The iab-7 Polycomb Response Element Maps to a Nucleosome-Free Region of Chromatin and Requires Both GAGA and Pleiohomeotic for Silencing Activity

Abstract

In the work reported here we have undertaken a functional dissection of a Polycomb response element (PRE) from the iab-7 cis-regulatory domain of the Drosophila melanogaster bithorax complex (BX-C). Previous studies mapped the iab-7 PRE to an 860-bp fragment located just distal to the Fab-7 boundary. Located within this fragment is an~230-bp chromatin-specific nuclease-hypersensitive region called HS3. We have shown that HS3 is capable of functioning as a Polycomb-dependent silencer in vivo, inducing pairing-dependent silencing of a mini-white reporter. The HS3 sequence contains consensus binding sites for the GAGA factor, a protein implicated in the formation of nucleosome-free regions of chromatin, and Pleiohomeotic (Pho), a Polycomb group protein that is related to the mammalian transcription factor YY1. We show that GAGA and Pho interact with these sequences in vitro and that the consensus binding sites for the two proteins are critical for the silencing activity of the iab-7 PRE in vivo.Segment identity in the posterior two-thirds of the Drosophila melanogaster embryo, from parasegment 5 (PS5) to PS14, is determined by the pattern of expression of the bithorax complex (BX-C) homeotic genes, Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) (13,32,39,46). These three homeotic genes are regulated by an elaborate cis-regulatory region that spans a DNA segment of over 300 kb. This large cis-regulatory region is subdivided into nine functionally autonomous domains, abx/bx, bxd/pbx, and iab-2 to iab-8 (2, 9, 29, 32). Each domain specifies the identity of a specific parasegment by activating one of the BX-C homeotic genes in a pattern appropriate for that parasegment. For example, the iab-5 cis-regulatory domain regulates Abd-B expression in a pattern that confers PS10 identity to the cells in this parasegment. Similarly, the iab-6, iab-7, and iab-8 cis-regulatory domains activate Abd-B expression in patterns appropriate for PS11, PS12, and PS13 identity, respectively (5, 9, 45). When one of the BX-C cis-regulatory domains is inactivated, the parasegment specified by the affected regulatory domain is transformed into a copy of the parasegment immediately anterior. Thus, in a deletion that inactivates iab-7, iab-7 Sz , PS12 is transformed into a duplicate copy of PS11 (16). In this case, Abd-B expression in both PS11 and PS12 is driven by the iab-6 cis-regulatory domain.

Dissecting the regulatory landscape of theAbd-Bgene of the bithorax complex

“…We believe that maintenance elements respond to the activity state of initiator elements and modify the chromatin structure of each domain accordingly. Consistent with this, maintenance elements (MEs) have generally been found to contain binding sites for Polycomb-Group (Pc-G) and trithorax-Group proteins (Trx-G) (Strutt et al, 1997;Brown et al, 1998;Fritsch et al, 1999;Tillib et al, 1999;Horard et al, 2000;Busturia et al, 2001;Mishra et al, 2001;Hur et al, 2002;Dejardin et al, 2005). Both Pc-G and trx-G proteins are known to be involved in modifying chromatin structure, with Pc-G proteins being involved in compacting chromatin (silencing) and Trx-G proteins being involved in opening chromatin (for reviews, see Ringrose and Paro, 2004;Brock and Fisher, 2005).…”

Developmental modulation ofFab-7boundary function

“…As shown in Fig. 1A, it extends from the minor nuclease hypersensitive site (*) on the proximal side to the iab-7 PRE (which corresponds HS3) (Hagstrom et al, 1997;Mishra et al, 2001) on the distal side and includes two major chromatin-specific nuclease hypersensitive regions, HS1 and HS2 . The largest hypersensitive region, HS1, contains six consensus GAGA factor binding sites arranged in three pairs, 1-2, 3-4 and 5-6.…”

The relationship between BCL6 bodies and nuclear sites of normal and halogenated DNA and RNA synthesis

“…Although only BCL6-mediated-transcriptional silencing has been reported to date, the implication of a single POK protein in various aspects of DNA metabolism is now increasingly clear. This is strikingly exemplified by the well-studied Drosophila GAGA POK protein that acts in anti-repression and nucleosome disruption, mediates stable repression, counteracts heterochromatinmediated repression, modulates enhancer-promoter communication, and is required for chromosome condensation and separation (Bhat et al, 1996;Busturia et al, 2001;Croston et al, 1991;Farkas et al, 1994;Horard et al, 2000;Mahmoudi et al, 2002;Mishra et al, 2001;Ohtsuki and Levine, 1998;Tsukiyama et al, 1994;Xiao et al, 2001). All these activities are likely mechanistically linked and depend on a "basic" property shared by other POK proteins, namely the ability to self-aggregate and bring together "distant" pieces of DNA (Americo et al, 2002;Katsani et al, 1999;Mahmoudi et al, 2002;West et al, 2002).…”