The PBAP remodeling complex is required for histone H3.3 replacement at chromatin boundaries and for boundary functions

Abstract

SUMMARYEstablishment and maintenance of epigenetic memories are essential for development. Replacement of canonical histone H3 by its variant H3.3 has been implicated in cellular memory. Drosophila sequence-specific DNA-binding protein GAGA factor and a chromatin factor FACT direct H3.3 replacement in conjunction with H3.3-specific chaperone HIRA at chromatin boundaries to counteract the spreading of silent chromatin. However, little is known about which ATP-driven chromatin remodeling factor is responsible for the H3.3 replacement at chromatin boundaries. Here, we report that GAGA factor associates with the Polybromoassociated Brm (PBAP) remodeling complex, which consists of many Trithorax group proteins, and recruits this complex to chromatin boundaries d1 (which is downstream of w), the Fab-7 DNase-hypersensitive site (HS) 1 of Abd-B and the bxd region of Ubx. Trlencoding GAGA factor, brm and polybromo/bap180 mutations compromise the H3.3 replacement and boundary functions in a synergistic manner. Furthermore, Polybromo is necessary for generation of the DNase HS at d1, and HIRA functions to restore the alteration. Taken together, we propose that FACT and PBAP complexes are recruited to chromatin boundaries in a GAGA factordependent manner, and are needed for H3.3 replacement to execute boundary functions. Our results provide new insight into the function of the trithorax group during development.

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex

“…Though there is the caveat that the GAF antibody does not distinguish between GAF in the LBC complex and bulk GAF, this would nevertheless be consistent with the notion that the LBC is bound to the Fab-7 insulator in vivo. The involvement of GAF also fits with other experiments that have implicated this protein in Fab-7 boundary activity (56,84,85). However, it was previously thought that the primary function of GAF was to generate a nucleosome-free region that would permit the binding of proteins that function as insulators.…”

Integrated molecular mechanism directing nucleosome reorganization by human FACT

“…Such disruption of local DNA-histone contacts may promote the exposure of the HBR, which triggers the FACT invasion into nucleosomes. Indeed, an ATP-dependent remodeler is required for the FACT-dependent histone H3.3 replacement at chromatin boundaries (Nakayama et al 2012). Third, DNA lesions may be removed through complex pathways involving disruptions of local DNAhistone contacts.…”

Earmuff restricts progenitor cell potential by attenuating the competence to respond to self-renewal factors

“…Larval brains were fixed in 100 mM PIPES (pH 6.9), 1 mM EGTA, 0.3% Triton X-100, 1 mM MgSO 4 containing 4% formaldehyde for 23 minutes and processed for immunofluorescence staining according to a previously published protocol (Weng et al, 2012). Antibodies used in this study include rabbit anti-Erm (1:100; this study), guinea pig anti-Ase (1:1000; this study), rat anti-Dpn (1:2) (Xiao et al, 2012), rat anti-Wor (1:2) (Lee et al, 2006a), rabbit antiAse (1:400) (Weng et al, 2010), mouse anti-Pros (MR1A, 1:100) (Lee et al, 2006b), mouse anti-Elav [1:100; 9F8A9, Developmental Studies Hybridoma Bank (DSHB)], mouse anti-Dlg (1:50; 4F3E3E9, DSHB), mouse anti-Osa (1:2) (Treisman et al, 1997), rabbit anti-Brm (1:2000) (Nakayama et al, 2012), chicken anti-GFP (1:2000; cat. no.…”