InteractiveFly: GeneBrief

Nipped-B: Biological Overview | References


Gene name - Nipped-B

Synonyms -

Cytological map position-2R

Function - transcription factor

Keywords - mitosis, regulation of sister chromatid cohesion, regulation of gene expression by controlling cohesin dynamics

Symbol - Nipped-B

FlyBase ID: FBgn0026401

Genetic map position 2R: 579,193..616,515 [-] -

Classification - chromosomal adherins

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Wu, Y., Gause, M., Xu, D., Misulovin, Z., Schaaf, C. A., Mosarla, R. C., Mannino, E., Shannon, M., Jones, E., Shi, M., Chen, W. F., Katz, O. L., Sehgal, A., Jongens, T. A., Krantz, I. D. and Dorsett, D. (2015). Drosophila Nipped-B mutants model Cornelia de Lange Syndrome in growth and behavior. PLoS Genet 11: e1005655. PubMed ID: 26544867
Summary:
Individuals with Cornelia de Lange Syndrome (CdLS) display diverse developmental deficits, including slow growth, multiple limb and organ abnormalities, and intellectual disabilities. Severely-affected individuals most often have dominant loss-of-function mutations in the Nipped-B-Like (NIPBL) gene, and milder cases often have missense or in-frame deletion mutations in genes encoding subunits of the cohesin complex. Cohesin mediates sister chromatid cohesion to facilitate accurate chromosome segregation, and NIPBL is required for cohesin to bind to chromosomes. Individuals with CdLS, however, do not display overt cohesion or segregation defects. Rather, studies in human cells and model organisms indicate that modest decreases in NIPBL and cohesin activity alter the transcription of many genes that regulate growth and development. Sister chromatid cohesion factors, including the Nipped-B ortholog of human NIPBL, are also critical for gene expression and development in Drosophila melanogaster. This study describes how a modest reduction in Nipped-B activity alters growth and neurological function in Drosophila. These studies reveal that Nipped-B heterozygous mutant Drosophila show reduced growth, learning, and memory, and altered circadian rhythms. Importantly, the growth deficits are not caused by changes in systemic growth controls, but reductions in cell number and size attributable in part to reduced expression of myc (diminutive) and other growth control genes. The learning, memory and circadian deficits are accompanied by morphological abnormalities in brain structure. These studies confirm that Drosophila Nipped-B mutants provide a useful model for understanding CdLS, and provide new insights into the origins of birth defects.
Swain, A., Misulovin, Z., Pherson, M., Gause, M., Mihindukulasuriya, K., Rickels, R. A., Shilatifard, A. and Dorsett, D. (2016). Drosophila TDP-43 RNA-binding protein facilitates association of sister chromatid cohesion proteins with genes, enhancers and polycomb response elements. PLoS Genet 12: e1006331. PubMed ID: 27662615
Summary:
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. This study shows 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.

BIOLOGICAL OVERVIEW

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).

Drosophila Nipped-B mutants model Cornelia de Lange syndrome in growth and behavior

Individuals with Cornelia de Lange Syndrome (CdLS) display diverse developmental deficits, including slow growth, multiple limb and organ abnormalities, and intellectual disabilities. Severely-affected individuals most often have dominant loss-of-function mutations in the Nipped-B-Like (NIPBL) gene, and milder cases often have missense or in-frame deletion mutations in genes encoding subunits of the cohesin complex. Cohesin mediates sister chromatid cohesion to facilitate accurate chromosome segregation, and NIPBL is required for cohesin to bind to chromosomes. Individuals with CdLS, however, do not display overt cohesion or segregation defects. Rather, studies in human cells and model organisms indicate that modest decreases in NIPBL and cohesin activity alter the transcription of many genes that regulate growth and development. Sister chromatid cohesion factors, including the Nipped-B ortholog of NIPBL, are also critical for gene expression and development in Drosophila melanogaster. This study describes how a modest reduction in Nipped-B activity alters growth and neurological function in Drosophila. These studies reveal that Nipped-B heterozygous mutant Drosophila show reduced growth, learning, and memory, and altered circadian rhythms. Importantly, the growth deficits are not caused by changes in systemic growth controls, but reductions in cell number and size attributable in part to reduced expression of myc (diminutive) and other growth control genes. The learning, memory and circadian deficits are accompanied by morphological abnormalities in brain structure. These studies confirm that Drosophila Nipped-B mutants provide a useful model for understanding CdLS, and provide new insights into the origins of birth defects (Wu, 2015).

Prior studies have revealed that Nipped-B heterozygous mutant Drosophila display subtle and latent external morphological phenotypes that become overt in adults only when combined with mutations in key developmental regulatory genes such as cut, Ultrabithorax, Notch, mastermind, hedgehog, and genes encoding cohesin or Polycomb silencing complex subunits. This contrasts with mice and humans, where similar deficiencies in NIPBL cause multiple specific and obvious morphological changes. There are also differences between mice and humans. For instance, limb abnormalities are largely absent in Nipbl(+/-) mice, but heart defects are significantly more frequent than in individuals with CdLS. Extrapolating from Drosophila genetic interaction data, this study posits that the individual physical birth defects in vertebrates stem from altered expression of specific sets of developmental genes, and that the variability between individuals with CdLS reflects differences in genetic background (Wu, 2015).

Although the external morphological changes in Nipped-B mutant Drosophila are minimal in an otherwise wild-type background, they share the reduced size with Nipbl(+/-) mice and CdLS. The data argue that the reduced size reflects decreases in both total cell number and size, and not changes in the systemic control of growth that sense critical body mass, deficits in the utilization of nutrition, or increased cell death. The decrease in size likely stems in part from modestly reduced expression of the myc (dm), Tor, InR and other genes that promote cell proliferation, division and growth, and not increased apoptosis. Indeed, one of the most intriguing findings is the reduced ability of excess dm expression to induce apoptosis in Nipped-B heterozygous mutants. This may stem in part from reduced function of a Dm-dependent enhancer that drives expression of the grim and reaper pro-apoptosis genes. This region binds Nipped-B and cohesin in wing discs, and deletion of this region permits excess dm expression using the tub-myc driver to increase wing size more than in wild-type flies because it reduced apoptosis (Wu, 2015).

It remains to be seen to what extent these findings in Drosophila might explain the reduced growth in CdLS and in Nipbl(+/-) mice. It seems likely that similar mechanisms at least make a significant contribution to the reduced growth in mammals because Nipbl(+/-) mice and cells from individuals with CdLS also show reduced c-myc expression. Organisms use a variety of mechanisms to sense body size and regulate growth and developmental transitions. Drosophila transitions are timed by pulses of the ecdysone steroid hormone produced by the prothoracic gland located between the two lobes of the brain. Specific neurons in the brain secrete a peptide hormone, prothoracicotropic hormone (PTTH) that stimulates ecdysone production. Insulin signaling, nutrition and many other factors, which are not all well understood, control the hormonal pathways and timing of the ecdysone pulses that determine absolute body size. For example, nutrient deprivation substantially delays the ecdysone pulses, but not enough to fully restore maximal body size. What is particularly striking is how close Nipped-B mutants are to wild type in their developmental timing and in how their developmental staging responds to nutrient deprivation. This argues that the systemic hormonal pathways that regulate body size and hormonal pulses that induce developmental stages are largely unaffected, and that the reduced size of Nipped-B mutants stems primarily from a small but significant reduction in the number of cell divisions, and in the mechanisms that determine final cell size (Wu, 2015).

It is more difficult to precisely time the developmental staging in mice and humans than in Drosophila, and thus to determine whether or not developmental timing or systemic growth pathways are significantly altered with decreased NIPBL function. Although some individuals with CdLS show slightly delayed puberty, puberty occurs at the normal age in many. The slight delays, and incomplete pubertal changes might all be attributed to causes other than a general developmental delay, such as structural abnormalities or changes in hormone levels. The relatively normal timing of puberty, therefore seems to indicates that the more extreme reductions in overall size observed in CdLS are also more likely to stem from changes in cell number and size than changes in systemic body size controls (Wu, 2015).

It was also found that Drosophila Nipped-B heterozygotes display many behavioral and neurological features resembling those seen in CdLS patients: they are deficient in learning and short-term memory, and display disruptive sleep patterns and abnormal circadian rhythms. Intellectual disability is the most common clinical phenotype seen in individuals with CdLS. The average IQ score of typical CdLS cases, mostly with NIPBL mutations, is 53 (range 30–86). It was found that although Nipped-B heterozygous mutant flies are capable of learning, their learning capacity is significantly lower than that of the wiso31 controls. Furthermore, these Nipped-B mutants are accompanied by pleiotropic structural abnormalities in mushroom bodies, the major brain structures controlling learning and memory. While it is conceivable that the morphological defects in the mushroom bodies could contribute to the learning and memory deficits observed in the Nipped-B mutants, how these structural and functional deficiencies correlate with each other warrant further detailed studies (Wu, 2015).

A striking similarity was observed in the sleep patterns of fly Nipped-B heterozygotes and those seen in CdLS patients. Sleep disturbances are common in CdLS and seen in up to 55% of CdLS individuals. Insomnia (difficulty in initiating sleep), difficulty staying asleep, frequent night wakenings, and sleepiness during the daytime are the most common sleep problems reported in CdLS. Frequent but dramatically shortened sleep episodes was the characteristic sleep pattern observed in Nipped-B heterozygote flies. In fact, the reduced length of sleep episodes lead to a dramatic loss in daytime and nighttime sleep, which was unable to be compensated for by significant increases in the number of sleep bouts (Wu, 2015).

While it has been speculated that sleep disturbances in CdLS individuals may be in part attributable to a circadian rhythm disorder, strict and objective studies to confirm this suggestion have not been undertaken. The locomotor activity-based circadian rhythm assay is a well-established measure of circadian rhythm in Drosophila. It was demonstrated that an aberrant circadian rhythm exists in a large fraction of Nipped-B haploinsufficient male and female flies. For Nipped-B mutants that still display rhythmic circadian patterns, their free-running activity rhythms are maintained at around 24 hours, similar to the periodicity of wiso31 controls. A remarkable consistency between the circadian defects and sleep aberrance was observed in these Nipped-B heterozygous mutants; flies that were profoundly arrhythmic were the ones that showed the most disturbed sleep patterns. Nevertheless, the circadian rhythm alterations are more apparent than the sleep disturbances in males, suggesting that additional sex-specific factors are involved in determining the sleep patterns. Taken together, these data suggest that at least in fly Nipped-B mutants, intrinsic circadian rhythm defects likely contribute to their aberrant sleep patterns (Wu, 2015).

Overall, this study on fly Nipped-B mutants demonstrates a strikingly analogous growth and neurocognitive/behavioral phenotype between heterozygous Nipped-B mutants and human CdLS individuals, including small body size, learning and memory deficits, disruptive sleep patterns and circadian rhythm defects. Drosophila Nipped-B heterozygotes are thus a valuable resource, with multiple objective and readily measureable metrics, for modeling human CdLS. Studies on the function of Nipped-B and cohesin components in CdLS patient cell lines, Nipbl(+/-) mouse and zebrafish models, and Drosophila have contributed substantially to our understanding of the roles of Nipped-B and cohesin components in development and gene regulation. The presence of a sophisticated genetic tool kit and economical availability of fruit flies will make it possible to explore developmental deficits in a tissue- and stage-specific manner, as well as to test their relevance to human development and the pathogenesis of CdLS. Drosophila is an ideal model organism to address these issues, given its short life cycle, lower degree of genomic redundancy and the available genetic tools. Nipped-B mutants can also be utilized to search for and test new pharmacologic therapeutic modalities, towards amelioration of the growth and neurodevelopmental functioning of individuals with CdLS (Wu, 2015).

Cohesin, gene expression and development: lessons from Drosophila: A review

The cohesin complex (see The cohesin complex and regulatory factors), 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 (Dorsett, 2009; full text of article).

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-BNC71 and Nipped-BNC78 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-BNC71 and Nipped-BNC78 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-BNC71 and Nipped-BNC78 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-BNC71 and Nipped-BNC78 affect HEAT repeats 2 and 3, respectively. Indeed, Nipped-BNC71 and Nipped-BNC78 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-BNC71 and Nipped-BNC78. The structural similarity between Nipped-BNC71 and Nipped-BNC78 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).

The Drosophila cohesin subunit Rad21 is a trithorax group (trxG) protein

The cohesin complex is a key player in regulating cell division. Cohesin proteins SMC1, SMC3, Rad21, and stromalin (SA), along with associated proteins Nipped-B, Pds5, and EcoI, maintain sister chromatid cohesion before segregation to daughter cells during anaphase. Recent chromatin immunoprecipitation (ChIP) data reveal extensive overlap of Nipped-B and cohesin components with RNA polymerase II binding at active genes in Drosophila. These and other data strongly suggest a role for cohesion in transcription; however, there is no clear evidence for any specific mechanisms by which cohesin and associated proteins regulate transcription. This study reports a link between cohesin components and trithorax group (trxG) function, thus implicating these proteins in transcription activation and/or elongation. The Drosophila Rad21 protein is encoded by verthandi (vtd), a member of the trxG gene family that is also involved in regulating the hedgehog (hh) gene. In addition, mutations in the associated protein Nipped-B show similar trxG activity i.e., like vtd, they act as dominant suppressors of Pc and hhMrt without impairing cell division. These results provide a framework to further investigate how cohesin and associated components might regulate transcription (Hallson, 2008).

In eukaryotic mitosis, accurate chromosome segregation requires paired sister chromatids to attach to opposite spindle poles. Sister chromatids are held together by the cohesin protein complex, which consists of four core subunits, Rad21/SCC1, stromalin (SA) and structural maintenance of chromosome (SMC) proteins SMC1 and SMC3. A widely accepted model postulates that cohesin forms a ring-like structure via interaction of the N- and C-termini of Rad21 with a SMC1/SMC3 heterodimer. With the participation of SCC2/Nipped-B, SCC4, EcoI/Ctf7, and Pds5 proteins, sister-chromatid cohesion is maintained until the onset of mitosis. Cleavage of Rad21 and the resulting removal of cohesin then allow separation of sister chromatids in anaphase. Mutation of genes encoding these subunits leads to errors in chromosome segregation and aneuploidy, which are hallmarks of cancer and a leading cause of birth defects in humans (Hallson, 2008).

Given the highly conserved role for cohesin in sister chromatid cohesion, it was unexpected to discover that cohesin and associated proteins might also play a distinct, independent role in regulating gene expression. Reduction in Nipped-B expression in Drosophila affects expression of the cut and Ultrabithorax genes, and mutations in the human orthologue, NIPBL, result in Cornelia de Lange Syndrome. In zebrafish, mutations in rad21 or Smc3 affect embryonic runx gene transcription in heterozygous mutant animals without compromising cell division, suggesting that these proteins may have functions in transcription that are distinct from a mitotic role. Recently, extensive overlap has been found of Nipped-B and cohesin components with RNA polymerase II binding at active genes and apparent exclusion from genes silenced by Polycomb group (PcG) genes. This intriguing chromatin immunoprecipitation (ChIP) result strongly suggests a role in transcription for cohesin and Nipped-B, although the mechanisms are unknown (Hallson, 2008).

Trithorax group (trxG) genes encode proteins implicated in transcriptional regulation. These genes were initially characterized as regulators of homeotic genes in Drosophila. The trxG genes are required to maintain activation of homeotic and other genes; many that have been molecularly characterized encode members of multimeric complexes with roles in transcriptional initiation and/or elongation. Typically, mutations in trxG genes suppress the phenotypes of mutations in PcG genes, whose function is to maintain the repressed state of homeotic genes and other developmentally important genes like hedgehog (hh), a gene required for cell signaling (Hallson, 2008).

As part of work toward a functional annotation of heterochromatin of Drosophila, the verthandi (vtd) locus, a member of the trxG gene family with Suppressor of Polycomb [Su(Pc)] activity, was characterized (Kennison, 1988; Schulze, 2001). The vtd locus also affects hh expression; vtd mutations are dominant suppressors of Moonrat (Mrt), a dominant gain of function allele of hh (Schulze, 2001; Felsenfeld, 1995). However, because of its location deep within the centric heterochromatin of the left arm of chromosome, vtd has resisted characterization at the molecular level (Hallson, 2008).

This study reports that vtd mutations, isolated on the basis of their trxG phenotypes, map to the gene encoding the cohesin subunit Rad21 and exhibit corresponding defects in mitosis and sister chromatid cohesion. Mutations in Nipped-B also show trxG phenotypes, and as is the case for vtd, heterozygous mutant flies show trxG phenotypes without significantly affecting cell division. These results provide a link between sister chromatid cohesion proteins and trxG functions, thus suggesting that cohesion factors may act by facilitating transcription activation and/or elongation (Hallson, 2008).

Alleles of vtd have lesions in rad21, mutations or knockdowns of rad21 have vtd phenotypes, and vice versa, and a transgene containing rad21 rescues the lethality of vtd. It is also noteworthy that reductions in Rad21 or Nipped-B dosage alter gene expression without seriously affecting chromatid cohesion, suggesting that these may be separable functions for cohesin and associated proteins. Evidence has accumulated that cohesin and associated proteins have important roles in gene regulation, but the functional basis for this has been unclear. The simplest model that explains the existing data is that Rad21, like most other trxG proteins, facilitates transcription (Hallson, 2008).

In Drosophila, many trxG proteins are subunits of complexes with diverse roles in transcriptional activation. Trx and Ash1 encode SET domain proteins that methylate lysine 4 of histone H3 (H3K4), and Ash2 is a member of a complex that also methylates H3K4. Other trxG proteins (e.g., Brahma, Osa, Moira, Kismet) are members of ATP-dependent nucleosome remodeling complexes. However, despite concerted efforts from many laboratories, the precise mechanisms by which trxG proteins regulate transcription remain unclear. In addition to chromatin modification, trxG proteins appear to be directly involved in recruiting factors required for transcription elongation, and noncoding RNAs may also play a role in regulating some of the affected genes (Hallson, 2008).

The hypothesis that cohesin facilitates transcription is supported by the results of a recent genome-wide ChIP study, which shows preferential binding of Nipped-B and the cohesin subunits SMC1 and SA to transcribed regions, overlapping with RNA polymerase II (Pol II) binding sites (Misulovin, 2008). The colocalization of Nipped-B with cohesin on chromosomes, and physical association with SA and Rad21 in extracts further suggests that Nipped-B and cohesin act together (Hallson, 2008).

There are strong correlations between binding of cohesin components and active gene expression. The dosage sensitive suppression of the hhMrt gain of function allele by both vtd and Nipped-B mutations suggests that Nipped-B and cohesin both promote expression of hh. It is unknown, however, if this effect is direct. Cohesin or Nipped-B do not bind to the hh gene in any of the three cell lines examined, however, in at least two of these, PcG proteins actively silence hh. Genome-wide, PcG silencing and the resulting histone H3 lysine 27 trimethylation strongly anti-correlates with Nipped-B and cohesin binding. Thus, it would not be expected that cohesin binds hh in these cell lines even if it directly regulates hh. For example, although Nipped-B regulates Ubx expression in vivo, Nipped-B and cohesin are excluded from the silenced Ubx and Abd-A genes in Sg4 cells, but bind to the transcribed Abd-B gene. In cells in which Abd-B is silenced, cohesin does not bind to the Abd-B promoter region. Thus, it remains possible that Nipped-B and cohesin directly stimulate hh transcription in vivo (Hallson, 2008).

Identification of loss of function zebrafish rad21 alleles in a genetic screen for mutations that reduce expression of runx genes also suggests that cohesin promotes gene expression, but again, it is unknown if this effect is direct (Horsfield, 2007). Stronger evidence supporting the idea that cohesin directly stimulates transcription arises from a recent study on axon pruning in the Drosophila mushroom body (Schuldiner, 2008). In this study, loss of function alleles of the Smc1 and SA genes were isolated in a screen for mutations that block pruning. The lack of pruning correlated with reduced expression of the ecdysone receptor (EcR) gene, and could be partially rescued by ectopic EcR expression. Nipped-B and cohesin bind to the transcribed portion of the EcR gene in all three cell lines examined, including the ML-DmBG3 line derived from third instar central nervous system, suggesting that they directly facilitate EcR expression (Hallson, 2008).

The question remains as to whether the same cohesin complexes required for cohesion of sister chromatids also function in transcription regulation, or whether, analogous to trxG proteins, different cohesin subunits have different functions in transcription, presumably because they are members of different complexes. One might conclude the latter based upon the observation that reductions in Rad21, SA, or SMC1 all increase cut expression, whereas decreases in Nipped-B reduce cut expression. These effects are likely direct because cohesin and Nipped-B bind to a 180 kb region that encompasses the entire upstream regulatory and transcribed regions of cut in ML-DmBG3 cells. The expression of RNAi transgenes encoding for SA and Rad21 decreases the severity of the cutK allele, whereas Nipped-B mutations enhance the cutK phenotype, also suggesting that they have opposite effects at cut. Finally, in contrast to results with Nipped-B mutations, no consistent effects on cut expression were observed for vtd mutant heterozygotes; moreover, mutations in vtd and Nipped-B both suppress the phenotypes of Pc4 and Mrt, but mutations in Smc1 or pds5 did not. Similarly, Dorsett (2005) has reported that null alleles of the cohesion factors sans and deco have no effect on the expression of cut when a functional chromosomal copy is present. Based on all of the above evidence, one might therefore conclude that different cohesin components may act differentially, possibly because, like trxG proteins, they are members of different regulatory complexes (Hallson, 2008).

However, it is also possible that the same cohesin complex involved in chromatid cohesion also regulates transcription, if binding at different loci results in different, gene specific consequences. Thus, in cut, which is activated by a remote wing margin enhancer located >80 kb upstream of the promoter, it has been proposed that cohesin could inhibit long range activation, and that Nipped-B facilitates activation by maintaining a dynamic cohesin binding equilibrium (Dorsett, 2005). In other genes, such as EcR or hh, cohesin might help maintain open chromatin to facilitate transcription by encircling a 10-nm fiber and preventing refolding to a higher order structure. As for the differences observed in the genetics of cohesin components, there are likewise other plausible explanations: differences in genetic background of mutant lines tested, differences in maternal expression/loading of required gene products in different heterozygous flies, or the possibility that the cutK or Pc alleles are less sensitive to changes in rad21/vtd, SMC1, or pds5 gene dosage than they are to the gene dosage of Nipped-B. Consistent with this idea, effects of rad21 dosage on cut expression were observed when RNAi was used to deplete rad21 mRNA, presumably to levels lower than those available in vtd(+) heterozygotes. It was also reported that Nipped-B expression is not directly proportional to gene dosage. The data in this study also show that reductions in Nipped-B and rad21 dosage act in the same direction i.e., suppress Mrt and Pc, suggesting that both genes may contribute to gene activation. The fact that both the rad21 and Nipped-B genes are resident within a late-replicating, heterochromatic environment may also explain some differences in outcomes of genetics tests of cohesin subunit function (Hallson, 2008).

These results provide a link between cohesin binding and trxG gene function. It will be an interesting challenge for the future to determine how components involved in chromatid cohesion act at the molecular level to regulate transcription, particularly given other very recent evidence implicating cohesin in gene regulation. The discovery that vtd encodes the Rad21 cohesin subunit expands the known roles of cohesin and Nipped-B in Drosophila development to include regulation of hh, which like cut, Ubx, and EcR, has many developmental roles. Similar modulation of key developmental regulators in humans, each with multiple roles, could explain why Cornelia de Lange syndrome patients have multiple diverse developmental deficits (Hallson, 2008).

Cohesin organizes chromatin loops at DNA replication factories

Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. This study demonstrates that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication (Guillou, 2010).

The first part of this study describes a physical interaction between cohesin and the MCM complex in human cells that is consistent with a previous report of an interaction between Smc1 and Mcm7. Whether the association of cohesin with chromatin depends on the previous formation of pre-RCs at origins has been a matter of discussion. This study shows that cohesin associates normally with chromatin after the down-regulation of ORC or MCM, arguing that cohesin loading is independent of pre-RC formation in human cells, as it happens in yeast or Drosophila cells. Therefore, the requirement of pre-RCs for cohesin loading that has been reported in Xenopus extracts could be a particularity of this system. Xenopus extracts recapitulate the early embryonic cycles, a quick succession of chromosome duplication and segregation events with no active transcription. In this context, the genomic positions where pre-RCs are assembled may constitute the only 'entry points' for cohesin. In addition, considering the results of this study, the loading of cohesin at pre-RC sites in Xenopus would ensure its physical presence around origins, where it would contribute to the dynamics of DNA replication (Guillou, 2010).

Cohesin can be detected at thousands of sites along the genome. While a complete genome-wide correlation between CBSs and replication origins cannot be established because of the lack of a comprehensive map of the latter, using a bioinformatics approach, an enrichment of cohesin at the origins located within the ENCODE representation of the genome has indeed been identified. When data from the cohesin ChIP-chip assay were compared with the genomic positions of origins mapped within ENCODE by nascent strand analyses in the same cell line, it became clear that origins are preferential sites for cohesin binding. This observation, further validated by cohesin ChIP assays, seems a conserved feature through evolution because it has also been reported in yeast, Drosophila (MacAlpine, 2009), and even Bacillus subtilis, and suggests a role for cohesin in origin activity. Actually, it was found that cohesin down-regulation slows down S-phase progression by a mechanism that is independent of sister chromatid cohesion, regulation of gene expression, and checkpoint responses. Instead, single-molecule analyses revealed that cohesin down-regulation reduced the number of active origins and increased the average interfork distance, without affecting fork speed. These results imply that the presence of cohesin at origins modulates their activity, providing a novel link between the DNA replication and cohesion machineries, which is independent from the reported effect of cohesin acetylation on fork progression (Terret, 2009; Guillou, 2010 and references therein).

The assembly of DNA replication factories in human cells entails the physical association of a cluster of origins and the formation of chromatin loops. This study has shown that cohesin down-regulation leads to a significant increase in the length of DNA loops in which chromatin is organized. This result, combined with the negative impact of cohesin loss on DNA replication, leads to a proposal that cohesin is required for the formation and/or stabilization of loops at replication foci. In this model, cohesin would mediate the long-range intrachromosomal interactions necessary to bring together a cluster of replication origins. Loop formation would occur at late mitosis and during G1, at the time of origin selection and licensing. In the resultant structures, origins would be located at the bases of the loops, where they are more prone to fire (Courbet, 2008). Upon cohesin down-regulation, replication foci would be structured in a different manner, with fewer origins, longer loops, and, therefore, larger replicon units. This alternative arrangement explains the S-phase phenotypes and the fact that cohesin down-regulation reduces the average intensity of each replication factory without reducing the total number of replication foci (Guillou, 2010).

Interestingly, down-regulation of CTCF neither delayed DNA replication nor affected halo size. The latter observation may seem surprising, but it could be explained because the 'DNA halo' technique allows the visualization of chromatin loops anchored to insoluble nuclear structures, such as those in replication factories, rather than DNA loops that are formed transiently to regulate transcription. In any case, it is possible that other proteins cooperate with cohesin to organize loops at replication factories, much as CTCF, the mediator complex, or tissue-specific transcription factors cooperate with cohesin to regulate gene expression in different contexts (Guillou, 2010).

Enhancer of Split Gene Complex: architecture and coordinate regulation by Notch, Cohesin and Polycomb group proteins

The cohesin protein complex functionally interacts with Polycomb group (PcG) silencing proteins to control expression of several key developmental genes, such as the Drosophila Enhancer of split gene complex [E(spl)-C]. The E(spl)-C contains twelve genes that inhibit neural development. In a cell line derived from central nervous system, cohesin and the PRC1 PcG protein complex bind and repress E(spl)-C transcription, but the repression mechanisms are unknown. The genes in the E(spl)-C are directly activated by the Notch receptor. This study shows that depletion of cohesin or PRC1 increases binding of the Notch intracellular fragment (NICD) to genes in the E(spl)-C, correlating with increased transcription. The increased transcription likely reflects both direct effects of cohesin and PRC1 on RNA polymerase activity at the E(spl)-C, and increased expression of Notch ligands. By chromosome conformation capture this study found that the E(spl) C is organized into a self-interactive architectural domain that is co-extensive with the region that binds cohesin and PcG complexes. The self-interactive architecture is formed independently of cohesin or PcG proteins. It is posited that the E(spl)-C architecture dictates where cohesin and PcG complexes bind and act when they are recruited by as yet unidentified factors, thereby controlling the E(spl)-C as a coordinated domain (Schaaf, 2013).

These studies investigated the regulation of the E(spl)-C complex by cohesin, PRC1, and the

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 smc1exc46 allele is recessive lethal and chromosome squashes show precocious sister chromatid separation (Dorsett, 2005).

The effects of smc1exc46 on the mutant phenotype displayed by the ctK gypsy transposon insertion allele of cut were used to determine changes in cut expression. The ctK 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 smc1exc46 mutation reduced the number of ctK 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 ctK 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}CG17509EY06473 transposon insertion in the first exon is homozygous viable. It was mobilized to generate two recessive lethal mutations, pds5e3 and pds5e6, 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 pds5e3/+ 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 pds5e3 and pds5e6 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 ctK phenotype was tested relative to the viable P element insertion used to generate them. Unexpectedly, the two mutations had different effects. The pds5e3 mutation slightly increased the number of wing margin nicks, indicating that it decreased cut expression, whereas pds5e6 increased cut expression. Although a small increase in wing margin nicks was consistently observed with pds5e3, the wing nicking was not significantly different from that seen with the parental chromosome. The decreased nicking associated with the pds5e6 allele, however, was significantly different from that of the parental chromosome. It is concluded that the pds5e6 mutation dominantly increases cut expression, and that the pds5e3 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 pds5e3 and pds5e6 second instar larvae were examined. No pds5 transcripts were detected in homozygous pds5e3 mutants, but a shorter transcript (3.65 kb) was seen at levels similar to those of wild type in both heterozygous and homozygous pds5e6 mutants. A wild-type sized transcript was present in heterozygous pds5e6 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 pds5e3 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 pds5e6 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 pds5e6 genomic DNA revealed that the region from the P insertion site through exon 5 is missing. 5' RACE analysis of the pds5e6 transcript shows that it starts 67 nucleotides upstream of the wild-type start site predicted by EST analysis. The pds5e6 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 pds5e6 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 pds5e3 mutant chromosomes were thicker than wild type, and the pds5e6 mutant chromosomes were thinner. The pds5e3 null allele did not reduce staining for Smc1 or Stromalin, although the pattern appeared less discrete. By contrast, pds5e6 mutant chromosomes showed strongly reduced staining for Smc1 and Stromalin. Loss of cohesin staining was observed in multiple nuclei from multiple pds5e6 salivary glands. These results indicate that the pds5e6 mutant either blocks loading of cohesin onto chromosomes, or facilitates removal. The reduction of cohesin binding caused by pds5e6, 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 pds5e6 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 pds5e6 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 pds5e3 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 pds5e3 and pds5e6 (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 pds5e3 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 pds5e6 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 pds5e6 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).


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