The effect of Nipped-B-like (Nipbl) haploinsufficiency on genome-wide cohesin binding and target gene expression: modeling Cornelia de Lange syndrome.

BACKGROUND: Cornelia de Lange syndrome (CdLS) is a multisystem developmental disorder frequently associated with heterozygous loss-of-function mutations of Nipped-B-like (NIPBL), the human homolog of Drosophila Nipped-B. NIPBL loads cohesin onto chromatin. Cohesin mediates sister chromatid cohesion important for mitosis but is also increasingly recognized as a regulator of gene expression. In CdLS patient cells and animal models, expression changes of multiple genes with little or no sister chromatid cohesion defect suggests that disruption of gene regulation underlies this disorder. However, the effect of NIPBL haploinsufficiency on cohesin binding, and how this relates to the clinical presentation of CdLS, has not been fully investigated. Nipbl haploinsufficiency causes CdLS-like phenotype in mice. We examined genome-wide cohesin binding and its relationship to gene expression using mouse embryonic fibroblasts (MEFs) from Nipbl+/- mice that recapitulate the CdLS phenotype. RESULTS: We found a global decrease in cohesin binding, including at CCCTC-binding factor (CTCF) binding sites and repeat regions. Cohesin-bound genes were found to be enriched for histone H3 lysine 4 trimethylation (H3K4me3) at their promoters; were disproportionately downregulated in Nipbl mutant MEFs; and displayed evidence of reduced promoter-enhancer interaction. The results suggest that gene activation is the primary cohesin function sensitive to Nipbl reduction. Over 50% of significantly dysregulated transcripts in mutant MEFs come from cohesin target genes, including genes involved in adipogenesis that have been implicated in contributing to the CdLS phenotype. CONCLUSIONS: Decreased cohesin binding at the gene regions is directly linked to disease-specific expression changes. Taken together, our Nipbl haploinsufficiency model allows us to analyze the dosage effect of cohesin loading on CdLS development.

Conditional Creation and Rescue of Nipbl-Deficiency in Mice Reveals Multiple Determinants of Risk for Congenital Heart Defects.

Elucidating the causes of congenital heart defects is made difficult by the complex morphogenesis of the mammalian heart, which takes place early in development, involves contributions from multiple germ layers, and is controlled by many genes. Here, we use a conditional/invertible genetic strategy to identify the cell lineage(s) responsible for the development of heart defects in a Nipbl-deficient mouse model of Cornelia de Lange Syndrome, in which global yet subtle transcriptional dysregulation leads to development of atrial septal defects (ASDs) at high frequency. Using an approach that allows for recombinase-mediated creation or rescue of Nipbl deficiency in different lineages, we uncover complex interactions between the cardiac mesoderm, endoderm, and the rest of the embryo, whereby the risk conferred by genetic abnormality in any one lineage is modified, in a surprisingly non-additive way, by the status of others. We argue that these results are best understood in the context of a model in which the risk of heart defects is associated with the adequacy of early progenitor cell populations relative to the sizes of the structures they must eventually form.

Using mouse and zebrafish models to understand the etiology of developmental defects in Cornelia de Lange Syndrome.

Cornelia de Lange Syndrome (CdLS) is a multisystem birth defects disorder that affects every tissue and organ system in the body. Understanding the factors that contribute to the origins, prevalence, and severity of these developmental defects provides the most direct approach for developing screens and potential treatments for individuals with CdLS. Since the majority of cases of CdLS are caused by haploinsufficiency for NIPBL (Nipped-B-like, which encodes a cohesin-associated protein), we have developed mouse and zebrafish models of CdLS by using molecular genetic tools to create Nipbl-deficient mice and zebrafish (Nipbl(+/-) mice, zebrafish nipbl morphants). Studies of these vertebrate animal models have yielded novel insights into the developmental etiology and genes/gene pathways that contribute to CdLS-associated birth defects, particularly defects of the gut, heart, craniofacial structures, nervous system, and limbs. Studies of these mouse and zebrafish CdLS models have helped clarify how deficiency for NIPBL, a protein that associates with cohesin and other transcriptional regulators in the nucleus, affects processes important to the emergence of the structural and physiological birth defects observed in CdLS: NIPBL exerts chromosome position-specific effects on gene expression; it influences long-range interactions between different regulatory elements of genes; and it regulates combinatorial and synergistic actions of genes in developing tissues. Our current understanding is that CdLS should be considered as not only a cohesinopathy, but also a "transcriptomopathy," that is, a disease whose underlying etiology is the global dysregulation of gene expression throughout the organism. © 2016 Wiley Periodicals, Inc.

Genetic enhancement of limb defects in a mouse model of Cornelia de Lange syndrome.

Cornelia de Lange Syndrome (CdLS) is characterized by a wide variety of structural and functional abnormalities in almost every organ system of the body. CdLS is now known to be caused by mutations that disrupt the function of the cohesin complex or its regulators, and studies of animal models and cell lines tell us that the effect of these mutations is to produce subtle yet pervasive dysregulation of gene expression. With many hundreds of mostly small gene expression changes occurring in every cell type and tissue, identifying the etiology of any particular birth defect is very challenging. Here we focus on limb abnormalities, which are commonly seen in CdLS. In the limb buds of the Nipbl-haploinsufficient mouse (Nipbl(+/-) mouse), a model for the most common form of CdLS, modest gene expression changes are observed in several candidate pathways whose disruption is known to cause limb abnormalities, yet the limbs of Nipbl(+/-) mice develop relatively normally. We hypothesized that further impairment of candidate pathways might produce limb defects similar to those seen in CdLS, and performed genetic experiments to test this. Focusing on Sonic hedgehog (Shh), Bone morphogenetic protein (Bmp), and Hox gene pathways, we show that decreasing Bmp or Hox function (but not Shh function) enhances polydactyly in Nipbl(+/-) mice, and in some cases produces novel skeletal phenotypes. However, frank limb reductions, as are seen in a subset of individuals with CdLS, do not occur, suggesting that additional signaling and/or gene regulatory pathways are involved in producing such dramatic changes. © 2016 Wiley Periodicals, Inc.

Clinical, developmental and molecular update on Cornelia de Lange syndrome and the cohesin complex: abstracts from the 2014 Scientific and Educational Symposium.

Cornelia de Lange Syndrome (CdLS) is the most common example of disorders of the cohesin complex, or cohesinopathies. There are a myriad of clinical issues facing individuals with CdLS, particularly in the neurodevelopmental system, which also have implications for the parents and caretakers, involved professionals, therapists, and schools. Basic research in developmental and cell biology on cohesin is showing significant progress, with improved understanding of the mechanisms and the possibility of potential therapeutics. The following abstracts are presentations from the 6th Cornelia de Lange Syndrome Scientific and Educational Symposium, which took place on June 25-26, 2014, in conjunction with the Cornelia de Lange Syndrome Foundation National Meeting in Costa Mesa, CA. The Research Committee of the CdLS Foundation organizes the meeting, reviews and accepts abstracts, and subsequently disseminates the information to the families through members of the Clinical Advisory Board. In addition to the scientific and clinical discussions, there were educationally focused talks related to practical aspects of behavior and development. AMA CME credits were provided by Greater Baltimore Medical Center, Baltimore, MD.

Cohesin mediates chromatin interactions that regulate mammalian ß-globin expression.

The ß-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5' and 3' boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.

Multiple organ system defects and transcriptional dysregulation in the Nipbl(+/-) mouse, a model of Cornelia de Lange Syndrome.

Cornelia de Lange Syndrome (CdLS) is a multi-organ system birth defects disorder linked, in at least half of cases, to heterozygous mutations in the NIPBL gene. In animals and fungi, orthologs of NIPBL regulate cohesin, a complex of proteins that is essential for chromosome cohesion and is also implicated in DNA repair and transcriptional regulation. Mice heterozygous for a gene-trap mutation in Nipbl were produced and exhibited defects characteristic of CdLS, including small size, craniofacial anomalies, microbrachycephaly, heart defects, hearing abnormalities, delayed bone maturation, reduced body fat, behavioral disturbances, and high mortality (75-80%) during the first weeks of life. These phenotypes arose despite a decrease in Nipbl transcript levels of only approximately 30%, implying extreme sensitivity of development to small changes in Nipbl activity. Gene expression profiling demonstrated that Nipbl deficiency leads to modest but significant transcriptional dysregulation of many genes. Expression changes at the protocadherin beta (Pcdhb) locus, as well as at other loci, support the view that NIPBL influences long-range chromosomal regulatory interactions. In addition, evidence is presented that reduced expression of genes involved in adipogenic differentiation may underlie the low amounts of body fat observed both in Nipbl+/- mice and in individuals with CdLS.

The role of foxg1 in the development of neural stem cells of the olfactory epithelium.

The olfactory epithelium (OE) of the mouse is an excellent model system for studying principles of neural stem cell biology because of its well-defined neuronal lineage and its ability to regenerate throughout life. To approach the molecular mechanisms of stem cell regulation in the OE, we have focused on Foxg1, also known as brain factor 1, which is a member of the Forkhead transcription factor family. Foxg1(-/-) mice show major defects in the OE at birth, suggesting that Foxg1 plays an important role in OE development. We find that Foxg1 is expressed in cells within the basal compartment of the OE, the location where OE stem and progenitor cells are known to reside. Since FoxG1 is known to regulate proliferation of neuronal progenitor cells during telencephalon development, we performed bromodeoxyuridine pulse-chase labeling of Sox2-expressing neural stem cells during primary OE neurogenesis. We found the percentage of Sox2-expressing cells that retained bromodeoxyuridine was twice as high in Foxg1(-/-) OE cells as in the wild type, suggesting that these cells are delayed and/or halted in their development in the absence of Foxg1. Our findings suggest that the proliferation and/or subsequent differentiation of Sox2-expressing neural stem cells in the OE is regulated by Foxg1.

Foxg1 promotes olfactory neurogenesis by antagonizing Gdf11.

Foxg1, a winged-helix transcription factor, promotes the development of anterior neural structures; in mice lacking Foxg1, development of the cerebral hemispheres and olfactory epithelium (OE) is severely reduced. It has been suggested that Foxg1 acts by positively regulating the expression of growth factors, such as Fgf8, which support neurogenesis. However, Foxg1 also binds Smad transcriptional complexes, allowing it to negatively regulate the effects of TGFbeta family ligands. Here, we provide evidence that this latter effect explains much of the ability of Foxg1 to drive neurogenesis in the OE. We show that Foxg1 is expressed in developing OE at the same time as the gene encoding growth differentiation factor 11 (Gdf11), a TGFbeta family member that mediates negative-feedback control of OE neurogenesis. Mutations in Gdf11 rescue, to a considerable degree, the major defects in Foxg1(-/-) OE, including the early, severe loss of neural precursors and olfactory receptor neurons, and the subsequent collapse of both neurogenesis and nasal cavity formation. Rescue is gene-dosage dependent, with loss of even one allele of Gdf11 restoring substantial neurogenesis. Notably, we find no evidence for a disruption of Fgf8 expression in Foxg1(-/-) OE. However, we do observe both a failure of expression of follistatin (Fst), which encodes a secreted Gdf11 antagonist normally expressed in and around OE, and an increase in the expression of Gdf11 itself within the remaining OE in these mutants. Fst expression is rescued in Foxg1(-/-);Gdf11(-/-) and Foxg1(-/-);Gdf11(+/-) mice. These data suggest that the influence of Foxg1 on Gdf11-mediated negative feedback of neurogenesis may be both direct and indirect. In addition, defects in development of the cerebral hemispheres in Foxg1(-/-) mice are not rescued by mutations in Gdf11, nor is Gdf11 expressed at high levels within these structures. Thus, the pro-neurogenic effects of Foxg1 are likely to be mediated through different signaling pathways in different parts of the nervous system.

Fgf8 expression defines a morphogenetic center required for olfactory neurogenesis and nasal cavity development in the mouse.

In vertebrate olfactory epithelium (OE), neurogenesis proceeds continuously, suggesting that endogenous signals support survival and proliferation of stem and progenitor cells. We used a genetic approach to test the hypothesis that Fgf8 plays such a role in developing OE. In young embryos, Fgf8 RNA is expressed in the rim of the invaginating nasal pit (NP), in a small domain of cells that overlaps partially with that of putative OE neural stem cells later in gestation. In mutant mice in which the Fgf8 gene is inactivated in anterior neural structures, FGF-mediated signaling is strongly downregulated in both OE proper and underlying mesenchyme by day 10 of gestation. Mutants survive gestation but die at birth, lacking OE, vomeronasal organ (VNO), nasal cavity, forebrain, lower jaw, eyelids and pinnae. Analysis of mutants indicates that although initial NP formation is grossly normal, cells in the Fgf8-expressing domain undergo high levels of apoptosis, resulting in cessation of nasal cavity invagination and loss of virtually all OE neuronal cell types. These findings demonstrate that Fgf8 is crucial for proper development of the OE, nasal cavity and VNO, as well as maintenance of OE neurogenesis during prenatal development. The data suggest a model in which Fgf8 expression defines an anterior morphogenetic center, which is required not only for the sustenance and continued production of primary olfactory (OE and VNO) neural stem and progenitor cells, but also for proper morphogenesis of the entire nasal cavity.