InteractiveFly: GeneBrief

Nipped-B: Biological Overview | References

The cohesin complex is a chromosomal component required for sister chromatid cohesion that is conserved from yeast to man. The similarly conserved Nipped-B protein is needed for cohesin to bind to chromosomes. In higher organisms, Nipped-B and cohesin regulate gene expression and development by unknown mechanisms. Using chromatin immunoprecipitation, it was found that Nipped-B and cohesin bind to the same sites throughout the entire non-repetitive Drosophila genome. They preferentially bind transcribed regions and overlap with RNA polymerase II. This contrasts sharply with yeast, where cohesin binds almost exclusively between genes. Differences in cohesin and Nipped-B binding between Drosophila cell lines often correlate with differences in gene expression. For example, cohesin and Nipped-B bind the Abd-B homeobox gene in cells in which it is transcribed, but not in cells in which it is silenced. They bind to the Abd-B transcription unit and a downstream regulatory region and thus could regulate both transcriptional elongation and activation. It is proposed transcription facilitates cohesin binding, perhaps by unfolding chromatin, and that Nipped-B then regulates gene expression by controlling cohesin dynamics. These mechanisms are likely involved in the etiology of Cornelia de Lange syndrome, in which mutation of one copy of the NIPBL gene encoding the human Nipped-B ortholog causes diverse structural and mental birth defects (Misulovin, 2008).

Development of higher organisms requires tissue-specific activation and silencing of genes. Tissue-specific regulation is often mediated by sequences located several kilobases away from a gene and the combined actions of transcriptional activators, silencing proteins, and factors that modify chromatin structure. Studies in Drosophila reveal that chromosomal proteins required for sister chromatid cohesion also play critical roles in control of gene expression during development. The Drosophila Nipped-B protein was discovered in a screen for factors that facilitate expression of the cut homeobox gene in the developing wing margin that is driven by a distant transcriptional enhancer located more than 80 kb upstream of the transcription start site (Rollins, 1999). Nipped-B is essential, and homozygous Nipped-B mutants die as second instar larvae, while heterozygous Nipped-B mutations decrease expression of the cut and Ultrabithorax (Ubx) genes. These two genes and some unknown developmental processes are exquisitely sensitive to Nipped-B dosage: heterozygous Nipped-B null mutations reduce Nipped-B messenger RNA (mRNA) levels by only 25% and a 50% reduction induced by RNAi is lethal (Rollins, 2004; Misulovin, 2008 and references therein).

Homozygous Nipped-B mutants show sister chromatid cohesion defects just before death as the maternally provided Nipped-B wanes (Rollins, 2004). Studies on Nipped-B orthologs in other organisms indicate that these defects result from a failure of the cohesin protein complex that mediates cohesion to bind to chromosomes (Arumugam, 2003; Ciosk, 2000; Gillespie, 2004; Seitan, 2006; Takahashi, 2004; Tomonaga, 2000; Watrin, 2006; Misulovin, 2008 and references therein).

Cohesin binds to chromosomes throughout interphase when gene expression occurs. It contains four subunits, Smc1, Smc3, Rad21, and Stromalin (SA), which form a ring-like structure. In most organisms, cohesin is loaded along chromosomes during telophase and is removed from the arms at the subsequent prophase. A leading idea is that cohesin mediates cohesion by encircling both sister chromatids (Misulovin, 2008).

Heterozygous Nipped-B mutants do not show cohesion defects, indicating that their effects on gene expression are unlikely to be caused by a significant reduction in binding of cohesin to chromosomes. Changes in cohesin dosage, however, also affect cut expression, suggesting that Nipped-B's role in gene expression involves its ability to regulate cohesin binding. Although Nipped-B and cohesin are both needed for sister chromatid cohesion, they have opposite effects on cut expression. Reducing cohesin dosage increases cut expression in the developing wing margin while reducing Nipped-B decreases expression (Rollins, 1999; Rollins, 2004; Dorsett, 2005). This gave rise to the idea that cohesin binds to cut and inhibits expression, possibly by interfering with enhancer-promoter communication, and that Nipped-B maintains a dynamic cohesin-binding equilibrium to alleviate these effects (Dorsett, 2004). Consistent with this idea, cohesin binds directly to cut regulatory sequences in cultured cells and to the cut locus in salivary gland chromosomes (Dorsett, 2005; Misulovin, 2008 and references therein).

Cornelia de Lange syndrome (CdLS) is caused by heterozygous loss-of-function mutations in the Nipped-B-Like (NIPBL) ortholog of Nipped-B and, in a few cases, by viable missense mutations in the Smc1A or Smc3 cohesin subunit genes (Deardorff; 2007; Krantz; 2004; Musio; 2006; Tonkin; 2004). CdLS patients display slow growth, mental retardation, and defects in limbs and organs (Dorsett; 2007; Jackson; 1993; Strachan; 2005). Most do not show cohesion defects (Kaur; 2005; Vrouwe; 2007), suggesting that the diverse developmental deficits are caused by gene expression changes similar to those in Drosophila. The similar effects of reduced NIPBL activity and cohesin subunit missense mutations on human development in the absence of obvious effects on sister chromatid cohesion further suggest that Nipped-B/NIPBL are likely to dynamically regulate cohesin (Misulovin, 2008).

Another potential link between the effects of sister chromatid cohesion factors on development and effects on gene expression is provided by the finding that mice homozygous for a knockout of the Pds5B gene show developmental deficits reminiscent of some that occur in CdLS patients (Zhang, 2007). The Pds5 protein, which is also conserved from fungi to man, interacts with cohesin and plays roles in establishment and/or maintenance of sister chromatid cohesion. In mammals, there are two Pds5 proteins, and the mice lacking Pds5B that show developmental abnormalities do not have cohesion defects. In Drosophila, there is a single pds5 gene, and heterozygous pds5 mutations alter cut gene expression without the effects on cohesion seen in homozygous mutants (Dorsett, 2005), suggesting that changes in gene expression also likely underlie the effects of Pds5B on mouse development (Misulovin, 2008).

The binding of cohesin and the Scc2 ortholog of Nipped-B have been mapped genome-wide in the yeast Saccharomyces cerevisiae (Glynn; 2004; Lengronne; 2004). Cohesin binds almost exclusively between genes in yeast, and most binding sites are between convergent transcription units. Coupled with the finding that Scc2 does not colocalize with cohesin, this led to the idea that cohesin loads onto chromosomes at Scc2 binding sites and then is pushed to the ends of genes by RNA polymerase (Misulovin, 2008).

The intergenic localization of cohesin in yeast, where it rarely overlaps regulatory sequences, and the lack of co-localization with Scc2, which is inconsistent with dynamic control by Scc2, are incompatible with the models for how Nipped-B/NIPBL and cohesin regulate Drosophila gene expression. The yeast genome, however, is much more compact than that of higher eukaryotes, with smaller intergenic regions, few introns, and rare long-range regulation. Thus, the mechanisms that determine the location of cohesin binding sites are likely to differ in higher organisms. This study mapped the Nipped-B and cohesin binding sites in the entire non-repetitive Drosophila genome to gain insights into how they interact with genes. Strikingly, it was found that in contrast their orthologs in yeast, Nipped-B and cohesin colocalize and bind preferentially, but not exclusively, to active transcription units (Misulovin, 2008).

The studies reported in this paper represent the first large-scale mapping of cohesin binding to a metazoan genome. The cohesin binding regions in Drosophila are much larger on average than in yeast, extending from a few kilobases up to 100 kb or so in length, and cohesin-free regions can extend from several kilobases in size up to a megabase or so. The reasons for the differences in cohesin localization between yeast and Drosophila are unknown, but multiple speculative possibilities can be considered. One is that, in Drosophila, transcription might be needed in many cases to provide a 10-nm chromatin fiber that fits into the 35-nm internal diameter of the cohesin ring (Anderson, 2002), while in yeast, much of the chromosome already has an accessible structure. For instance, the H1 linker histone that helps form higher order chromatin structures is likely present at most nucleosomes in metazoan organisms, while in yeast, the related Hho1 linker histone is present at low levels and does not globally regulate chromatin structure or gene expression. It is also feasible that in yeast, which has a small compact genome, the positions of cohesin binding sites have been evolutionarily optimized to avoid interference with transcription. It is also worth noting that in Drosophila, cohesin peaks occur three- to eightfold less frequently in coding sequences than in intergenic sequences or introns. In yeast, where most genes lack introns, similar preferences would favor binding to intergenic sequences. It is unclear why cohesin prefers noncoding over coding sequences in Drosophila, but it is possible that differences in DNA sequence or binding of other proteins could be critical factor (Misulovin, 2008).

In yeast, cohesin binds more densely around centromeres. In Drosophila, the centromeres are in heterochromatin that consists largely of repetitive sequences. Thus, the studies reported in this paper provide no information regarding the binding of cohesin or Nipped-B binding to centromeres. By immunostaining, cohesin binds to both mitotic and meiotic centromeres in Drosophila (Warren; 2000; Valdeolmillos; 2004; Khetani; 2007; Gause; 2007). Immunostaining with Nipped-B antibody indicates that Nipped-B colocalizes with cohesin along chromosome arms in both polytene and meiotic chromosomes, but not at centromeres in meiotic chromosomes (Gause, 2007). Thus, Nipped-B might not be involved in regulating association of cohesin with centromeres during meiosis (Misulovin, 2008).

Based on effects of Nipped-B and cohesin on cut expression in vivo, it was originally proposed that cohesin binding to the cut regulatory region hinders enhancer-promoter interactions and that Nipped-B alleviates this effect by dynamic control of cohesin binding (Dorsett, 2004). The finding that Nipped-B colocalizes with cohesin supports the idea that it dynamically regulates binding. The preferential association of cohesin with transcribed regions suggests additional mechanisms by which cohesin binding might affect transcription, and vice versa. As a general model, it is envisioned that transcription facilitates cohesin binding and that the cohesin that binds affects subsequent transcription. Nipped-B then regulates these effects on transcription by dynamic control of cohesin binding or subunit interaction (Misulovin, 2008).

Features of the cohesin binding to the active Abd-B gene in Sg4 cells raise the possibility that in some cases, cohesin could interfere with both transcriptional elongation and activation. Some cohesin and PolII peaks coincide in both the Abd-B transcription unit and 3' regulatory region, which contains intergenic transcription units likely involved in Abd-B regulation. The cohesin in the regulatory region could hinder Abd-B activation by affecting this intergenic transcription. For instance, in the human β-globin gene, blocking intergenic transcription between the enhancer and promoter by insertion of a transcription terminator or an insulator reduces activation. Genes with distant regulatory elements, such as cut and Ubx, may be more sensitive to Nipped-B dosage because of combined effects on activation and elongation (Misulovin, 2008).

Cohesin might also have positive effects on gene expression in some cases. Although it is unknown if the effect is direct, reduction of Rad21 dosage decreases runx gene expression during early zebrafish development. Similarly, Smc1 homozygous mutant clones in the Drosophila mushroom body show reduced ecdysone receptor (EcR) gene expression, and cohesin binds EcR in all three cell lines examined in this study. These findings do not provide an obvious explanation for how cohesin could directly facilitate gene expression, except the possibility that it might help maintain the chromatin in an unfolded state that is more conducive to transcription. Another possibility is that, in specific cases, cohesin might contribute to chromatin boundary function to block the spread of silencing factors as it does at the HMR silent locus in yeast. There is a cohesin/Nipped-B peak at the Fab-7 boundary element flanking the active Abd-B domain in Sg4 cells, and thus the possibility cannot be ruled out that cohesin plays a role in defining chromatin domains permissive for gene expression (Misulovin, 2008).

The data indicate that cohesin and Nipped-B bind preferentially, but not exclusively, to active genes. It is speculated that transcription facilitates cohesin binding by unfolding chromatin to a 10-nm fiber that can be encircled by cohesin. Based on the anti-correlation with histone H3 lysine 27 trimethylation, it also appears likely that silencing, either by preventing transcription or through an independent effect on chromatin structure, inhibits cohesin binding (Misulovin, 2008).

Transcription is neither necessary nor sufficient for cohesin binding because some poorly expressed genes, such as cut, bind cohesin, and some active genes, such as SA, do not. In the case of cut, PolII binds primarily at the promoter in both Sg4 and BG3 cells. There is little downstream polymerase in the cut transcription unit in either cell type, yet there is substantially more cohesin binding to this region in BG3 cells. Thus, there must be additional factors besides transcription that regulate cohesin binding (Misulovin, 2008).

Association of cohesin and Nipped-B with many genes suggests that the diversity of CdLS phenotypes stems from effects on multiple genes. Many of the genes bound by cohesin in Drosophila cells encode evolutionarily conserved transcription factors and receptors that control limb, organ, peripheral, and central nervous system development. These include the genes encoding the Notch receptor, its Serrate and Delta ligands and Mastermind coactivator, the Thickvein transforming growth factor beta (TGFβ) receptor and the Mad DNA-binding protein that mediates TGFβ signaling, the Patched hedgehog receptor, the Ecdysone receptor, and the Epidermal growth factor receptor. Homeobox genes bound by cohesin include cut, Lim1, Distal-less (Dll), homeobrain (hbn), Abd-B, invected (inv), homothorax (hth), and C15, among others. There are also multiple zinc finger protein genes that bind cohesin, including the pannier (pnr) GATA1 ortholog and its interaction partner u-shaped (ush). In BG3 cells, the entire Enhancer of split gene complex encoding multiple bHLH transcription factors involved in nervous system development is bound by cohesin and Nipped-B (Misulovin, 2008).

The finding that cohesin binding to Abd-B correlates with Abd-B expression and the variation in cohesin binding between the three cell lines indicate that many other genes are also likely to bind cohesin in other cell types. Thus, identification of target genes that cause specific CdLS phenotypes will require mapping cohesin binding and gene expression patterns in affected tissues at critical stages of development. Because many genes are bound by cohesin in each cell type, it is speculated that some of the individual patient phenotypes might stem from simultaneous effects on the expression of multiple genes (Misulovin, 2008).

Functional links between Drosophila Nipped-B and cohesin in somatic and meiotic cells

Drosophila Nipped-B is an essential protein that has multiple functions. It facilitates expression of homeobox genes and is also required for sister chromatid cohesion. Nipped-B is conserved from yeast to man, and its orthologs also play roles in deoxyribonucleic acid repair and meiosis. Mutation of the human ortholog, Nipped-B-Like (NIPBL), causes Cornelia de Lange syndrome (CdLS), associated with multiple developmental defects. The Nipped-B protein family is required for the cohesin complex that mediates sister chromatid cohesion to bind to chromosomes. A key question, therefore, is whether the Nipped-B family regulates gene expression, meiosis, and development by controlling cohesin. To gain insights into Nipped-B's functions, the effects were examined of several Nipped-B mutations on gene expression, sister chromatid cohesion, and meiosis. Association of Nipped-B and cohesin with somatic and meiotic chromosomes was examined by immunostaining. Missense Nipped-B alleles affecting the same HEAT repeat motifs as CdLS-causing NIPBL mutations have intermediate effects on both gene expression and mitotic chromatid cohesion, linking these two functions and the role of NIPBL in human development. Nipped-B colocalizes extensively with cohesin on chromosomes in both somatic and meiotic cells and is present in soluble complexes with cohesin subunits in nuclear extracts. In meiosis, Nipped-B also colocalizes with the synaptonemal complex and contributes to maintenance of meiotic chromosome cores. These results support the idea that direct regulation of cohesin function underlies the diverse functions of Nipped-B and its orthologs (Gause, 2008; full text of article).

Relative to null alleles, the missense Nipped-B^NC71 and Nipped-B^NC78 mutations that alter HEAT repeats have weaker effects on both gene expression and sister chromatid cohesion. This agrees with prior studies suggesting that Nipped-B's role in gene expression is to regulate cohesin chromosome binding. The prior studies showed that Nipped-B facilitates activation of the cut gene by a distant transcriptional enhancer and that cohesin has an opposite effect to Nipped-B on cut expression. It has also been shown that cohesin binds to cut in multiple cell types and that a unique pds5 mutation that reduces binding of cohesin to chromosomes increases cut expression. Combined, these findings and the genetic linkage between Nipped-B's roles in gene expression and sister chromatid cohesion reported in this study support the hypothesis that Nipped-B facilitates cut expression by alleviating negative effects of cohesin. This model suggests that cohesin binding to cut inhibits expression and by maintaining a dynamic cohesin chromosome-binding equilibrium, Nipped-B facilitates cohesin removal or relocation (Gause, 2008).

Based on this model, it is posited that the Nipped-B^NC71 and Nipped-B^NC78 missense mutations in the HEAT repeats are hypomorphic and reduce but do not abolish the ability of Nipped-B to control cohesin binding to chromosomes. In this scenario, the homozygous HEAT repeat mutants show milder cohesion defects than null alleles because loading of cohesin onto chromosomes is only partially reduced, and the heterozygous mutants have weaker effects on gene expression because they only slightly alter the cohesin chromosome-binding equilibrium (Gause, 2008).

The Nipped-B^NC71 and Nipped-B^NC78 missense mutations provide the first evidence that some of the HEAT repeats are important for gene expression and sister chromatid cohesion, and they also link Nipped-B's role in gene expression to the function of NIPBL and cohesin in human development. There are CdLS-causing missense mutations in all seven HEAT repeats of NIPBL, and Nipped-B^NC71 and Nipped-B^NC78 affect HEAT repeats 2 and 3, respectively. Indeed, Nipped-B^NC71 and Nipped-B^NC78 both affect conserved residues adjacent or very close to conserved residues affected by CdLS-causing mutations, and thus the CdLS mutations are likely to have dominant effects on gene expression similar to Nipped-B^NC71 and Nipped-B^NC78. The structural similarity between Nipped-B^NC71 and Nipped-B^NC78 and the NIPBL mutations provides a link to cohesin in human development because like the NIPBL HEAT repeat mutations, missense mutations in the Smc1 and Smc3 cohesin subunits also cause CdLS (Gause, 2008).

The functional connections between cohesin and the Nipped-B protein family in sister chromatid cohesion, gene expression, and development that have been revealed genetically likely reflect direct regulation of cohesin function by Nipped-B. This is supported by the finding that Nipped-B colocalizes with cohesin on polytene and meiotic chromosomes and the presence of soluble complexes containing Nipped-B and the Rad21 and SA cohesin subunits in cell nuclear extracts. It is not known why soluble complexes containing Nipped-B and whole cohesin are not detected, but it is possible that the antibodies used for immunoprecipitation disrupt these interactions, that the epitopes are masked in such complexes, that such interactions are transitory, or that they are stable only when Nipped-B and cohesin are both bound to DNA (Gause, 2008).

In S. cerevisiae, cohesin does not colocalize with the Scc2 ortholog of Nipped-B on chromosomes. Nevertheless, purification of yeast Scc2-Scc4 complex brings along small amounts of cohesin subunits, and thus it has also been proposed that the Scc2-Scc4 complex also directly regulates binding of cohesin to chromosomes. It is suggested that cohesin loads at Scc2-binding sites and translocates away. In contrast, chromatin immunoprecipitation experiments using cultured Drosophila cells reveals that Nipped-B colocalizes with cohesin in the entire nonrepetitive genome, confirming that their colocalization is not unique to polytene and meiotic chromosomes. Thus, the separate localization of the Nipped-B/Scc2 cohesin loader and cohesin may be unique to yeast chromosomes (Gause, 2008).

The results raise the possibility that Nipped-B is not critical for the function of cohesin at centromeres. Cohesin binds densely to pericentric heterochromatin, and cohesin at centromeres is protected in both mitotic and meiotic cells by a member of the Shugoshin/Mei-S332 protein family. Although Nipped-B completely colocalizes with Smc1 and Smc3 along meiotic chromosome cores, no Nipped-B staining is seen around the centromeres, where cohesin staining is the strongest. No Nipped-B was seen at the centromeres of polytene chromosomes, but this might be explained by the under-replication of pericentric heterochromatin in these cells. Consistent with this idea, no strong cohesin staining was seen at polytene centromeres. Nipped-B, however, also does not colocalize with the HP1 heterochromatin protein, whose Swi6 ortholog in S. pombe is required for cohesion and interacts with cohesin, at positions that are not under-replicated. In cultured Drosophila cells, Rad21 and SA persist at the centromeres until the metaphase-anaphase transition. Metaphase chromosome spreads from Kc cells were immunostained and Smc1 and SA can be seen that colocalizes with the CID centromere-specific histone at centromeres, but Nipped-B has not been detected. In this case, however, given the relatively weak signals, lack of staining elsewhere in the same nucleus, and the different fixation conditions needed to get good chromosome morphology, it is difficult to rule out the possibility that Nipped-B cannot be detected because of insufficient sensitivity (Gause, 2008).

Although the lack of pericentric Nipped-B staining in meiotic chromosomes might be caused by epitope masking, other evidence indicates that Nipped-B function is also not critical at meiotic centromeres. In particular, the retention of Smc1 and Smc3 protein at the meiotic chromosome centromeres in heterozygous Nipped-B mutants and the normal meiotic transmission of the J21A minichromosome with a weak centromere indicate that centromeric cohesion is not altered, although the chromosome cores disintegrate early when the dosage of Nipped-B protein is reduced. This contrasts sharply with the chromosome nondisjunction and lack of cohesin binding to the meiotic centromeres in orientation disrupter (ord) mutants. Thus, the evidence suggests that either Nipped-B does not play a critical role in the function of cohesin at meiotic centromeres or that its role at centromeres is not as dosage sensitive as its function along the chromosome cores. Given the lack of Nipped-B staining, the colocalization of Ord and cohesin, and the lack of cohesin at centromeres in ord mutants, it is possible that Ord functionally substitutes for Nipped-B at meiotic centromeres (Gause, 2008).

The data provide evidence for a functional link between the Nipped-B protein family and cohesin in meiosis. The C. coprinus Rad9 hypomorphic mutant, in addition to having meiotic cohesion defects, fails to complete synaptonemal complex (SC) formation, indicating that the Nipped-B family is involved in SC assembly (Cummings, 2002; Seitz, 1996). The findings that Nipped-B colocalizes with cohesin subunits along meiotic chromosome cores and that cores disassemble prematurely when Nipped-B dosage is reduced indicates that the Nipped-B family is also involved in the maintenance of SC structure (Gause, 2008).

The Nipped-B family functions in SC assembly and maintenance are both likely to involve cohesin. Cohesin colocalizes with SC proteins and is involved in homolog pairing, SC formation, and SC structure in diverse organisms. The C(2)M protein, which contains kleisin motifs similar to those in Rad21 and which interacts with Smc3, is needed for SC formation and the coalescence of Smc1 and Smc3 into chromosome cores (Khetani, 2007). C(2)M, however, is not required for binding of the Smc1 and Smc3 cohesin subunits to either centromeres or chromosome arms. Taken together, these data suggest a model in which Nipped-B and C(2)M collaborate in formation of the cohesin chromosome cores and that Nipped-B and Ord then cooperate to maintain this structure. Thus, the Nipped-B protein family plays a role beyond simply loading cohesin onto chromosomes and is involved directly in regulating the higher-order meiosis-specific organization of cohesin (Gause, 2008).

Nipped-B, a Drosophila homolog of chromosomal adherins, participates in activation by remote enhancers in the cut and Ultrabithorax genes

Enhancers are able to activate promoters located several kilobases away, but how this is accomplished is not known. Activation by the wing margin enhancer in the cut gene, located 85 kb from the promoter, requires several genes that participate in the Notch receptor pathway in the wing margin, including scalloped, vestigial, mastermind, Chip, and the Nipped locus. Nipped mutations disrupt one or more of four essential complementation groups: l(2)41Ae, l(2)41Af, Nipped-A, and Nipped-B. Heterozygous Nipped mutations modify Notch mutant phenotypes in the wing margin and other tissues, and magnify the effects that mutations in the cis regulatory region of cut have on cut expression. Nipped-A and l(2)41Af mutations further diminish activation of a wing margin enhancer that has been partly impaired due to a small deletion. In contrast, Nipped-B mutations do not diminish activation by the impaired enhancer, but increase the inhibitory effect of a gypsy transposon insertion between the enhancer and promoter. Nipped-B mutations also magnify the effect of a gypsy insertion in the Ultrabithorax gene. Gypsy binds the Suppressor of Hairy-wing insulator protein [Su(Hw)] that blocks enhancer-promoter communication. Increased insulation by Su(Hw) in Nipped-B mutants suggests that Nipped-B products structurally facilitate enhancer-promoter communication. Compatible with this idea, Nipped-B protein is homologous to a family of chromosomal adherins with broad roles in sister chromatid cohesion, chromosome condensation, and DNA repair (Rollins, 1999).

Database searches reveal homologs of the Nipped-B protein in fungi, worms, and mammals. Only short expressed-sequence tags (ESTs) of Caenorhabditis elegans, mouse, and humans were identified. The human ESTs are from a variety of tissue-specific libraries, suggesting that the human homologs are widely expressed. The combined human ESTs, which do not represent a complete sequence, encode 411 amino acids. Residues 2-232 of the partial human protein overlap Nipped-B residues 1744-1994 with 34% identity and 52% similarity. In order of decreasing homology, the 2157-amino acid Rad9 protein of Coprinus cinereus, the 1583-amino acid Mis4 protein of Schizosaccharomyces pombe, and the 1493-amino acid Scc2 protein of Saccharomyces cerevisiae, are more distantly related. Rad9 residues 669-2071 display 21% identity and 41% similarity to Nipped-B residues 576-1887; Mis4 residues 780-1492 have 19% identity and 41% similarity to Nipped-B residues 1110-1818, and Scc2 residues 697-1291 display 19% identity with 39% similarity to Nipped-B residues 1103-1704. The three fungal homologs show similar levels of homology among themselves, but it is evident that there is a large conserved domain shared by all three proteins. Consistent with the idea that Nipped-B plays an architectural role in enhancer-promoter communication, the fungal homologs of Nipped-B all participate in regulating chromosome structure, with roles in DNA repair, meiotic chromosome condensation, or sister chromatid cohesion. It has been proposed that these three fungal proteins define a new class of chromosomal proteins and have been named adherins to distinguish them from the cohesins that have similar functions (Rollins, 1999 and references).

Although the data do not yet distinguish whether the heterochromatic Nipped locus is a single complex transcription unit or a cluster of distinct genes, several conclusions may be drawn about Nipped functions and their roles relative to the other cut regulators. To summarize, Nipped mutations define three separable essential functions that regulate cut in the wing margin, provided by the Nipped-A, Nipped-B, and l(2)41Af lethal complementation groups. Dosage-sensitive genetic interactions indicate that Nipped-A and l(2)41Af cooperate closely with mam and vg in the regulation of cut. Similar to mam and unlike sd and vg, Nipped-A and l(2)41Af also modulate Notch receptor signaling or expression in multiple tissues. Nipped-B has the most unique function. Like Chip, Nipped-B regulates both cut and Ubx and is antagonistic to insulation by Su(Hw). Together, the antagonism to Su(Hw) and the homology to chromosomal adherins lead to a proposal that Nipped-B protein performs an architectural role in enhancer-promoter communication (Rollins, 1999).

The primary evidence that Nipped-B is antagonistic to Su(Hw) insulator activity is that Nipped-B activity is only strongly limiting for cut expression when there is a gypsy insertion between the wing margin enhancer and promoter. Strikingly, in contrast to mutations disrupting any of the other cut regulators (including sd, mam, Chip, vg, Nipped-A, and l(2)41Af), heterozygous Nipped-B mutations do not detectably reduce activation by the partially crippled wing margin enhancer in ct^53d. Compared with sd, mam, or Nipped-A mutations, heterozygous Nipped-B mutations also only slightly reduce activation of cut expression by the solo wild-type wing margin enhancer present in ct^2s heterozygotes. Therefore, with both ct^53d and ct^2s, Nipped-B products are less limiting for wing margin enhancer activity than are Nipped-A products. Remarkably, the opposite is true when there is a gypsy insulator insertion in cut. Heterozygous Nipped-B mutations are severalfold more effective than Nipped-A mutations in magnifying the effect of the Su(Hw) insulator in ct^L-32; su(Hw)^e2 flies. Furthermore, of the known cut regulators, only Chip and Nipped-B mutations magnify the effect of the Su(Hw) insulator in su(Hw)^e2 bx^34e flies. The antagonism between Nipped-B and Su(Hw) is unlikely to be specific to the Su(Hw)^e2 protein. Su(Hw)^e2 has an amino acid substitution in a zinc finger that reduces DNA-binding activity but contains a wild-type enhancer-blocking domain. Moreover, Nipped-B mutations also reduce cut expression in the absence of a gypsy insertion, indicating that the increased effectiveness of Su(Hw)^e2 in Nipped-B mutants reflects a change in cut regulation rather than a change in Su(Hw)^e2 protein activity (Rollins, 1999).

The available data are insufficient to determine with absolute certainty whether or not Nipped-B directly regulates cut. However, direct regulation provides the simplest explanation for several observations. The ability of Nipped-B mutations to exacerbate different cut mutant phenotypes differs from all other cut regulators such as sd, vg, and mam. Therefore, Nipped-B does not regulate cut indirectly by altering expression of any of the other known cut regulators. Moreover, the effects of the Nipped-B⁴⁰⁷ mutation on cut and Ubx mutant phenotypes are dominant, although Nipped-B⁴⁰⁷ only partially reduces Nipped-B mRNA levels. A partial loss of Nipped-B activity is unlikely to cause a similar or greater loss of activity of another cut regulator. Therefore, in light of the observation that Nipped-B mutations magnify insulation by gypsy insertions in both cut and Ubx, the idea that Nipped-B products directly support enhancer-promoter communication in cut and Ubx is strongly favored. Because Nipped-B is essential and Nipped-B mRNA is expressed at all developmental stages, it may play a similar role in other genes (Rollins, 1999).

The hypothesis that Nipped-B protein plays an architectural role to facilitate enhancer-promoter interactions in cut and Ubx is supported by the diverse effects that the fungal adherin homologs of the Nipped-B protein have on chromosome structure and function. The Rad9 protein of Coprinus was identified in a screen for radiation-sensitive mutants. rad9 mutants were subsequently observed to display defects in synaptonemal complex formation and chromosome condensation during meiosis. Mutations in the Scc2 gene of budding yeast were identified as lethal temperature-sensitive mutants that display defects in sister chromatid cohesion during mitosis. In scc2 mutants, sister chromatids separate prematurely, just after formation of the bipolar spindle. Mutations in the Mis4 gene of fission yeast were identified as temperature-sensitive lethal mutants that missegregate minichromosomes. mis4 mutants also missegregate regular chromosomes and are radiation sensitive. The Mis4 protein is required during S phase and associates with chromosomes during the entire cell cycle. These diverse mutant phenotypes indicate that adherins play fundamental roles in chromosome structure. Although it is not yet known if Nipped-B also participates in mitotic or meiotic chromosome structure, its homology to adherins suggests explanations for how Nipped-B could architecturally facilitate enhancer-promoter communication. It is tempting to speculate, for example, that the biochemical activity of Nipped-B is to recognize and stabilize chromatin loops that hold distant chromosomal sites closer together. The chromatin loops could be created by other factors involved in enhancer-promoter interactions (Rollins, 1999 and references).

Drosophila Nipped-B protein supports sister chromatid cohesion and opposes the Stromalin/Scc3 cohesion factor to facilitate long-range activation of the cut gene

The Drosophila Nipped-B protein facilitates transcriptional activation of the cut and Ultrabithorax genes by remote enhancers. Sequence homologues of Nipped-B, Scc2 of Saccharomyces cerevisiae, and Mis4 of Schizosaccharomyces pombe are required for sister chromatid cohesion during mitosis. The evolutionarily conserved Cohesin protein complex mediates sister chromatid cohesion, and Scc2 and Mis4 are needed for Cohesin to associate with chromosomes. This study shows that Nipped-B is also required for sister chromatid cohesion but that, opposite the effect of Nipped-B, the stromalin/Scc3 component of Cohesin inhibits long-range activation of cut. To explain these findings, a model is proposed based on the chromatin domain boundary activities of Cohesin in which Nipped-B facilitates cut activation by alleviating Cohesin-mediated blocking of enhancer-promoter communication (Rollins, 2004).

These experiments addressed two questions: (1) does Nipped-B, in addition to facilitating remote activation of cut and Ultrabithorax, participate in mitotic sister chromatid cohesion, and (2) does Cohesin participate in long-range activation of cut? The first was motivated by the sequence similarity of Nipped-B to yeast adherins required for sister chromatid cohesion, and the second was motivated by the published observations that the yeast adherins are required for the Cohesin complex to associate with chromosomes. The results indicate that the cooperation between adherins and Cohesin that occurs in yeast is conserved in Drosophila. The findings also indicate, however, that the SA/Scc3 subunit of Cohesin opposes Nipped-B in long-range activation of the cut gene (Rollins, 2004).

The high rate of precocious sister chromatid separation (PSCS) in homozygous and heteroallelic Nipped-B mutants and the increased lethality of Rad21/Scc1 RNAi in flies heterozygous for a Nipped-B mutation observed in this study are consistent with the findings that the yeast Scc2 and Mis4 homologues of Nipped-B are required for Cohesin to associate with chromosomes. Although PSCS was detected in third-instar neuroblasts in RNAi experiments, it is unlikely that the synthetic lethality of a heterozygous Nipped-B mutation and Rad21/Scc1 RNAi is caused by changes in long range gene activation, because as discussed below, Nipped-B and Cohesin appear to have opposing roles in gene activation. In the RNAi experiments, it is possible that the pupal lethality is caused by PSCS in a subpopulation of critical cells, but the possibility cannot be ruled out that Nipped-B and Cohesin cooperate with each other in other essential functions (Rollins, 2004).

No physical association between the yeast adherins and Cohesin has been detected, nor do they colocalize on chromosomes. No chromosomal population of Nipped-B could be detected, but if Nipped-B acts primarily as a chaperone for loading Cohesin onto chromosomes, only a small fraction of Nipped-B may be transiently interacting with chromosomes at any time. The mechanisms by which yeast adherins facilitate Cohesin chromosome binding are unclear, but the synthetic lethality between a heterozygous Nipped-B mutation and Rad21/Scc1 RNAi observed here indicates that this functional connection is conserved in metazoans. In budding yeast, Cohesin begins to associate with chromosomes in late G₁, while in fission yeast, Caenorhabditis elegans, Drosophila, and mammalian cells it begins to associate in telophase. Nipped-B is detected in the nucleus at all stages that have a nuclear membrane, indicating that it could be involved in Cohesin chromosomal association beginning in telophase and thus could influence all potential interphase functions of Cohesin, in addition to sister chromatid cohesion (Rollins, 2004).

The finding that SA/Scc3 RNAi reduces the severity of the ct^K wing-nicking phenotype indicates that the SA/Scc3 component of Cohesin inhibits cut expression. This is the opposite to the role of Nipped-B at cut. Multiple Nipped-B mutations were recovered in a screen for mutations that increase the severity of a wing-nicking phenotype displayed by a cut allele with a weak gypsy insulator insertion. Reduced mRNA levels indicated that some of these Nipped-B mutations are loss-of-function alleles, and viability of homozygous Nipped-B mutants was rescued by a transgene expressing a Nipped-B cDNA from a Chip gene promoter. Thus, Nipped-B protein facilitates activation of cut by the wing margin enhancer (Rollins, 2004).

The effect of Nipped-B on cut expression is likely direct. Nipped-B does not regulate cut by altering the activities of known cut regulators because it is most limiting for cut expression when there is a gypsy insertion at cut while the other known regulators are more limiting with other types of cut mutations. Moreover, heterozygous Nipped-B loss-of-function alleles reduce cut expression, and partial reduction of Nipped-B is unlikely to cause an equal or greater change in the expression of another cut regulator. Although the effects of Nipped-B on gene expression were most apparent with gypsy insertion alleles of cut, a measurable effect was observed in heterozygous females with a wild-type cut allele and an allele in which the wing margin enhancer is deleted. Thus, Nipped-B also facilitates the activation of wild-type cut (Rollins, 2004).

All three SA/Scc3 RNAi insertions and one of three Rad21/Scc1 insertions reduced the number of nicks displayed by the ct^K gypsy insertion. It is thought likely that the Cohesin complex, and not just one or two of its subunits, is responsible for reducing cut expression. Scc1 and Scc3 operate together as a unit in both Drosophila and C. elegans. Thus, it is unlikely that they work independently of each other in regulating gene expression. Indeed, Rad21/Scc1 RNAi in cultured Drosophila cells reduces both Rad21/Scc1 and SA/Scc3 proteins, and data presented here indicate that Rad21/Scc1 and SA/Scc3 may regulate each other's transcript levels. However, the possibility cannot be ruled out that Rad21/Scc1 and SA/Scc3 work independently of the Smc1 and Cap/Smc3 Cohesin subunits, which form another stable subcomplex. Initial attempts to reduce expression of the SMC subunits by RNAi were unsuccessful (Rollins, 2004).

It is unlikely that the effects of SA/Scc3 on cut expression occur by reducing the expression of a cut activator. The small reductions in SA/Scc3 expression in these experiments are unlikely to cause equal or larger changes in the activities of other cut regulators. Also effects are not seen of Cohesin RNAi on ct^53d, which has a small deletion in the enhancer and is affected by all known cut regulators except Nipped-B. It is most likely, therefore, that SA/Scc3 acts directly at cut or by reducing the ability of Nipped-B to facilitate activation (Rollins, 2004).

The possibility that the negative effect of SA/Scc3 on cut expression may be specific to gypsy insertion alleles cannot be ruled out, it is thought improbable. The negative effect is likely to be related to the positive effect of Nipped-B, and Nipped-B facilitates the expression of wild-type cut. If SA/Scc3 does specifically affect gypsy insertion alleles, however, it may interact with the gypsy insulator and contribute to enhancer blocking. This is consistent with evidence that Cohesin functions at chromosomal boundaries in yeast. Certain Smc1 and Smc3 mutations reduce the ability of a boundary that flanks the HMR silent mating-type locus to block the spread of gene-silencing Sir protein complexes, and Scc1 associates with this boundary. It has also been proposed that Cohesin binding sites are boundaries that control the extent of chromosome loop formation by Condensin. This proposal is based in part on the observation that Cohesin is needed to reestablish chromosome condensation upon returning temperature-sensitive Condensin mutants to the permissive temperature. In Drosophila, the gypsy insulator partially blocks the negative effects of heterochromatin on the expression of a euchromatic gene, suggesting that it has boundary activity, and in yeast, the Su(Hw) protein that binds the gypsy insulator also blocks the spread of gene-silencing complexes. If SA/Scc3 or Cohesin increases insulation by gypsy, Nipped-B could facilitate activation by reducing their association with the insulator (Rollins, 2004).

A more general version of the 'Cohesin insulator' model is preferred, in which native Cohesin binding sites in the 85-kb region separating the wing margin enhancer from the cut promoter act as insulators and impede the formation of structures needed to bring the wing margin enhancer close to the promoter. In yeast, Cohesin binds every 10 kb or so along the chromosomes. The spacing of Cohesin in Drosophila has not been investigated, but multiple complexes could bind in the 85-kb interval between the wing margin enhancer and the cut promoter. Assuming that Nipped-B, perhaps by opening the Cohesin ring, facilitates both the loading and the removal of Cohesin from chromosomes, could explain how Nipped-B facilitates the activation of wild-type cut. By opening the Cohesin ring, Nipped-B would help achieve equilibrium between the bound and unbound states by providing opportunities to load or remove Cohesin from chromosomes. This mechanism would be distinct from proteolytic removal of Cohesin by separase at the metaphase-to-anaphase transition but could be involved in the removal of Cohesin from chromosome arms in prophase. In heterozygous Nipped-B mutants, which retain substantial Nipped-B activity, the equilibrium endpoint would not be altered, but it might take longer to achieve equilibrium. Thus, reduced Cohesin binding to chromosomes would not be expected, but the lower Nipped-B levels would reduce the windows of opportunity for removal of Cohesin needed to allow long-range activation. This model also predicts that Nipped-B does not have to stably associate with chromosomes, which could explain why no chromosomally bound Nipped-B was detected by immunostaining (Rollins, 2004).

Finally, in a simple indirect model it could be supposed that, similar to its role in loading Cohesin, Nipped-B could also facilitate chromosomal binding of another protein complex that assists long-range enhancer-promoter interactions. In this case, there would be competition between Cohesin and the long-range activation complex for Nipped-B, and reduction of Cohesin would make Nipped-B more available to facilitate long-range activation. This and the insulator model described above are not mutually exclusive, but both explain how Nipped-B cooperates with Cohesin in sister chromatid cohesion but opposes the effect of Cohesin proteins on cut expression (Rollins, 2004).

Effects of sister chromatid cohesion proteins on cut gene expression during wing development in Drosophila

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. This study tests 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. Cohesin binds between a remote wing margin enhancer and the promoter at the cut locus in cultured cells, and reducing the dosage of the Smc1 cohesin subunit increases cut expression in the developing wing margin. cut expression is increased by a unique pds5 gene mutation (see CG17509) that reduces the binding of cohesin to chromosomes. On the basis of these results, it is posited 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 (Dorsett, 2005).

To identify general facilitators of enhancer-promoter communication, genetic screens were conducted to isolate factors that support activation of the cut gene by a wing margin-specific enhancer located 85 kbp upstream of the promoter. The region between this enhancer and the promoter contains many enhancers that activate cut in specific tissues during embryogenesis and larval development. In addition to tissue-specific activators that bind to the wing margin enhancer, these screens identified two proteins, Chip and Nipped-B, that are expressed in virtually all cells, and facilitate the expression of diverse genes. Chip interacts with many DNA-binding proteins, and likely supports the cooperative binding of proteins to enhancers and to sites between enhancers and promoters (Dorsett, 2005).

Nipped-B functions by a different mechanism. Unlike other cut regulators, Nipped-B is more limiting for cut expression when enhancer-promoter communication is partially compromised by a weak gypsy insulator than it is when the enhancer is partially inactivated by a small deletion, leading to the idea that Nipped-B specifically facilitates enhancer-promoter communication (Rollins, 1999). Nipped-B homologs in Saccharomyces cerevisiae, S. pombe and Xenopus (Scc2, Mis4 and Xscc2), known collectively as adherins, load the cohesin protein complex onto chromosomes (Ciosk, 2000; Tomonaga, 2000; Gillespie, 2004; Takahashi, 2004; reviewed by Dorsett, 2004), Nipped-B is required for sister chromatid cohesion, and thus is a functional adherin (Rollins, 2004). The fact that Nipped-B is an adherin raises the critical question, addressed here, of whether or not cohesin plays a role in enhancer-promoter communication. In all metazoans examined, cohesin loading starts in late anaphase, and it is not removed from the chromosome arms until prophase. Cohesin, therefore, is a structural component of chromosomes during interphase, when gene expression occurs (Dorsett, 2005).

Cohesin consists of two Smc proteins, Smc1 and Smc3, and two accessory subunits, Rad21 (Mcd1/Scc1) and Stromalin (Scc3/SA). Cohesin forms a ring-like structure. One idea is that adherins, such as Nipped-B, temporarily open the ring and allow it to encircle the chromosome (Arumugam, 2003). It is proposed that cohesin encircles both sister chromatids after DNA replication to establish cohesion. Cohesin binds every 10 kbp or so along the chromosome arms in yeast. If it binds at a similar density in metazoans, it could potentially affect the expression of many genes (Dorsett, 2005).

Determining if the effects of Nipped-B on gene expression are mediated through cohesin is pertinent to Cornelia de Lange syndrome (CdLS, OMIM #122470), which is caused by heterozygous loss-of-function mutations in the human homolog of Nipped-B, Nipped-B-Like (NIPBL, GenBank Accession Number NM_133433 (Krantz, 2004; Tonkin, 2004). CdLS results in numerous birth defects, including slow physical and mental growth, upper limb deformities, gastroesophageal and cardiac abnormalities. These developmental deficits likely reflect changes in gene expression similar to those caused by heterozygous Nipped-B mutations (Dorsett, 2005).

This study examines binding of cohesin to the cut gene, and the effects that the Pds5 sister chromatid cohesion factor has on cut expression and cohesin binding to chromosomes. The results are consistent with the idea that cohesin inhibits the activation of cut by the wing margin enhancer (Dorsett, 2005).

RNAi experiments have shown that slightly reducing the Stromalin (Scc3) and Rad21 (Mcd1/Scc1) subunits of cohesin increased expression of the cut gene in the developing wing margin (Rollins, 2004). To determine if the Smc1 cohesin subunit plays a similar role, a null allele of the smc1 gene was generated by excision of a viable P transposon insertion near the transcription start site. The smc1^exc46 allele is recessive lethal and chromosome squashes show precocious sister chromatid separation (Dorsett, 2005).

The effects of smc1^exc46 on the mutant phenotype displayed by the ct^K gypsy transposon insertion allele of cut were used to determine changes in cut expression. The ct^K gypsy insulator partially blocks activation of cut by the wing margin enhancer, causing a scalloped wing phenotype sensitive to the dosage of factors that regulate cut (Gause, 2001; Rollins, 2004). A decrease in cut expression increases nicks in the wing margin, and an increase in expression leads to fewer nicks. The wing-nicking assay is a highly specific and sensitive measure of the activation of cut by the wing margin enhancer, in the developing margin cells of the wing discs during the 24-hour period centered around pupariation (Dorsett, 2005).

In repeated experiments, the heterozygous smc1^exc46 mutation reduced the number of ct^K wing margin nicks relative to the number observed with the heterozygous parental chromosome. The difference was significant. It is concluded that, similar to the effects of reducing the Stromalin (Scc3) and Rad21 (Mcd1/Scc1) cohesin subunits, reducing the levels of the Smc1 subunit increases cut expression. Because all three cohesin subunits have a similar effect, it is concluded that the cohesin complex inhibits cut expression (Dorsett, 2005).

Mutations in genes that modulate cohesin activity for effects on cut expression were tested. The separation anxiety (san) and deco (eco, as listed in FlyBase) genes encode putative acetyltransferase proteins that are required for sister chromatid cohesion and the association of cohesin with centromeric regions (Williams, 2003). Separase (Sse) encodes a protease that cleaves cohesin to permit sister chromatid separation. Mutations in these genes did not have significant effects on the ct^K mutant phenotype. It is possible that heterozygosity for these mutations do not sufficiently alter cohesin activity to change cut expression. Alternatively, these proteins may affect cohesin only at the centromere (Dorsett, 2005).

If cohesin directly regulates cut, it would be expected to bind to the cut locus. Salivary gland polytene chromosomes were immunostained with anti-Smc1 and anti-Stromalin (Scc3) to determine whether cohesin binds to cut. In wild type, Stromalin and Smc1 co-localize on polytene chromosomes as expected. Distinct regions of cohesin staining were seen in both bands and interbands. It is concluded that cohesin binds many sites along all chromosome arms. Staining was observed in some chromosomal puffs, suggesting that cohesin associates with transcribed loci. To test this, it was determined whether cohesin binds to heat-shock puffs. After 20 minutes of heat shock at 37°C, cohesin staining was observed in the 93D puff, but not in the others. Thus, cohesin localization does not correlate with transcription (Dorsett, 2005).

The examination of several nuclei showed that the chromosome band containing the cut locus (7B3-4) consistently displayed cohesin staining. This band contains 150 kbp of DNA, and four genes other than cut, in the regulatory region between the wing margin enhancer and the cut promoter. At least three of these genes are testis specific. Salivary glands do not express cut, but because cohesin is a constitutive chromosomal component, and probably binds to cut in most cells, these data are consistent with the view that the effects of cohesin on cut expression in the wing margin are direct (Dorsett, 2005).

Further support is provided by chromatin immunoprecipitation experiments, which show that cohesin binds to the regulatory region of cut in Drosophila cultured Kc cells of embryonic origin. An 85-kbp region was examined encompassing the wing margin enhancer and the promoter; four cohesin-binding sites were detected. Two binding sites were centered 0.5- and 4-kbp upstream of the promoter, one was centered about 30.5-kbp upstream of the promoter, and another small broad peak was 68-kbp upstream of the promoter. The same sites were seen with both antisera, and neither pre-immune serum showed enrichment of any sequences. Thus, in addition to the non-dividing polytene salivary cells, cohesin also binds cut in predominantly diploid dividing cells of embryonic origin. Based on the assumption that cohesin is a constitutive chromosomal component, and the finding that it binds to the cut locus in two very different cell types, it is posited that cohesin also binds cut in the developing wing margin cells and that the effects of cohesin on cut expression in the developing wing are direct (Dorsett, 2005).

The possibility was considered that other factors recruited by cohesin could inhibit cut activation. In fungi, the Pds5 (Spo76) protein is required for sister chromatid cohesion. Pds5 associates with cohesin sites on chromosomes, and requires cohesin for association (Dorsett, 2005 and references therein).

By sequence analysis, the CG17509 gene was identified as the likely pds5 homolog. The P{EPgy2}CG17509^EY06473 transposon insertion in the first exon is homozygous viable. It was mobilized to generate two recessive lethal mutations, pds5^e3 and pds5^e6, that fail to complement each other, and a deletion of the region [Df(2R)BSC39]. Both homozygous mutants and the heteroallelic combination are lethal in late third instar to early pupal stages of development. Late third instar larvae of both mutants display small or missing imaginal discs, and the larval brains are approximately half the volume of wild type, consistent with a mitotic defect (Dorsett, 2005).

Neuroblast metaphase nuclei from mutant third instar larvae were examined for cohesion defects. Despite examining more than 30 metaphases from each, no normal metaphases were found in the pds5 mutants. Nearly all displayed aneuploidy, and most displayed precocious sister chromatid separation. By contrast, 15.4% of the pds5^e3/+ heterozygote metaphases showed aneuploidy and 12.8% showed sister chromatid separation, similar to the frequencies observed with wild-type neuroblasts. It is concluded that the pds5^e3 and pds5^e6 mutations affect chromosome segregation and sister chromatid cohesion, and that CG17509 encodes a functional Pds5 homolog (Dorsett, 2005).

The effect of the pds5 mutations on the ct^K phenotype was tested relative to the viable P element insertion used to generate them. Unexpectedly, the two mutations had different effects. The pds5^e3 mutation slightly increased the number of wing margin nicks, indicating that it decreased cut expression, whereas pds5^e6 increased cut expression. Although a small increase in wing margin nicks was consistently observed with pds5^e3, the wing nicking was not significantly different from that seen with the parental chromosome. The decreased nicking associated with the pds5^e6 allele, however, was significantly different from that of the parental chromosome. It is concluded that the pds5^e6 mutation dominantly increases cut expression, and that the pds5^e3 mutation may cause a small decrease (Dorsett, 2005).

pds5 expression was examined in the two pds5 mutants to determine why they have different effects on cut expression. Northern blots revealed a pds5 transcript of the expected size (4.6 kb) in embryos prior to zygotic gene expression, indicating that it is maternal. The transcript was present at 10- to 25-fold lower levels in larvae. To avoid detecting maternal pds5 mRNA, the transcripts produced in pds5^e3 and pds5^e6 second instar larvae were examined. No pds5 transcripts were detected in homozygous pds5^e3 mutants, but a shorter transcript (3.65 kb) was seen at levels similar to those of wild type in both heterozygous and homozygous pds5^e6 mutants. A wild-type sized transcript was present in heterozygous pds5^e6 mutants, but was undetectable in homozygotes. The viable parental P insertion line used to generate both lethal pds5 alleles produced wild-type levels of a wild-type sized transcript (Dorsett, 2005).

The Northern blots indicate that pds5^e3 is a null allele, and PCR analysis of mutant genomic DNA revealed that sequences starting at the P insertion site and extending upstream of the transcription start site are missing. Thus, a reduction in Pds5 dosage slightly decreases cut expression. It is concluded, therefore, that wild-type Pds5 does not contribute to the inhibition of cut expression by cohesin, but may slightly decrease the inhibitory effect (Dorsett, 2005).

The presence of a new transcript in the pds5^e6 mutant suggested that it could produce a mutant protein lacking an activity crucial for sister chromatid cohesion that somehow interferes with the inhibition of cut by cohesin. PCR analysis of pds5^e6 genomic DNA revealed that the region from the P insertion site through exon 5 is missing. 5' RACE analysis of the pds5^e6 transcript shows that it starts 67 nucleotides upstream of the wild-type start site predicted by EST analysis. The pds5^e6 transcript extends from the start site to the P insertion site. The next 17 nucleotides are from the end of the P insertion, followed by 12 nucleotides of internal P sequence fused to the pds5 sequence 11 nucleotides downstream of the exon 6 5' splice site. The exon 6 sequences present in the mutant transcript contain six in-frame AUG codons, two of which match a consensus (RNVATGR) for Drosophila translation initiation sites. Thus the pds5^e6 mutant transcript encodes a protein lacking the N terminus (Dorsett, 2005).

The effects of the two pds5 mutations on cut expression correlate with a difference in cohesin binding to chromosomes. Although the salivary glands of the homozygous pds5 mutants are substantially reduced, polytene chromosomes were obtained from both. Morphology was altered enough to make it difficult to identify specific loci. Nevertheless, individual chromosome tips could be identified, and developmental puffs, including the puff at 2B, were present in both mutants, indicating that the chromosomes are transcribed. In size-matched third instars, the pds5^e3 mutant chromosomes were thicker than wild type, and the pds5^e6 mutant chromosomes were thinner. The pds5^e3 null allele did not reduce staining for Smc1 or Stromalin, although the pattern appeared less discrete. By contrast, pds5^e6 mutant chromosomes showed strongly reduced staining for Smc1 and Stromalin. Loss of cohesin staining was observed in multiple nuclei from multiple pds5^e6 salivary glands. These results indicate that the pds5^e6 mutant either blocks loading of cohesin onto chromosomes, or facilitates removal. The reduction of cohesin binding caused by pds5^e6, which dominantly increases cut expression, is consistent with the hypothesis that cohesin inhibits cut expression (Dorsett, 2005).

The demonstration that cohesin binds to the cut locus in polytene chromosomes, and to multiple sites between the remote wing margin enhancer and the promoter in cultured cells, supports the hypothesis that the effects of cohesin on cut expression in the wing margin are direct. The wing margin enhancer does not activate cut in salivary glands or Kc cells, but it is not technically feasible to examine the association of cohesin with cut in the developing margin cells in which the wing margin enhancer functions. Based on the association of cohesin with cut in two diverse cell types, it is posited that cohesin also binds cut in the developing wing margin cells, and inhibits activation by the wing margin enhancer. Such a direct effect of cohesin could explain why small reductions (<20%) in cohesin subunits induced by RNAi have detectable effects on cut expression (Dorsett, 2005).

Both pds5 mutations tested cause similar sister chromatid cohesion defects, but only pds5^e6 reduces the binding of cohesin to chromosomes and increases cut expression. This provides additional evidence that binding of cohesin to chromosomes is required for it to inhibit cut activation, and also shows that Pds5 itself is not required to inhibit gene expression (Dorsett, 2005).

The negative effects of cohesin on cut expression raise the possibility that cohesin contributes to the silencing of euchromatic genes placed in heterochromatin. Cohesin binds more densely in centromeric heterochromatin in yeasts and metazoans, and, in S. pombe, heterochromatin proteins recruit cohesin. Moreover, RNAi-mediated silencing of a non-centromeric gene in S. pombe causes the recruitment of heterochromatin proteins and cohesin to the silenced gene (Dorsett, 2005).

The idea is favored that cohesin inhibits enhancer-promoter communication in cut. This idea originates from the allele-specific effects of Nipped-B mutations on cut expression. The known cut regulators required for activation by the wing margin enhancer, including scalloped, vestigial, mastermind, Chip, Nipped-A and l(2)41Af, all display different cut allele specificities than Nipped-B. In contrast to these factors, Nipped-B is most limiting when enhancer-promoter communication is partially compromised by a weak gypsy insulator, suggesting that Nipped-B facilitates long-range communication (Dorsett, 2005).

Binding of cohesin to multiple sites between the wing margin enhancer and the cut promoter is consistent with the hypothesis that Nipped-B facilitates enhancer-promoter communication by regulating the binding of cohesin to chromosomes. To explain how Nipped-B aids activation, it is theorized that Nipped-B can remove cohesin from chromosomes. In a simple model, Nipped-B facilitates the cohesin-binding equilibrium. When Nipped-B is partially reduced but not abolished, it takes longer to achieve equilibrium, but the extent of cohesin binding is not altered and there is little effect on sister chromatid cohesion. The reduced cohesin on-off rates, however, would diminish the opportunities for gene activation that require cohesin removal or repositioning (Dorsett, 2005).

It is not known how cohesin inhibits long-range activation, but mechanisms can be envisioned for various long-range activation models. For example, cohesin could inhibit the folding or looping of the chromosome that is required to bring the enhancer into contact with the promoter. Alternatively, cohesin could block a Chip-mediated spread of protein binding between the enhancer and promoter in linking models for long-range activation, or block the transfer or tracking of RNA polymerase from the enhancer to the promoter, as appears to occur in the chicken beta-globin gene locus (Dorsett, 2005).

This study found that Drosophila Pds5 is required for sister chromatid cohesion, consistent with studies on fungal Pds5. To explain the effects of the pds5^e6 mutation on cohesin chromosome binding and cut expression, it is proposed that it produces a mutant protein that blocks cohesin binding, or causes cohesin to be released from chromosomes. This agrees with observations on vertebrate and S. pombe Pds5 suggesting that Pds5 has both positive and negative effects on cohesion, possibly by regulating the association of cohesin with chromosomes (Dorsett, 2005).

Vertebrates contain two Pds5 isoforms that associate with chromosomal cohesin. Reduction of Pds5 partially decreases sister chromatid cohesion. Consistent with the finding that the pds5^e3 null mutation does not reduce the binding of cohesin to polytene chromosomes, and with previous work on S. cerevisiae and S. pombe Pds5, Xenopus Pds5 is not required for the binding of cohesin to chromosomes. One report suggested that S. cerevisiae Pds5 is required for the association of cohesin with chromosomes, but it is possible that this discrepancy might be caused by differences in the mutant alleles, similar to the differences found between pds5^e3 and pds5^e6 (Dorsett, 2005 and references therein).

Depletion of Pds5 from Xenopus extracts increases the amount of cohesin associated with chromatin. A similar increase in cohesin binding could explain the slight decrease in cut expression caused by the pds5^e3 null allele. Consistent with the idea that wild-type Pds5 partially reduces cohesin binding, deletion of S. pombe pds5 partially suppresses a temperature-sensitive mutation in mis4, which encodes the homolog of the Nipped-B and Scc2 cohesin-loading factors (Dorsett, 2005 and references therein).

Because wild-type Pds5 appears to partially reduce the binding of cohesin to chromosomes, it is speculated that the pds5^e6 mutation increases this activity, which may be related to the cohesin-loading function of Nipped-B/Scc2. Scc2 interacts with cohesin, and is thought to open the cohesin ring. In synchronized yeast cells, cohesin loads at Scc2-binding sites and translocates away. Like Nipped-B/Scc2, Pds5 contains several HEAT repeats, and thus might also open the cohesin ring during DNA replication to allow it to encompass both sister chromatids. It could play a similar role in the snap model, in which cohesin complexes bound to the two sisters interlock to hold the sisters together. If the pds5^e6 mutant protein interacts with cohesin non-productively, it could block access to Nipped-B and prevent loading. Alternatively, when the mutant Pds5 attempts to establish cohesion, it might fail, releasing cohesin from the chromosome. Wild-type Pds5 might partially reduce cohesin binding by competing with Nipped-B for cohesin, or by occasionally failing to establish cohesion (Dorsett, 2005).

The effects of cohesin on cut expression are likely pertinent to the etiology of Cornelia de Lange syndrome (CdLS). CdLS is caused by mutations in the Nipped-B-Like (NIPBL) human homolog of Nipped-B. Most missense mutations that cause CdLS affect residues conserved in Nipped-B. CdLS is characterized by several physical and mental deficits, including slow growth, mental retardation, and upper limb, gastroesophageal and cardiac deformities. Heterozygous loss-of-function NIPBL mutations cause CdLS, and thus the developmental changes likely reflect gene expression effects similar to those caused by heterozygous Nipped-B mutations. At least some birth defects in CdLS, such as limb truncations and cardiac abnormalities, could be caused by changes in expression of the known homeotic genes. The observations presented here indicate that cohesin likely plays a role in CdLS by inhibiting the long-range gene control of homeotic genes (Dorsett, 2005).

The possibility that some developmental changes in CdLS reflect reduced sister chromatid cohesion cannot be ruled out. A recent study found evidence for cohesion deficits in 41% of CdLS patients compared with 9% of controls. Also, the autosomal recessive Roberts syndrome has some similarities to CdLS, and is caused by mutations in a human homolog of the Eco1/Eso1/Deco cohesion factor. Cells from Roberts patients display defects in sister chromatid cohesion. Homozygous Drosophila deco¹ mutants appear to affect cohesin binding only at centromeric regions, and, as described above, dominant effects of deco mutations on cut gene expression are not seen, leading to the the idea that most CdLS developmental deficits reflect changes in gene expression instead of in sister chromatid cohesion (Dorsett, 2005).

Search PubMed for articles about Drosophila Nipped-B

Anderson, D. E., Losada, A., Erickson, H. P. and Hirano, T. (2002). Condensin and cohesin display different arm conformations with characteristic hinge angles. J. Cell Biol. 156: 419-424. Medline abstract: 11815634

Arumugam, P., Gruber, S., Tanaka, K., Haering, C. H., Mechtler, K. and Nasmyth, K. (2003). ATP hydrolysis is required for cohesin's association with chromosomes. Curr. Biol. 13: 1941-1953. 14614819

Ciosk, R., Shirayama, M., Shevchenko, A., Tanaka, T., Toth, A., Shevchenko, A. and Nasmyth, K. (2000). Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol. Cell 5: 243-254. 10882066

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date revised: 5 February 2008

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