The POZ domain is a conserved protein-protein interaction motif present in a variety of transcription factors involved in development, chromatin remodelling and human cancers. Here, we study the role of the POZ domain of the GAGA transcription factor in promoter recognition. Natural target promoters for GAGA typically contain multiple GAGA-binding elements. Our results show that the POZ domain mediates strong cooperative binding to multiple sites but inhibits binding to single sites. Protein cross-linking and gel filtration chromatography experiments established that the POZ domain is required for GAGA oligomerization into higher order complexes. Thus, GAGA oligomerization increases binding specificity by selecting only promoters with multiple sites. Electron microscopy revealed that GAGA binds to multiple sites as a large oligomer and induces bending of the promoter DNA. Our results indicate a novel mode of DNA binding by GAGA, in which a large GAGA complex binds multiple GAGA elements that are spread out over a region of a few hundred base pairs. We suggest a model in which the promoter DNA is wrapped around a GAGA multimer in a conformation that may exclude normal nucleosome formation.

Nuclear pore complexes (NPCs) are multisubunit protein entities embedded into the nuclear envelope (NE). Here, we examine the in vivo dynamics of the essential Drosophila nucleoporin Nup107 and several other NE-associated proteins during NE and NPCs disassembly and reassembly that take place within each mitosis. During both the rapid mitosis of syncytial embryos and the more conventional mitosis of larval neuroblasts, Nup107 is gradually released from the NE, but it remains partially confined to the nuclear (spindle) region up to late prometaphase, in contrast to nucleoporins detected by wheat germ agglutinin and lamins. We provide evidence that in all Drosophila cells, a structure derived from the NE persists throughout metaphase and early anaphase. Finally, we examined the dynamics of the spindle checkpoint proteins Mad2 and Mad1. During mitotic exit, Mad2 and Mad1 are actively imported back from the cytoplasm into the nucleus after the NE and NPCs have reformed, but they reassociate with the NE only later in G1, concomitantly with the recruitment of the basket nucleoporin Mtor (the Drosophila orthologue of vertebrate Tpr). Surprisingly, Drosophila Nup107 shows no evidence of localization to kinetochores, despite the demonstrated importance of this association in mammalian cells. INTRODUCTIONIn eukaryotes, the nuclear envelope (NE) defines the limits between the nucleus and the cytoplasm. The outer NE membrane is considered to be structurally and functionally part of the endoplasmic reticulum network, whereas the inner membrane, with its distinct protein composition, provides anchoring points for the chromatin and nuclear lamina. Nuclear pore complexes (NPCs) are embedded at the points of fusion between the inner and outer NE membrane and represent the sole channels of transport across the NE. NPCs are composed of multiple copies of ϳ30 different proteins termed nucleoporins (Nups), most of which are organized into subcomplexes that associate with each other to build up the mature NPCs (for reviews, see Hetzer et al., 2005;Schwartz, 2005;Lim and Fahrenkrog, 2006;Tran and Wente, 2006). During cell division, the NE and NPCs are subjected to major rearrangements. However, the extent to which the NE and NPCs disassemble at mitotic entry varies among organisms (for reviews, see Margalit et al., 2005;Prunuske and Ullman, 2006). Unlike in most yeast and fungi, characterized by a "closed mitosis," NE disassembly is required in animal cells to allow spindle microtubule access to chromosomes. In vertebrates, cell division leads to complete NE breakdown at the prophase-prometaphase transition. During this "open mitosis," integral membrane proteins of the NE and the soluble subcomplexes of the NPCs redistribute throughout the endoplasmic reticulum and the mitotic cytoplasm (for reviews, see Hetzer et al., 2005;Margalit et al., 2005;Prunuske and Ullman, 2006). In Drosophila and Caenorhabditis elegans embryos, however, the NE only partially disassembles near spindle poles in early mitosis. NPCs disassemble during prometapha...

Trithorax (TRX) is a Drosophila SET domain protein that is required for the correct expression of homeotic genes. Here, we show that the TRX SET domain efficiently binds to core histones and nucleosomes. The primary target for the SET domain is histone H3 and binding requires the N-terminal histone tails. The previously described trx Z11 mutation changes a strictly conserved glycine in the SET domain to serine and causes homeotic transformations in the fly. We found that this mutation selectively interferes with histone binding, suggesting that histones represent a critical target during developmental gene regulation by TRX. The Polycomb group (PcG) of repressors and trithorax group (trxG) of activators target chromatin in order to "freeze" a mitotically stable pattern of gene expression and determined cell fate (Pirrotta 1998;Lyko and Paro 1999;Mahmoudi and Verrijzer 2001). The founding member of the trxG, the Drosophila trx gene, is required throughout development and controls the expression of several developmental regulators, including the homeotic genes (Ingham and Whittle 1980;Ingham 1985;Breen 1999). trx is related to the human Mixed Lineage Leukemia (MLL) gene, which is involved in translocations associated with the majority of cases of infant leukemias (Waring and Cleary 1997). TRX and MLL are part of a highly conserved regulatory network that is required for the correct expression of the homeotic selector genes and determination of segment identity in both mammals and Drosophila. They are very large proteins that contain structural motifs common to chromatin-associated factors such as PHD fingers and a C-terminal SET domain ( Fig. 1A; Mazo et al. 1990;Stassen et al. 1995).The SET domain is a highly conserved 130-150 amino acids motif initially recognized as a common element in chromatin regulators with opposing activities: the suppressor of position affect variegation Su(var)3-9, the PcG protein Enhancer of Zeste [E(z)], and TRX (Jenuwein et al. 1998). The SET domain has been implicated in a multitude of different protein-protein interactions and functions. The SET domains of MLL, yeast Set1p, and E(z) bind to myotubularin-related dual-specificity phosphatases and anti-phosphatases that modulate growth control (Cui et al. 1998). The TRX and MLL SET domains bind to the SNF5 component of the ATP-dependent remodeler SWI/SNF (Rozenblatt-Rosen et al. 1998) and mediate self-association (Rozovskaia et al. 2000). Furthermore, the SET domain of yeast Set1p binds the Mec3p checkpoint protein and has been implicated in DNA repair and telomere function (Corda et al. 1999). The Set1p SET domain alone suffices to mediate telomeric silencing, suggesting that it forms a functional unit (Nislow et al. 1997). Recently, it was shown that SUV39H1, the mammalian homolog of Su(var)3-9, selectively methylates lysine 9 of histone H3 (Rea et al. 2000;Jenuwein 2001). This modification creates a binding site for HP1 and thus can contribute to the propagation of a heterochromatin domain (Bannister et al. 2001;Lachner et al. 2001;...

There is growing evidence for the involvement of Y-complex nucleoporins (Y-Nups) in cellular processes beyond the inner core of nuclear pores of eukaryotes. To comprehensively assess the range of possible functions of Y-Nups, we delimit their structural and functional properties by high-specificity sequence profiles and tissue-specific expression patterns. Our analysis establishes the presence of Y-Nups across eukaryotes with novel composite domain architectures, supporting new moonlighting functions in DNA repair, RNA processing, signaling and mitotic control. Y-Nups associated with a select subset of the discovered domains are found to be under tight coordinated regulation across diverse human and mouse cell types and tissues, strongly implying that they function in conjunction with the nuclear pore. Collectively, our results unearth an expanded network of Y-Nup interactions, thus supporting the emerging view of the Y-complex as a dynamic protein assembly with diverse functional roles in the cell.