An interdependent network of functional enhancers regulates transcription and EZH2 loading at the INK4a/ARF locus

1. Download : Download high-res image (135KB)
2. Download : Download full-size image

     

Two novel EGFP insertion alleles reveal unique aspects of Pax2 function in embryonic and adult kidneys

The Pax2 gene encodes a DNA binding protein with multiple functions in the developing intermediate mesoderm and urogenital tract. Loss of Pax2 in mice results in the complete absence of kidneys, ureters, and sex specific epithelial structures derived from the intermediate mesoderm in both males and females. In this report, we describe two new alleles of Pax2 created by inserting the enhanced green fluorescent protein coding region into the 5' untranslated leader sequence. One allele is a hypomorph that generates less protein and exhibits structural defects in kidneys and ureters upon homozygosity. A second allele is a true null that can be used to image Pax2 expressing cells in a mutant background. Organ culture and embryo analyses point to a loss of epithelial cell polarity and increased mobility in cells that have deleted Pax2 function. These experiments provide new insight into the role of Pax2 protein levels in determining correct renal architecture and cell fate. These new Pax2 alleles are valuable genetic reagents for in vivo studies of urogenital development.     

Pax6 and Pdx1 form a functional complex on the rat somatostatin gene upstream enhancer

The somatostatin upstream enhancer (SMS-UE) is a highly complex enhancer element. The distal A-element contains overlapping Pdx1 and Pbx binding sites. However, a point mutation in the A-element that abolishes both Pdxl and Pbx binding does not impair promoter activity. In contrast, a point mutation that selectively eliminates Pdx1 binding to a proximal B-element reduces the promoter activity. The B-element completely overlaps with a Pax6 binding site, the C-element. A point mutation in the C-element demonstrates that Pax6 binding is essential for promoter activity. Interestingly, a block mutation in the A-element reduces both Pax6 binding and promoter activity. In heterologous cells, Pdx1 potentiated Pax6 mediated activation of a somatostatin reporter. We conclude that the beta/delta-cell-specific activity of the SMS-UE is achieved through simultaneous binding of Pdx1 and Pax6 to the B- and C-elements, respectively. Furthermore, the A-element appears to stabilise Pax6 binding.     

Nodal cis-regulatory elements reveal epiblast and primitive endoderm heterogeneity in the peri-implantation mouse embryo

Nodal, a secreted factor known for its conserved functions in cell-fate specification and the establishment of embryonic axes, is also required in mammals to maintain the pluripotency of the epiblast, the tissue that gives rise to all fetal lineages. Although Nodal is expressed as early as E3.5 in the mouse embryo, its regulation and functions at pre- and peri-implantation stages are currently unknown. Sensitive reporter transgenes for two Nodal cis-regulatory regions, the PEE and the ASE, exhibit specific expression profiles before implantation. Mutant and inhibitor studies find them respectively regulated by Wnt/β-catenin signaling and Activin/Nodal signaling, and provide evidence for localized and heterogeneous activities of these pathways in the inner cell mass, the epiblast and the primitive endoderm. These studies also show that Nodal and its prime effector, FoxH1, are not essential to preimplantation Activin/Nodal signaling. Finally, a strong upregulation of the ASE reporter in implanting blastocysts correlates with a downregulation of the pluripotency factor Nanog in the maturing epiblast. This study uncovers conservation in the mouse blastocyst of Wnt/β-catenin and Activin/Nodal-dependent activities known to govern Nodal expression and the establishment of polarity in the blastula of other deuterostomes. Our results indicate that these pathways act early on to initiate distinct cell-specification processes in the ICM derivatives. Our data also suggest that the activity of the Activin/Nodal pathway is dampened by interactions with the molecular machinery of pluripotency until just before implantation, possibly delaying cell-fate decisions in the mouse embryo.     

Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.     

The glucocorticoid receptor recruits the COMPASS complex to regulate inflammatory transcription at macrophage enhancers

1. Download : Download high-res image (131KB)
2. Download : Download full-size image

     

Analysis of Pax6 Contiguous Gene Deletions in the Mouse,Mus musculus,Identifies Regions Distinct from Pax6 Responsible for Extreme Small-Eye and Belly-Spotting Phenotypes

In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca⁺²-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.CONTIGUOUS gene deletions account for a significant portion of human genetic syndromes. The application of fluorescence in situ hybridization (FISH) cytogenetics and array comparative genome hybridization (array-CGH) technologies have enabled more accurate localization of deletion breakpoints. This deletion information combined with the annotation of the human genome structure provides critical information to identify genes responsible for particular phenotypes associated with a syndrome. For example, deletions of the 11p11p12 and 11p13 regions on the short arm of human chromosome (Chr) 11 have been identified in the Potocki–Shaffer syndrome (Shaffer et al. 1993; Bartsch et al. 1996; Potocki and Shaffer 1996) and the Wilm''s tumor- aniridia- genitourinary abnormalities- mental retardation (WAGR) syndrome (Riccardi et al. 1978; Francke et al. 1979; Hittner et al. 1979; Fryns et al. 1981), respectively. Deletion analyses were important in identifying genes associated with clinical features of the syndromes: EXT2 for multiple exostoses and ALX4 for parietal foramina in Potocki–Shaffer syndrome (Ligon et al. 1998; Wu et al. 2000; Wakui et al. 2005), WT1 for Wilm''s tumor, and PAX6 for aniridia in WAGR syndrome (van Heyningen et al. 1985; Glaser et al. 1986, 1992; Fantes et al. 1992). Deletion analyses have also defined the extent of the deleted region in patients with combined Potocki–Shaffer and WAGR syndromes (McGaughran et al. 1995; Brémond-Gignac et al. 2005) as well as microdeletions 3′ to PAX6, which prevent expression of PAX6 and cause aniridia (Lauderdale et al. 2000; D''elia et al. 2007; Davis et al. 2008).The mouse Chr 2 region homologous to the human WAGR region contains the genes Wt1, Rcn1, Pax6, and Elp4. An extensive allelic series at Pax6 has been identified (Bult et al. 2008). Heterozygote Pax6 intragenic null mutants express microphthalmia, iris anomalies, corneal opacities, lens opacities, and lens-corneal adhesions. Homozygotes are anophthalmic and die shortly after birth (Roberts 1967; Hogan et al. 1986). Five deletions in the region have been identified: Pax6^Sey-Dey, Pax6^Sey-H, Pax6^Sey-2H, Pax6^Sey-3H, Pax6^Sey-4H of which two, Pax6^Sey-H (Hogan et al. 1986; Kent et al. 1997; Kleinjan et al. 2002; Webb et al. 2008) and Pax6^Sey-Dey (Theiler et al. 1978; Hogan et al. 1987; Glaser et al. 1990), have been well characterized. Heterozygotes for both deletions express belly spotting and a more extreme eye phenotype than that observed for heterozygotes of intragenic Pax6 null mutations. Homozygotes for both deletions are lethal at an early embryonic stage.We were particularly interested in the extreme eye phenotype associated with the Pax6 deletions and considered two alternative hypotheses. Either Pax6 deletions are functionally different from Pax6 intragenic null mutations or deletion of a region linked to but distinct from the Pax6 structural gene affects the eye phenotype.In the present study we identify three new deletions encompassing the Pax6 region of the mouse. They have been assigned the mutant allele symbols Del(2)Pax6^11Neu/1Neu, Del(2)Pax6^12Neu/2Neu, and Del(2)Pax6^13Neu/3Neu and will be referred to throughout this publication as Pax6^11Neu, Pax6^12Neu, and Pax6^13Neu, respectively. All three deletions are homozygous lethal at an early embryonic stage. The deletions differentiate for the extent of the eye abnormality expressed: Pax6^11Neu heterozygotes express extreme microphthalmia similar to that observed in the Pax6^Sey-Dey and Pax6^Sey-H deletions. Pax6^12Neu and Pax6^13Neu heterozygotes express the milder eye abnormality seen in heterozygous intragenic null mutants. For all three deletions, heterozygotes do not express belly spotting. Genetic, phenotypic, and molecular characterization of the deletions allowed us to identify regions associated with the array of phenotypes in these contiguous gene deletions.     

DNaseI hypersensitive sites 1, 2 and 3 of the human beta-globin dominant control region direct position-independent expression.

The human beta-globin dominant control region (DCR) which flanks the multigene beta-globin locus directs high level, site of integration independent, copy number dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukemia (MEL) cells. We have assayed each of the individual DNaseI hypersensitive regions present in the full 15kb DCR for position independence and copy number dependence of a linked beta-globin gene in transgenic mice. The results show that at least three of the individual DNaseI hypersensitive site regions (sites 1, 2 and 3), though expressing at lower levels than the full DCR, are capable of position independent, copy number dependent expression. Site 2 alone directs the highest level of expression of the single site constructs, producing nearly 70% of the level of the full DCR. Sites 1 and 3 each provide 30% of the full activity. Deletion of either site 2 or 3 from the complete set significantly reduces the level of expression, but does not effect position independence or copy number dependence. This demonstrates that sites 2 and 3 are required for full expression and suggests that all the sites are required for the full expression of even a single gene from this multigene locus.