Sister Chromatid Cohesion Protein Research Articles

Characterization of the core region of grape VvHOS1 promoter activity and its upstream regulatory proteins

The tropical origins of the grape (Vitis vinifera L.) make it vulnerable to low temperatures, which constitute a serious threat to grape production. Our knowledge of the molecular basis of cold tolerance in perennial grape species is limited. The E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1 (HOS1) has been demonstrated to negatively regulate cold responsiveness, but the understanding of the HOS1 in grape remains fragmentary. Here, we initially evaluated the expression of HOS1 in cold-tolerant Vitis amurensis (VaHOS1) and susceptible V. vinifera (VvHOS1) cultivars and discovered their responses to cold stress differed significantly. The two grape HOS1 genes exhibit a high degree of sequence similarity (98.77% at the amino acid level) but their lengths (313-bp insertion/deletion polymorphism) and promoter sequences are substantially different (91.96%). This 313-bp region significantly affects the differential expression of HOS1 between V. vinifera and V. amurensis. The 313-bp deletion abolished VvHOS1 promoter activity in promoter-GUS assay of transgenic Arabidopsis. Moreover, 313-bp fragment might function as a positive core region to affect promoter activity of VvHOS1 to involved in different developmental stages and cold stress. In addition, DNA pull-down and mass spectrometry (MS) assay revealed the potential regulatory proteins may bind to the 313-bp core promoter region. We show that the two linker histone H1 genes (designated as VvHH1: LOC100853898 and LOC100259836), and one sister chromatid cohesion protein PDS5 homolog B (designated as VvPDS5B: LOC100853350), which associates with the core promoter region, decreases or activates GUS expression. This work elucidates the expression features of the HOS1 promoter from genus Vitis and further identifies a core region required for the promoter activity via trans-activation or suppression of candidate regulatory proteins.

Maize Dek15 Encodes the Cohesin-Loading Complex Subunit SCC4 and Is Essential for Chromosome Segregation and Kernel Development.

Cohesin complexes maintain sister chromatid cohesion to ensure proper chromosome segregation during mitosis and meiosis. In plants, the exact components and functions of the cohesin complex remain poorly understood. Here, we positionally cloned the classic maize (Zea mays) mutant defective kernel 15 (dek15), revealing that it encodes a homolog of SISTER CHROMATID COHESION PROTEIN 4 (SCC4), a loader subunit of the cohesin ring. Developing dek15 kernels contained fewer cells than the wild type, but had a highly variable cell size. The dek15 mutation was found to disrupt the mitotic cell cycle and endoreduplication, resulting in a reduced endosperm and embryo lethality. The cells in the dek15 endosperm and embryo exhibited precocious sister chromatid separation and other chromosome segregation errors, including misaligned chromosomes, lagging chromosomes, and micronuclei, resulting in a high percentage of aneuploid cells. The loss of Dek15/Scc4 function upregulated the expression of genes involved in cell cycle progression and stress responses, and downregulated key genes involved in organic synthesis during maize endosperm development. Our yeast two-hybrid screen identified the chromatin remodeling proteins chromatin remodeling factor 4, chromatin remodeling complex subunit B (CHB)102, CHB105, and CHB106 as SCC4-interacting proteins, suggesting a possible mechanism by which the cohesin ring is loaded onto chromatin in plant cells. This study revealed biological functions for DEK15/SCC4 in mitotic chromosome segregation and kernel development in maize.

MiR-223 Regulates Cell Proliferation and Invasion via Targeting PDS5B in Pancreatic Cancer Cells

Emerging evidence has demonstrated that miR-223 is critically involved in the progression of pancreatic cancer (PC); however, the underlying mechanisms are not fully elucidated. In the present study, we explored the molecular basis of miR-223-mediated tumor progression in PC. We performed numerous approaches including MTT, FACS, transfection, RT-PCR, western blotting, Transwell, and animal studies to determine the physiological role of miR-223 in PC cells. We found that sister chromatid cohesion protein PDS5 homolog B (PDS5B) is a direct target of miR-223 in PC. Moreover, PDS5B exhibits tumor-suppressive function in PC cells. Consistently, ectopic overexpression of PDS5B reversed miR-223-mediated tumor progression in PC cells. These results suggest that the miR-223/PDS5B axis regulates cell proliferation and invasion in PC cells. Our findings indicated that downregulation of miR-223 could be a novel therapeutic approach for PC.

A Novel Role for α-Importins and Akirin in Establishment of Meiotic Sister Chromatid Cohesion in Caenorhabditis elegans

During meiotic prophase I, sister chromatid cohesion is established in a way that supports the assembly of the synaptonemal complex (SC). The SC connects homologous chromosomes, directing meiotic recombination to create crossovers. In this paper, we identify two proteins that cooperate to import and load meiotic cohesins, thus indirectly promoting SC assembly. AKIR-1 is a protein with a previously identified meiotic role in SC disassembly. akir-1 mutants have no obvious defects in sister chromatid cohesion. We identified ima-2, a gene encoding for an α-importin nuclear transport protein, as a gene interacting with akir-1 Analysis of akir-1;ima-2 double mutants reveals a decrease in the number of germline nuclei and the formation of polycomplexes (PCs) (an SC protein aggregate). These PCs contain proteins that are part of the two main substructures of the SC: the central region and the lateral element. Unlike typical PCs, they also contain sister chromatid cohesion proteins. In akir-1;ima-2 double mutants, PCs are located in both the nucleus and the cytoplasm. This suggests that the defects observed in the double mutants are both in nuclear import and in the assembly of sister chromatid cohesion. PC formation is also associated with recombination defects leading to reduced numbers of crossovers. Similarly to cohesion mutants, the pairing center protein HIM-8 is mislocalized in akir-1;ima-2 double mutants, forming multiple foci. We propose that AKIR-1 and IMA-2 operate in parallel pathways to import and load chromosomally associated cohesin complex proteins in meiotic nuclei, a novel finding for both of these conserved proteins.

Measuring Sister Chromatid Cohesion Protein Genome Occupancy in Drosophila melanogaster by ChIP-seq.

This chapter presents methods to conduct and analyze genome-wide chromatin immunoprecipitation of the cohesin complex and the Nipped-B cohesin loading factor in Drosophila cells using high-throughput DNA sequencing (ChIP-seq). Procedures for isolation of chromatin, immunoprecipitation, and construction of sequencing libraries for the Ion Torrent Proton high throughput sequencer are detailed, and computational methods to calculate occupancy as input-normalized fold-enrichment are described. The results obtained by ChIP-seq are compared to those obtained by ChIP-chip (genomic ChIP using tiling microarrays), and the effects of sequencing depth on the accuracy are analyzed. ChIP-seq provides similar sensitivity and reproducibility as ChIP-chip, and identifies the same broad regions of occupancy. The locations of enrichment peaks, however, can differ between ChIP-chip and ChIP-seq, and low sequencing depth can splinter broad regions of occupancy into distinct peaks.

Drosophila TDP-43 RNA-Binding Protein Facilitates Association of Sister Chromatid Cohesion Proteins with Genes, Enhancers and Polycomb Response Elements.

The cohesin protein complex mediates sister chromatid cohesion and participates in transcriptional control of genes that regulate growth and development. Substantial reduction of cohesin activity alters transcription of many genes without disrupting chromosome segregation. Drosophila Nipped-B protein loads cohesin onto chromosomes, and together Nipped-B and cohesin occupy essentially all active transcriptional enhancers and a large fraction of active genes. It is unknown why some active genes bind high levels of cohesin and some do not. Here we show that the TBPH and Lark RNA-binding proteins influence association of Nipped-B and cohesin with genes and gene regulatory sequences. In vitro, TBPH and Lark proteins specifically bind RNAs produced by genes occupied by Nipped-B and cohesin. By genomic chromatin immunoprecipitation these RNA-binding proteins also bind to chromosomes at cohesin-binding genes, enhancers, and Polycomb response elements (PREs). RNAi depletion reveals that TBPH facilitates association of Nipped-B and cohesin with genes and regulatory sequences. Lark reduces binding of Nipped-B and cohesin at many promoters and aids their association with several large enhancers. Conversely, Nipped-B facilitates TBPH and Lark association with genes and regulatory sequences, and interacts with TBPH and Lark in affinity chromatography and immunoprecipitation experiments. Blocking transcription does not ablate binding of Nipped-B and the RNA-binding proteins to chromosomes, indicating transcription is not required to maintain binding once established. These findings demonstrate that RNA-binding proteins help govern association of sister chromatid cohesion proteins with genes and enhancers.

Structural Studies Reveal the Functional Modularity of the Scc2-Scc4 Cohesin Loader.

The remarkable accuracy of eukaryotic cell division is partly maintained by the cohesin complex acting as a molecular glue to prevent premature sister chromatid separation. The loading of cohesin onto chromosomes is catalyzed by the Scc2-Scc4 loader complex. Here, we report the crystal structure of Scc4 bound to the N terminus of Scc2 and show that Scc4 is a tetratricopeptide repeat (TPR) superhelix. The Scc2 N terminus adopts an extended conformation and is entrapped by the core of the Scc4 superhelix. Electron microscopy (EM) analysis reveals that the Scc2-Scc4 loader complex comprises three domains: a head, body, and hook. Deletion studies unambiguously assign the Scc2N-Scc4 as the globular head domain, whereas in vitro cohesin loading assays show that the central body and the hook domains are sufficient to catalyze cohesin loading onto circular DNA, but not chromatinized DNA in vivo, suggesting a possible role for Scc4 as a chromatin adaptor.

Arabidopsis MZT1 homologs GIP1 and GIP2 are essential for centromere architecture

Centromeres play a pivotal role in maintaining genome integrity by facilitating the recruitment of kinetochore and sister-chromatid cohesion proteins, both required for correct chromosome segregation. Centromeres are epigenetically specified by the presence of the histone H3 variant (CENH3). In this study, we investigate the role of the highly conserved γ-tubulin complex protein 3-interacting proteins (GIPs) in Arabidopsis centromere regulation. We show that GIPs form a complex with CENH3 in cycling cells. GIP depletion in the gip1gip2 knockdown mutant leads to a decreased CENH3 level at centromeres, despite a higher level of Mis18BP1/KNL2 present at both centromeric and ectopic sites. We thus postulate that GIPs are required to ensure CENH3 deposition and/or maintenance at centromeres. In addition, the recruitment at the centromere of other proteins such as the CENP-C kinetochore component and the cohesin subunit SMC3 is impaired in gip1gip2. These defects in centromere architecture result in aneuploidy due to severely altered centromeric cohesion. Altogether, we ascribe a central function to GIPs for the proper recruitment and/or stabilization of centromeric proteins essential in the specification of the centromere identity, as well as for centromeric cohesion in somatic cells.

Cell cycle-specific cleavage of Scc2 regulates its cohesin deposition activity.

Sister chromatid cohesion (SCC), efficient DNA repair, and the regulation of some metazoan genes require the association of cohesins with chromosomes. Cohesins are deposited by a conserved heterodimeric loading complex composed of the Scc2 and Scc4 proteins in Saccharomyces cerevisiae, but how the Scc2/Scc4 deposition complex regulates the spatiotemporal association of cohesin with chromosomes is not understood. We examined Scc2 chromatin association during the cell division cycle and found that the affinity of Scc2 for chromatin increases biphasically during the cell cycle, increasing first transiently in late G1 phase and then again later in G2/M. Inactivation of Scc2 following DNA replication reduces cellular viability, suggesting that this post S-phase increase in Scc2 chromatin binding affinity is biologically relevant. Interestingly, high and low Scc2 chromatin binding levels correlate strongly with the presence of full-length or amino-terminally cleaved forms of Scc2, respectively, and the appearance of the cleaved Scc2 species is promoted in vitro either by treatment with specific cell cycle-staged cellular extracts or by dephosphorylation. Importantly, Scc2 cleavage eliminates Scc2-Scc4 physical interactions, and an scc2 truncation mutant that mimics in vivo Scc2 cleavage is defective for cohesin deposition. These observations suggest a previously unidentified mechanism for the spatiotemporal regulation of cohesin association with chromosomes through cell cycle regulation of Scc2 cohesin deposition activity by Scc2 dephosphorylation and cleavage.

Structure of the human cohesin inhibitor Wapl

Cohesin, along with positive regulators, establishes sister-chromatid cohesion by forming a ring to circle chromatin. The wings apart-like protein (Wapl) is a key negative regulator of cohesin and forms a complex with precocious dissociation of sisters protein 5 (Pds5) to promote cohesin release from chromatin. Here we report the crystal structure and functional characterization of human Wapl. Wapl contains a flexible, variable N-terminal region (Wapl-N) and a conserved C-terminal domain (Wapl-C) consisting of eight HEAT (Huntingtin, Elongation factor 3, A subunit, and target of rapamycin) repeats. Wapl-C folds into an elongated structure with two lobes. Structure-based mutagenesis maps the functional surface of Wapl-C to two distinct patches (I and II) on the N lobe and a localized patch (III) on the C lobe. Mutating critical patch I residues weaken Wapl binding to cohesin and diminish sister-chromatid resolution and cohesin release from mitotic chromosomes in human cells and Xenopus egg extracts. Surprisingly, patch III on the C lobe does not contribute to Wapl binding to cohesin or its known regulators. Although patch I mutations reduce Wapl binding to intact cohesin, they do not affect Wapl-Pds5 binding to the cohesin subcomplex of sister chromatid cohesion protein 1 (Scc1) and stromal antigen 2 (SA2) in vitro, which is instead mediated by Wapl-N. Thus, Wapl-N forms extensive interactions with Pds5 and Scc1-SA2. Wapl-C interacts with other cohesin subunits and possibly unknown effectors to trigger cohesin release from chromatin.

Synaptonemal Complex-Dependent Centromeric Clustering and the Initiation of Synapsis in Drosophila Oocytes

The pairing of homologous chromosomes and the intimate synapsis of the paired homologs by the synaptonemal complex (SC) are essential for subsequent meiotic processes including recombination and chromosome segregation. Here we show that the centromere clustering plays an important role in initiating homolog synapsis during meiosis in Drosophila females. Although centromeres are not clustered prior to the onset of meiosis, all four pairs of centromeres are actively clustered into one or two masses during early meiotic prophase. Within the 16-cell cyst, centromeric clustering appears to define the first step in the initiation of synapsis. Clustering is restricted to the nuclei that form the SC and is dependent on all known SC proteins. Surprisingly, both centromeric clusters and the SC components associated with them persist long after the disassembly of the euchromatic SC at the end of pachytene. The initiation of homologous recombination through the formation of programmed double-strand breaks (DSBs) is not required for either the formation or the maintenance of the centromeric clusters. Our data support a view in which the SC-mediated clustering at the centromeres is the initiating event for meiotic synapsis.

The cohesin-interacting protein, precocious dissociation of sisters 5A/sister chromatid cohesion protein 112, is up-regulated in human astrocytic tumors

Glioblastoma multiforme (GBM) is the most prevalent, highly malignant, invasive and difficult-to-treat primary brain tumor in adults. At the genetic level, it is characterized by a high degree of chromosomal instability and aneuploidy. It has been shown that defects in the mitotic spindle checkpoint could lead to the development of aneuploidy as well as tumorigenesis. Additional proteins regulating sister chromatid cohesion could also be involved in maintaining the fidelity of chromosome segregation. One such protein is the precocious dissociation of sisters 5A (Pds5A), also known as sister chromatid cohesion protein 112. It is a nuclear protein, expressed from the S right through to the mitotic phase. It is highly conserved from yeast to man and plays a role in the establishment, maintenance and dissolution of sister chromatid cohesion. The mutation of Pds5A orthologs in lower organisms results in chromosome missegregation, aneuploidy and DNA repair defects. It is considered that such defects can cause either cell death or contribute to the development of cancer cells. Indeed, altered expression levels of Pds5A have been observed in tumors of the breast, kidney, oesophagus, stomach, liver and colon. Malignant gliomas, however, have not been analysed so far. Herein, we report on the cloning of Rattus norvegicus Pds5A and on the analysis of its expression pattern in rat tissue. We also show that Pds5A is significantly overexpressed at both the mRNA and protein level and that this overexpression correlates positively with the WHO grade of human gliomas. However, functional assays show that the siRNA-mediated knockdown of Pds5A affects sister chromatid cohesion but does not influence mitotic checkpoint function or the proliferation and survival of GBM cells. Although the mechanism by which Pds5A functions in GBM cells remains unclear, its overexpression in high grade gliomas implies that it could play a pivotal role during the development and progression of astrocytic tumors.

Positive regulation of c- Myc by cohesin is direct, and evolutionarily conserved

Contact between sister chromatids from S phase to anaphase depends on cohesin, a large multi-subunit protein complex. Mutations in sister chromatid cohesion proteins underlie the human developmental condition, Cornelia de Lange syndrome. Roles for cohesin in regulating gene expression, sometimes in combination with CCCTC-binding factor (CTCF), have emerged. We analyzed zebrafish embryos null for cohesin subunit rad21 using microarrays to determine global effects of cohesin on gene expression during embryogenesis. This identified Rad21-associated gene networks that included myca (zebrafish c- myc), p53 and mdm2. In zebrafish, cohesin binds to the transcription start sites of p53 and mdm2, and depletion of either Rad21 or CTCF increased their transcription. In contrast, myca expression was strongly downregulated upon loss of Rad21 while depletion of CTCF had little effect. Depletion of Rad21 or the cohesin-loading factor Nipped-B in Drosophila cells also reduced expression of myc and Myc target genes. Cohesin bound the transcription start site plus an upstream predicted CTCF binding site at zebrafish myca. Binding and positive regulation of the c- Myc gene by cohesin is conserved through evolution, indicating that this regulation is likely to be direct. The exact mechanism of regulation is unknown, but local changes in histone modification associated with transcription repression at the myca gene were observed in rad21 mutants.

Regulation of the Drosophila Enhancer of split and invected-engrailed Gene Complexes by Sister Chromatid Cohesion Proteins

The cohesin protein complex was first recognized for holding sister chromatids together and ensuring proper chromosome segregation. Cohesin also regulates gene expression, but the mechanisms are unknown. Cohesin associates preferentially with active genes, and is generally absent from regions in which histone H3 is methylated by the Enhancer of zeste [E(z)] Polycomb group silencing protein. Here we show that transcription is hypersensitive to cohesin levels in two exceptional cases where cohesin and the E(z)-mediated histone methylation simultaneously coat the entire Enhancer of split and invected-engrailed gene complexes in cells derived from Drosophila central nervous system. These gene complexes are modestly transcribed, and produce seven of the twelve transcripts that increase the most with cohesin knockdown genome-wide. Cohesin mutations alter eye development in the same manner as increased Enhancer of split activity, suggesting that similar regulation occurs in vivo. We propose that cohesin helps restrain transcription of these gene complexes, and that deregulation of similarly cohesin-hypersensitive genes may underlie developmental deficits in Cornelia de Lange syndrome.

Heterochromatin-Mediated Association of Achiasmate Homologs Declines With Age When Cohesion Is Compromised

Normally, meiotic crossovers in conjunction with sister-chromatid cohesion establish a physical connection between homologs that is required for their accurate segregation during the first meiotic division. However, in some organisms an alternative mechanism ensures the proper segregation of bivalents that fail to recombine. In Drosophila oocytes, accurate segregation of achiasmate homologs depends on pairing that is mediated by their centromere-proximal heterochromatin. Our previous work uncovered an unexpected link between sister-chromatid cohesion and the fidelity of achiasmate segregation when Drosophila oocytes are experimentally aged. Here we show that a weak mutation in the meiotic cohesion protein ORD coupled with a reduction in centromere-proximal heterochromatin causes achiasmate chromosomes to missegregate with increased frequency when oocytes undergo aging. If ORD activity is more severely disrupted, achiasmate chromosomes with the normal amount of pericentric heterochromatin exhibit increased nondisjunction when oocytes age. Significantly, even in the absence of aging, a weak ord allele reduces heterochromatin-mediated pairing of achiasmate chromosomes. Our data suggest that sister-chromatid cohesion proteins not only maintain the association of chiasmate homologs but also play a role in promoting the physical association of achiasmate homologs in Drosophila oocytes. In addition, our data support the model that deterioration of meiotic cohesion during the aging process compromises the segregation of achiasmate as well as chiasmate bivalents.

Cohesin, gene expression and development: Lessons from Drosophila

The cohesin complex, discovered through its role in sister chromatid cohesion, also plays roles in gene expression and development in organisms from yeast to human. This review highlights what has been learned about the gene control and developmental functions of cohesin and the Nipped-B (NIPBL/Scc2) cohesin loading factor in Drosophila. The Drosophila studies have provided unique insights into the aetiology of Cornelia de Lange syndrome (CdLS), which is caused by mutations affecting sister chromatid cohesion proteins in humans. In vivo experiments with Drosophila show that cohesin and Nipped-B have dosage-sensitive effects on the functions of many evolutionarily conserved genes and developmental pathways. Genome-wide studies with Drosophila cultured cells show that Nipped-B and cohesin co-localize on chromosomes, and bind preferentially, but not exclusively, to many actively transcribed genes and their regulatory sequences, including many of the proposed in vivo target genes. In contrast, the cohesion factors are largely excluded from genes silenced by Polycomb group (PcG) proteins. Combined, the in vivo genetic data and the binding patterns of cohesin and Nipped-B in cultured cells are consistent with the hypothesis that they control the action of gene regulatory sequences, including transcriptional enhancers and insulators, and suggest that they might also help define active chromatin domains and influence transcriptional elongation.

Mice lacking sister chromatid cohesion protein PDS5B exhibit developmental abnormalities reminiscent of Cornelia de Lange syndrome

PDS5B is a sister chromatid cohesion protein that is crucial for faithful segregation of duplicated chromosomes in lower organisms. Mutations in cohesion proteins are associated with the developmental disorder Cornelia de Lange syndrome (CdLS) in humans. To delineate the physiological roles of PDS5B in mammals, we generated mice lacking PDS5B (APRIN). Pds5B-deficient mice died shortly after birth. They exhibited multiple congenital anomalies, including heart defects, cleft palate, fusion of the ribs, short limbs, distal colon aganglionosis, abnormal migration and axonal projections of sympathetic neurons, and germ cell depletion, many of which are similar to abnormalities found in humans with CdLS. Unexpectedly, we found no cohesion defects in Pds5B(-/-) cells and detected high PDS5B expression in post-mitotic neurons in the brain. These results, along with the developmental anomalies of Pds5B(-/-) mice, the presence of a DNA-binding domain in PDS5B in vertebrates and its nucleolar localization, suggest that PDS5B and the cohesin complex have important functions beyond their role in chromosomal dynamics.

Sequential Loading of Cohesin Subunits during the First Meiotic Prophase of Grasshoppers

The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I.

Effects of sister chromatid cohesion proteins oncutgene expression during wing development inDrosophila

The cohesin protein complex is a conserved structural component of chromosomes. Cohesin binds numerous sites along interphase chromosomes and is essential for sister chromatid cohesion and DNA repair. Here, we test the idea that cohesin also regulates gene expression. This idea arose from the finding that the Drosophila Nipped-B protein, a functional homolog of the yeast Scc2 factor that loads cohesin onto chromosomes, facilitates the transcriptional activation of certain genes by enhancers located many kilobases away from their promoters. We find that cohesin binds between a remote wing margin enhancer and the promoter at the cut locus in cultured cells, and that reducing the dosage of the Smc1 cohesin subunit increases cut expression in the developing wing margin. We also find that cut expression is increased by a unique pds5 gene mutation that reduces the binding of cohesin to chromosomes. On the basis of these results, we posit that cohesin inhibits long-range activation of the Drosophila cut gene, and that Nipped-B facilitates activation by regulating cohesin-chromosome binding. Such effects of cohesin on gene expression could be responsible for many of the developmental deficits that occur in Cornelia de Lange syndrome, which is caused by mutations in the human homolog of Nipped-B.

Caenorhabditis elegans EVL-14/PDS-5 and SCC-3 are essential for sister chromatid cohesion in meiosis and mitosis.

Sister chromatid cohesion is fundamental for the faithful transmission of chromosomes during both meiosis and mitosis. Proteins involved in this process are highly conserved from yeasts to humans. In screenings for sterile animals with abnormal vulval morphology, mutations in the Caenorhabditis elegans evl-14 and scc-3 genes were isolated. Defects in cell divisions were observed in germ line as well as in vulval and somatic gonad lineages. Through positional cloning of these genes, we have shown that EVL-14 and SCC-3 are likely the only C. elegans homologs of the yeast sister chromatid cohesion proteins Pds5 and Scc3, respectively. Both evl-14 and scc-3 mutants displayed defects in the meiotic germ line. In evl-14 mutants, synaptonemal complexes (SCs) were detectable but more than the usual six DAPI (4',6'-diamidino-2-phenylindole)-positive structures were seen at diakinesis, suggesting that EVL-14/PDS-5 is important for the maintenance of sister chromatid cohesion in late prophase. In scc-3 mutant animals, normal SCs were not visible and approximately 24 DAPI-positive structures were seen at diakinesis, indicating that SCC-3 is necessary for sister chromatid cohesion. Immunostaining revealed that localization of REC-8, a homolog of the yeast meiotic cohesin subunit Rec8, to the chromosomes depends on the presence of SCC-3 but not that of EVL-14/PDS-5. scc-3 RNA interference (RNAi)-treated embryos were 100% lethal and displayed defects in cell divisions. evl-14 RNAi caused a range of phenotypes. These results indicate that EVL-14/PDS-5 and SCC-3 have functions in both mitosis and meiosis.