( 33 , 40 , 42 , 46 , 87 , 112 , 121 ).…”