Study of FoxA Pioneer Factor at Silent Genes Reveals Rfx-Repressed Enhancer at Cdx2 and a Potential Indicator of Esophageal Adenocarcinoma Development

Understanding how silent genes can be competent for activation provides insight into development as well as cellular reprogramming and pathogenesis. We performed genomic location analysis of the pioneer transcription factor FoxA in the adult mouse liver and found that about one-third of the FoxA bound sites are near silent genes, including genes without detectable RNA polymerase II. Virtually all of the FoxA-bound silent sites are within conserved sequences, suggesting possible function. Such sites are enriched in motifs for transcriptional repressors, including for Rfx1 and type II nuclear hormone receptors. We found one such target site at a cryptic “shadow” enhancer 7 kilobases (kb) downstream of the Cdx2 gene, where Rfx1 restricts transcriptional activation by FoxA. The Cdx2 shadow enhancer exhibits a subset of regulatory properties of the upstream Cdx2 promoter region. While Cdx2 is ectopically induced in the early metaplastic condition of Barrett's esophagus, its expression is not necessarily present in progressive Barrett's with dysplasia or adenocarcinoma. By contrast, we find that Rfx1 expression in the esophageal epithelium becomes gradually extinguished during progression to cancer, i.e, expression of Rfx1 decreased markedly in dysplasia and adenocarcinoma. We propose that this decreased expression of Rfx1 could be an indicator of progression from Barrett's esophagus to adenocarcinoma and that similar analyses of other transcription factors bound to silent genes can reveal unanticipated regulatory insights into oncogenic progression and cellular reprogramming

gob-1 is a trehalose-6-phosphate phosphatase required for intestine development in the nematode caenorhabditis elegans

Bibliography: p. 194-217Some pages are in colour

gob-1 is a trehalose-6-phosphate phosphatase required for intestine development in the nematode caenorhabditis elegans

Bibliography: p. 194-217Some pages are in colour.A genetic screen was developed with the intention of finding C. elegans genes that are required for intestine development or differentiation. The screen was based on the gut obstructed phenotype of the elt-2 null mutant. elt-2 is a GATA transcription factor believed to be the major transcription factor driving intestine differentiation. It was anticipated that screening for a similar gut obstructed mutant phenotype could identify genes that were functioning redundantly to elt-2 or downstream of elt-2. One of the mutations identified in this screen, gob-1(ca17), was mapped to the right-hand side of the X chromosome. Positional cloning of the gob-1(ca17) allele showed that it was a deletion of approximately 15 open reading frames. RNA-mediated interference to a gene in this region, H13N06.3, phenocopied the early larval arrest and gut obstructed phenotype of gob-1(ca17) and was thus identified as the gene responsible for this phenotype. The gob-1 RNAi mutant causes an early defect in the intestine. The gob-1 gene and GOB-1 protein are expressed specifically in the intestine during its early development. GOB-1 is a member of the Haloacid dehalogenase (HAD)-like hydrolase superfamily and is the first identified trehalose-6-phosphate phosphatase in the nematode phylum. GOB-1’s function in trehalose metabolism is currently unclear but mounting evidence suggests that GOB-1 may have a function required for viability of the worm independent of trehalose synthesis. ELT-2 is sufficient but not necessary for early embryonic expression of GOB-1. The ability of ELT-2 to drive gob-1 expression suggests that loss of gob-1 iv function may be a component of the later elt-2 loss of function defect in the larval intestine

gob-1 is a trehalose-6-phosphate phosphatase required for intestine development in the nematode caenorhabditis elegans

Bibliography: p. 194-217Some pages are in colour

Foxi2 Is an Animally Localized Maternal mRNA in <em>Xenopus</em>, and an Activator of the Zygotic Ectoderm Activator Foxi1e

<div><p>Foxi1e is a zygotic transcription factor that is essential for the expression of early ectodermal genes. It is expressed in a highly specific pattern, only in the deep cell layers of the animal hemisphere, and in a mosaic pattern in which expressing cells are interspersed with non-expressing cells. Previous work has shown that several signals in the blastula control this expression pattern, including nodals, the TGFβ family member Vg1, and Notch. However, these are all inhibitory, which raises the question of what activates Foxi1e. In this work, we show that a related Forkhead family protein, Foxi2, is a maternal activator of Foxi1e. <em>Foxi2</em> mRNA is maternally encoded, and highly enriched in animal hemisphere cells of the blastula. ChIP assays show that it acts directly on upstream regulatory elements of Foxi1e. Its effect is specific, since animal cells depleted of Foxi2 are able to respond normally to mesoderm inducing signals from vegetal cells. Foxi2 thus acts as a link between the oocyte and the early pathway to ectoderm, in a similar fashion to the vegetally localized VegT acts to initiate endoderm and mesoderm formation.</p> </div

Expression of Foxi1e, as well as early ectodermal markers, requires Foxi2.

<p>(A–C) Show effect of Foxi2 depletion of <i>Foxi1e</i> mRNA levels at the late blastula and early gastrula stages (A), and on Foxi1e protein levels by western and by densitometric analysis of the western blot (A: middle panels) and immunostaining (B). (A) also shows by western blotting that the anti-Foxi1e antibody cross-reacts with <i>Xenopus</i> Foxi1e protein (lower panel). (C) Shows by RT-PCR the reduced levels of expression of ectodermal genes including <i>Xlim5</i>, <i>E-cadherin</i>, <i>Sox2</i> and <i>Cytokeratin</i> in control and Foxi2-depleted embryos at the late blastula and early gastrula stages. (D) RT-PCR shows that expression levels of the mesodermal marker <i>Xbra</i>, endodermal marker <i>Xsox17a</i> and ectodermal marker, <i>KLFn</i> are unaffected by Foxi2 depletion. (E–F) show that reduction in expression of the ectoderm marker genes <i>E-cadherin</i> (E-cad), <i>Foxi1e</i>, <i>FoxJ1</i>, and <i>grainyhead-like 3</i> (GHL3) caused by Foxi2 depletion are rescued by subsequent injection of <i>Foxi2</i> mRNA (10 pg dose) in both whole embryos (E) and animal caps (F). Scale bar in (B) = 50 µm.</p

Foxi2 protein directly binds to the Foxi1e promoter.

<p>(A) Luciferase activity in blastulae after injecting <i>Xenopus</i> tropicalis <i>Foxi1e</i> promoter-luciferase construct into either animal or vegetal blastomeres at 8-cell stage embryos. (B) Foxi1e promoter activity was measured in blastulae derived from control (uninj) or Foxi2-depleted (4 ng and 5 ng) oocytes. (C,D) Chromatin immunoprecipitation assay. (C) A schematic diagram of <i>Xenopus</i> tropicalis Foxi1e promoter shows potential Foxi2 binding sites and ChIP PCR primer amplicons. Real-time PCR shows significantly higher signal on primer pair −1982∶−1734 after immunoprecipation by 6-myc-tagged Foxi2 protein. (D) A schematic diagram of <i>Xenopus</i> laevis Foxi1e promoter shows potential Foxi2 binding sites and ChIP PCR primer amplicons. Real-time PCR shows significantly higher signal on primer pair −1540∶−1391 after immunoprecipation by myc-tagged Foxi2 protein. (E) Luciferase activity in blastulae after injecting either wild-type (WT) <i>Xenopus</i> tropicalis Foxi1e promoter constructs or a construct lacking the Foxi2 binding region (Deletion). While wild-type promoter showed its activity (WT Ani), the construct lacking the Foxi2 binding site mutated promoter showed a basal level of promoter activity (Deletion Ani). (F) Luciferase activity in blastulae after injecting Wild-type and Deletion <i>Xenopus tropicalis Foxi1e</i> promoter constructs into vegetal blastomeres. <i>Foxi1e</i> promoter construct lacking the Foxi2 binding site showed a basal level of promoter activity (Deletion Veg 8) while Foxi2 overexpression in vegetal blastomeres had no effect on this mutated construct (Deletion+Foxi2 Veg 8) while wild-type promoter showed increased luciferase activity upon overexpression of Foxi2 (WT+Foxi2 Veg 8).</p

Spatial expression of Foxi1e is dependent on Foxi2.

<p>(A) The level of expression of Foxi1e is reduced in Foxi2-depleted animal caps (Foxi2 depleted caps) compared to the caps from control siblings (Uninj caps st.10) at the gastrula stage. Both dissociated superficial and deep cells from Foxi2-depleted embryos expressed reduced levels of <i>Foxi1e</i> mRNA. Note that the expression level of Foxi1e in dissociated superficial cells is much higher than in intact animal caps (Uninj superficial cells). (B) Maternal depletion of Dishevelled2 (Dvl2−) showed upregulation of Foxi1e expression. Double depletion of Foxi2 and Dvl2 (Foxi2−/Dvl2−) showed similar level of expression to Foxi2-depleted embryos (Foxi2−). (C) Maternal depletion of Vg1 (Vg1−) showed upregulation of Foxi1e expression. Double depletion of Foxi2 and Vg1 (Foxi2−/Vg1−) showed a level of expression similar to Foxi2-depleted embryos (Foxi2−). (D) Upper panel; Maternal depletion of transcription factor Mastermind1 (Mam1−) caused upregulation of Foxi1e expression. Double depletion of Foxi2 and Mastermind1 (Foxi2−/Mam1−) showed a similar level of expression as Foxi2 depleted embryos (Foxi2−). Lower panel; The overexpression of Supressor of Hairless (SuH) DNA binding mutant (SuH DBM) mRNA caused upregulation of Foxi1e expression. Foxi1e expression in Foxi2 depleted+1.2 ng of <i>SuH DBM</i> mRNA injected embryos showed similar level of expression as Foxi2 depleted embryos (Foxi2−).</p

The spatial and temporal expression pattern of Foxi2 and its depletion at blastula stage.

<p>(A) RT-PCR to show relative expression levels of Foxi2 and VegT in either whole oocytes or dissected animal and vegetal halves. Foxi2 and VegT have reciprocal expression domains, with Foxi2 being highly enriched in the animal hemisphere. (B) In situ hybridization of tissue section at the late blastula stage to show distribution of <i>Foxi2</i> mRNA. (C) RT-PCR analysis of developmental time course to show absence of early zygotic expression of <i>Foxi2</i>. (D) RT-PCR to show degree of depletion of <i>Foxi2</i> mRNA by antisense oligo. (E–F) Degree of depletion of Foxi2 protein by mRNA depletion; a western blot of oocytes and early stages is shown in (E), and by immunostaining at the late blastula stage in (F). Scale bar in (F) = 50 µm. High magnification Foxi2 immunostaining images from Uninjected (F′) and Foxi2 depleted (F″) embryos. Scale bars in F′ and F″ = 20 µm.</p

Pax3 regulation of FGF signaling affects the progression of embryonic progenitor cells into the myogenic program

Pax3/7-dependent stem cells play an essential role in skeletal muscle development. We now show that Fgfr4 lies genetically downstream from Pax3 and is a direct target. In chromatin immunoprecipitation (ChIP)-on-chip experiments, Pax3 binds to a sequence 3′ of the Fgfr4 gene that directs Pax3-dependent expression at sites of myogenesis in transgenic mouse embryos. The activity of this regulatory element is also partially dependent on E-boxes, targets of the myogenic regulatory factors, which are expressed as progenitor cells enter the myogenic program. Other FGF signaling components, notably Sprouty1, are also regulated by Pax3. In vivo manipulation of Sprouty expression reveals that FGF signaling affects the balance between Pax-positive progenitor cells and committed myoblasts. These results provide new insight into the Pax-initiated regulatory network that modulates stem cell maintenance versus tissue differentiation