Chromosome segregation and X-chromosome gene regulation in Caenorhabditis elegans share the component MIX-1, a mitotic protein that also represses X-linked genes during dosage compensation. MIX-1 achieves its dual roles through interactions with different protein partners. To repress gene expression, MIX-1 acts in an X-chromosome complex that resembles the mitotic condensin complex yet lacks chromosome segregation function. Here we show that MIX-1 interacts with a mitotic condensin subunit, SMC-4, to achieve chromosome segregation. The SMC-4/MIX-1 complex positively supercoils DNA in vitro and is required for mitotic chromosome structure and segregation in vivo. Thus, C. elegans has two condensin complexes, one conserved for mitosis and another specialized for gene regulation. SMC-4 and MIX-1 colocalize with centromere proteins on condensed mitotic chromosomes and are required for the restricted orientation of centromeres toward spindle poles. This cell cycle-dependent localization requires AIR-2/AuroraB kinase. Depletion of SMC-4/MIX-1 causes aberrant mitotic chromosome structure and segregation, but not dramatic decondensation at metaphase. Moreover, SMC-4/MIX-1 depletion disrupts sister chromatid segregation during meiosis II but not homologous chromosome segregation during meiosis I, although both processes require chromosome condensation. These results imply that condensin is not simply required for compaction, but plays a more complex role in chromosome architecture that is essential for mitotic and meiotic sister chromatid segregation.

Fab-7 deletions in the bithorax complex have a novel gain-of-function phenotype, typically transforming parasegment 11 (PSll) into PS12 identity. Genetic analysis indicates that removal of the Fab-7 element results in the fusion of the lab-6 (PSll) and iab-7 (PS12) cis-regulatory domains into a single regulatory domain that inappropriately regulates Abdominal-B in PSll. This has led to the hypothesis that Fab-7 is a chromatin domain boundary that normally functions to ensure the autonomous activity of the iab-6 and iab-7 cis-regulatory domains. We use several different enhancer blocking assays to demonstrate that Fab-7 has the insulating properties expected of a domain boundary. We define a minimal fragment of Fab-7 sufficient for enhancer blocking, and demonstrate that it is completely distinct from an adjacent Polycomb-dependent silencer. We compare Fab-7 to the su ( [Key Words: Bithorax complex; Fab-7; chromatin domain boundary; insulator; suppressor of Hairy-wing] Received September 19, 1996; revised version accepted November 1, 1996.Body patterning in many organisms involves the development of segments whose unique identities are specified by homeotic genes. The precise expression patterns of the homeotic genes are crucial for generating a normal body plan, and the misregulation of these genes can result in dramatic transformations of one body segment into another. In Drosophila, the identity of parasegments in the posterior of the fly is controlled by the three homeotic genes of the bithorax complex (BX-C), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B} (Lewis 1978;Duncan 1987). The parasegmentspecific expression patterns of these three genes are generated by a complicated cis-regulatory region that spans a DNA segment of 300 kb. This cis-regulatory region is organized in a series of nine parasegment-specific regulatory elements or domains, abx /bx, bxd/pbx, iab-2, iab-3, iab-4, iab-5, lab-6, iab-7, and iab-8 (Lewis 1978;Karch et al. 1985;Celniker et al. 1990;Sanchez-Herrero 1991). Each of these domains directs the expression of one of the three BX-C homeotic genes in a specific parasegment. For example, the lab-5, lab-6, and lab-7 cisregulatory domains direct Abd-B expression in parasegmerits (PS) 10, 11, and 12, respectively (Karch et al. 1985). ~Corresponding author. 2present address:

Summary Background Condensin complexes organize chromosome structure and facilitate chromosome segregation. Higher eukaryotes have two complexes, condensin I and condensin II, each essential for chromosome segregation. The nematode Caenorhabditis elegans was considered an exception, because it has a mitotic condensin II complex but appeared to lack mitotic condensin I. Instead, its condensin I-like complex (here called condensin IDC) dampens gene expression along hermaphrodite X chromosomes during dosage compensation. Results Here we report the discovery of a third condensin complex, condensin I, in C. elegans. We identify new condensin subunits and show that each complex has a conserved five-subunit composition. Condensin I differs from condensin IDC by only a single subunit. Yet condensin I binds to autosomes and X chromosomes in both sexes to promote chromosome segregation, whereas condensin IDC binds specifically to X chromosomes in hermaphrodites to regulate transcript levels. Both condensin I and II promote chromosome segregation, but associate with different chromosomal regions during mitosis and meiosis. Unexpectedly, condensin I also localizes to regions of cohesion between meiotic chromosomes before their segregation. Conclusions We demonstrate that condensin subunits in C. elegans form three complexes, one that functions in dosage compensation and two that function in mitosis and meiosis. These results highlight how the duplication and divergence of condensin subunits during evolution may facilitate their adaptation to specialized chromosomal roles and illustrate the versatility of condensins to function in both gene regulation and chromosome segregation.

Two related protein complexes, cohesin and condensin, are essential for separating identical copies of the genome into daughter cells during cell division. Cohesin glues replicated sister chromatids together until they split at anaphase, whereas condensin reorganizes chromosomes into their highly compact mitotic structure. Unexpectedly, mutations in the subunits of these complexes have been uncovered in genetic screens that target completely different processes. Exciting new evidence is emerging that cohesin and condensin influence crucial processes during interphase, and unforeseen aspects of mitosis. Each complex can perform several roles, and individual subunits can associate with different sets of proteins to achieve diverse functions, including the regulation of gene expression, DNA repair, cell-cycle checkpoints and centromere organization.

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.