Caudal Fgfr1 disruption produces localised spinal mis-patterning and a terminal myelocystocele-like phenotype in mice.

Closed spinal dysraphisms are poorly understood malformations classified as neural tube (NT) defects. Several, including terminal myelocystocele, affect the distal spine. We have previously identified a NT closure-initiating point, Closure 5, in the distal spine of mice. Here, we document equivalent morphology of the caudal-most closing posterior neuropore (PNP) in mice and humans. Closure 5 forms in a region of active FGF signalling, and pharmacological FGF receptor blockade impairs its formation in cultured mouse embryos. Conditional genetic deletion of Fgfr1 in caudal embryonic tissues with Cdx2Cre diminishes neuroepithelial proliferation, impairs Closure 5 formation and delays PNP closure. After closure, the distal NT of Fgfr1-disrupted embryos dilates to form a fluid-filled sac overlying ventrally flattened spinal cord. This phenotype resembles terminal myelocystocele. Histological analysis reveals regional and progressive loss of SHH- and FOXA2-positive ventral NT domains, resulting in OLIG2 labelling of the ventral-most NT. The OLIG2 domain is also subsequently lost, eventually producing a NT that is entirely positive for the dorsal marker PAX3. Thus, a terminal myelocystocele-like phenotype can arise after completion of NT closure with localised spinal mis-patterning caused by disruption of FGFR1 signalling.

Setd5 is required in cardiopharyngeal mesoderm for heart development and its haploinsufficiency is associated with outflow tract defects in mouse.

Congenital heart defects are a feature of several genetic haploinsufficiency syndromes, often involving transcriptional regulators. One property of haploinsufficient genes is their propensity for network interactions at the gene or protein level. In this article we took advantage of an online dataset of high throughput screening of mutations that are embryonic lethal in mice. Our aim was to identify new genes where the loss of function caused cardiovascular phenotypes resembling the 22q11.2 deletion syndrome models, that is, heterozygous and homozygous loss of Tbx1. One gene with a potentially haploinsufficient phenotype was identified, Setd5, thought to be involved in chromatin modification. We found murine Setd5 haploinsufficiency to be associated with double outlet right ventricle and perimembranous ventricular septal defect, although no genetic interaction with Tbx1 was detected. Conditional mutagenesis revealed that Setd5 was required in cardiopharyngeal mesoderm for progression of the heart tube through the ballooning stage to create a four-chambered heart.

A novel requirement for DROSHA in maintenance of mammalian CG methylation.

In mammals, faithful inheritance of genomic methylation patterns ensures proper gene regulation and cell behaviour, impacting normal development and fertility. Following establishment, genomic methylation patterns are transmitted through S-phase by the maintenance methyltransferase Dnmt1. Using a protein interaction screen, we identify Microprocessor component DROSHA as a novel DNMT1-interactor. Drosha-deficient embryonic stem (ES) cells display genomic hypomethylation that is not accounted for by changes in the levels of DNMT proteins. DNMT1-mediated methyltransferase activity is also reduced in these cells. We identify two transcripts that are specifically upregulated in Drosha- but not Dicer-deficient ES cells. Regions within these transcripts predicted to form stem-loop structures are processed by Microprocessor and can inhibit DNMT1-mediated methylation in vitro. Our results highlight DROSHA as a novel regulator of mammalian DNA methylation and we propose that DROSHA-mediated processing of RNA is necessary to ensure full DNMT1 activity. This adds to the DROSHA repertoire of non-miRNA dependent functions as well as implicating RNA in regulating DNMT1 activity and correct levels of genomic methylation.

Inactivation of Geminin in neural crest cells affects the generation and maintenance of enteric progenitor cells, leading to enteric aganglionosis.

Neural crest cells comprise a multipotent, migratory cell population that generates a diverse array of cell and tissue types, during vertebrate development. Enteric Nervous System controls the function of the gastrointestinal tract and is mainly derived from the vagal and sacral neural crest cells. Deregulation on self-renewal and differentiation of the enteric neural crest cells is evident in enteric nervous system disorders, such as Hirschsprung disease, characterized by the absence of ganglia in a variable length of the distal bowel. Here we show that Geminin is essential for Enteric Nervous System generation as mice that lacked Geminin expression specifically in neural crest cells revealed decreased generation of vagal neural crest cells, and enteric neural crest cells (ENCCs). Geminin-deficient ENCCs showed increased apoptosis and decreased cell proliferation during the early stages of gut colonization. Furthermore, decreased number of committed ENCCs in vivo and the decreased self-renewal capacity of enteric progenitor cells in vitro, resulted in almost total aganglionosis resembling a severe case of Hirschsprung disease. Our results suggest that Geminin is an important regulator of self-renewal and survival of enteric nervous system progenitor cells.

CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression.

AIMS: Haploinsufficiency of the chromo-domain protein CHD7 underlies most cases of CHARGE syndrome, a multisystem birth defect including congenital heart malformation. Context specific roles for CHD7 in various stem, progenitor, and differentiated cell lineages have been reported. Previously, we showed severe defects when Chd7 is absent from cardiopharyngeal mesoderm (CPM). Here, we investigate altered gene expression in the CPM and identify specific CHD7-bound target genes with known roles in the morphogenesis of affected structures. METHODS AND RESULTS: We generated conditional KO of Chd7 in CPM and analysed cardiac progenitor cells using transcriptomic and epigenomic analyses, in vivo expression analysis, and bioinformatic comparisons with existing datasets. We show CHD7 is required for correct expression of several genes established as major players in cardiac development, especially within the second heart field (SHF). We identified CHD7 binding sites in cardiac progenitor cells and found strong association with histone marks suggestive of dynamically regulated enhancers during the mesodermal to cardiac progenitor transition of mESC differentiation. Moreover, CHD7 shares a subset of its target sites with ISL1, a pioneer transcription factor in the cardiogenic gene regulatory network, including one enhancer modulating Fgf10 expression in SHF progenitor cells vs. differentiating cardiomyocytes. CONCLUSION: We show that CHD7 interacts with ISL1, binds ISL1-regulated cardiac enhancers, and modulates gene expression across the mesodermal heart fields during cardiac morphogenesis.

Geminin regulates cortical progenitor proliferation and differentiation.

During cortical development, coordination of proliferation and differentiation ensures the timely generation of different neural progenitor lineages that will give rise to mature neurons and glia. Geminin is an inhibitor of DNA replication and it has been proposed to regulate cell proliferation and fate determination during neurogenesis via interactions with transcription factors and chromatin remodeling complexes. To investigate the in vivo role of Geminin in the maintenance and differentiation of cortical neural progenitors, we have generated mice that lack Geminin expression in the developing cortex. Our results show that loss of Geminin leads to the expansion of neural progenitor cells located at the ventricular and subventricular zones of the developing cortex. Early cortical progenitors lacking Geminin exhibit a longer S-phase and a reduced ability to generate early born neurons, consistent with a preference on self-renewing divisions. Overexpression of Geminin in progenitor cells of the cortex reduces the number of neural progenitor cells, promotes cell cycle exit and subsequent neuronal differentiation. Our study suggests that Geminin has an important role during cortical development in regulating progenitor number and ultimately neuron generation.

The Dnmt3L ADD Domain Controls Cytosine Methylation Establishment during Spermatogenesis.

A critical aspect of mammalian gametogenesis is the reprogramming of genomic DNA methylation. The catalytically inactive adaptor Dnmt3L is essential to ensuring this occurs correctly, but the mechanism by which it functions is unclear. Using gene targeting to engineer a single-amino-acid mutation, we show that the Dnmt3L histone H3 binding domain (ADD) is necessary for spermatogenesis. Genome-wide single-base-resolution DNA methylome analysis of mutant germ cells revealed overall reductions in CG methylation at repetitive sequences and non-promoter CpG islands. Strikingly, we also observe an even more severe loss of non-CG methylation, suggesting an unexpected role for the ADD in this process. These epigenetic deficiencies were coupled with defects in spermatogonia, with mutant cells displaying marked changes in gene expression and reactivation of retrotransposons. Our results demonstrate that the Dnmt3L ADD is necessary for Dnmt3L function and full reproductive fitness.

DNA methylation is dispensable for suppression of the agouti viable yellow controlling element in murine embryonic stem cells.

The agouti viable (Avy) locus is considered a model to understand how retroelements function as controlling elements in mammals. Epigenetic factors, principally CpG methylation, are widely held to play a dominant regulatory role in controlling the locus' activity. The purpose of this study was to examine its behavior in ES cells and determine if this locus could be exploited for use in screen-based investigations. We have derived multiple Avy ES cell lines from the C57BL/6 strain and generated a cell line carrying a GFP-reporter gene (Avy/AGFP). Use of the DNA demethylating drug 5-azacitidine on various ES cell lines does not induce either agouti or GFP expression. Methylation analysis reveals that although most lines display normal methylation at IAP elements in general, the Avy IAP element is essentially unmethylated. In addition, we find that different repeat compartments are epigenetically unstable in a number of derived cell lines.

Cdt1 is differentially targeted for degradation by anticancer chemotherapeutic drugs.

BACKGROUND: Maintenance of genome integrity is crucial for the propagation of the genetic information. Cdt1 is a major component of the pre-replicative complex, which controls once per cell cycle DNA replication. Upon DNA damage, Cdt1 is rapidly targeted for degradation. This targeting has been suggested to safeguard genomic integrity and prevent re-replication while DNA repair is in progress. Cdt1 is deregulated in tumor specimens, while its aberrant expression is linked with aneuploidy and promotes tumorigenesis in animal models. The induction of lesions in DNA is a common mechanism by which many cytotoxic anticancer agents operate, leading to cell cycle arrest and apoptosis. METHODOLOGY/PRINCIPAL FINDING: In the present study we examine the ability of several anticancer drugs to target Cdt1 for degradation. We show that treatment of HeLa and HepG2 cells with MMS, Cisplatin and Doxorubicin lead to rapid proteolysis of Cdt1, whereas treatment with 5-Fluorouracil and Tamoxifen leave Cdt1 expression unaffected. Etoposide affects Cdt1 stability in HepG2 cells and not in HeLa cells. RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA. CONCLUSION/SIGNIFICANCE: Our data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis. Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficacy of chemotherapy.