Saturday, 22 December 2012

Medical applications II: transcriptional pathophysiology


Pax6, a pleiotropic player in development by Simpson and Price
Transcription factors
Drophosila Pax6  homologue eyeless retulates homeobox-containing gene sine oculis through a regulatory element in intron of so gene. Both genes are crucial in eye dev3elopment.

Pax6 regulates a TF, Maf, which is important in cellular differentiation of tissues. Pax6 activates Maf expression.

Pax6 controls bHLH TFs, Mash1, Math5 and Neurogenin2, Binds sequences in enhancers and promoters. bHLH proteins are important in cell differentiation during embryonic develpoment. May mediate effect of Pax6 on retinal progenitor cell fate.

Pax genes and eye organogenesis by Pichaud and Desplan
Pax6 encodes a nuclear TF from Pax family. Pax genes are defined by paired box, which encodes a paired domain (PD), a high conserved DNA binding domain. Pax proteins have other conserved domains eg homeodomain (HD). HD is another DNA-binding domain. Its specificity depends on a residue at position 50. Most homeoproteins have Gln at this position.

Pax 6 and eye organogenesis
Homozygous Pax6  mutants lack eyes and nose. Pax6 gene dosage is important. Heterozygous mutations in Pax6 cause eye defects, Aniridia or some of PEter's anomalies ni human, Small eye in mice or rats.  Extra copies of Pax6 in mice cause simialr eye defects to loss of one copy of locus in mice.

Pax6 is proposed to be the master control gene for eye development. Controls all aspects of eye development.

Presumptive mammalian eye tissue is regionalised and specified through autonomous and inductive processes between apposed neural (optic vesicle) and surface ectoderm (lens placode). Both require Pax6.

In flies, a single ectodermal epithelium forms retina and lens components of eye.

Pax6 mutants have no eyes. But Vertebrate eye cup and eye disc in flies forms in null Pax6 mutant mice, but then degenerates, Suggets that other genes are involved in parallel or upstream of Pax6.

Mouse knockout experiments suggest Pax6 is required in uncommitted retinal cells to retain pluripotency, allowing them to generate different cell types that compose retina. Pax6 is also required for lens development.

Mouse Pax6 interacts with Pax2.

Pax6 expression in the developing human eye by Nishina et al
To investigate changes in Pax6 expression in developing eye, they stained frozen human eyes (6-22 weeks gestation) immunohistochemically with monoclonal Ab to chick Pax6.

Pax6 is expressed on surface and neuroectoderms at early stage, then in differentiating cells in cornea, lens, ciliary body and retina through development.  Pax6 may play a role in determining cell fate in morphogenesis in various human ocular tissue.

Pax6 in sensory development by van Heyningen and Williamson
Pax6 gene: structure and evolution
PAX6 on chromosome 11p13 encodes a 422aa transcriptional regulator. It occupies 14 exons. It has 2 DNA-binding domains, a bipartite paired domain (PRD) and a paired-type homoedomain (HD).

N-terminal PRD has an inframe 14 aa alternatively spliced exon. It is separated from HD by a linker region.  Strong similarity in coding region among species. DNA-binding targets mat be highly conserved.
Figure 2. The genomic organization of the PAX6 gene with its long-range control elements. The 14 exons of the PAX6 gene are shown in red, with the alternatively spliced exon 5a in yellow. The neighbour gene, originally named PAXNEB, is now known to be the ELP4 subunit of a transcriptional elongation factor complex (83). Only the last three exons of ELP4 are shown (in blue). Direction of transcription is indicated by arrows. The table below the diagram details the regulatory elements shown in the diagram and discussed in the text.

PAx6 interactions and functional mechanisms
Figure 3. Upstream regulators and downstream targets, direct and indirect, of the mammalian PAX6/Pax6 gene. Many are discussed and referenced individually in the text and in (31) and (69). As most of the work is carried out in the mouse, official mouse nomenclature is used wherever available, in some cases with old gene names in parentheses.
Bmp4 and Bmp7 has roles in lens induction. Interaction with FGF signalling regulates Pax6.

Autoregulation by Pax6. Mutual regulation of Pax6/Six3 interactions and PAx6 and Pax2 modulation.

Some genes act at both DNA and protein level. Sox2 and Pax6 proteins form a co-DNA-binding complex with a key role in lens development. Control Sox2 expression by binding its promoter.

Figure 1. Domain structure of PAX6 with the paired domain structure shown in detail to illustrate the paired domain missense mutations identified in human and mouse. The protein consists of a paired domain (PRD) including the checkered alternatively spliced exon 5a; the homeodomain (HD), separated from the PRD by a linker region; and the proline, serine and threonine-rich transactivation domain (PST). The first three exons and most of the fourth exon constitute the 5′UTR; the 3′UTR is about 1 kb. The expanded paired domain includes α1–α3, denoting the N-terminal subdomain alpha helices, and α4–α6, C-terminal subdomain alpha helices. The amino acid sequence is shown, with residues in red contacting the DNA backbone and those in blue contacting the minor groove or the major groove (underlined). Human missense mutations are highlighted in pink, mouse mutations in green. Numbering for the alternatively spliced exon (chequered box) is from the start of that segment only. Mutations are referenced in the text, atwww.hgu.mrc.ac.uk/Softdata/PAX6/ and in (9); the two mutations in bold are our unpublished data.
Mutations of PAX6 gene detected in patients with a variety of optic nerve malformations by Azuma et al
Pax6 mutations have been detected in ocular anomalies eg aniridia, Peters anomaly, corneal dystrophy, congenital catarcts and foveal hypoplasia.

Novel mutations were found in 8 pedigrees with optic nerve malformations eg coloboma, morning glory disc anomaly, optic nerve hypoplasia/aplasia, persient hyperplastic primary vitreous. A functional assay showed that ecah mutation decreased transcriptional activation potential of PAX6 through paired DNA-binding domain. Pax6 and Pax2 are each thought to downregulate the expression of the other. 4 of detected mutations affected Pax6-mediated transcriptional repression of Pax2 promoter in a reporter assay. As Pax2 gene mutations were detected in papillorenal syndrome, alternation of PAx2 function by Pax6 mutations may affect phenotypic manifestations of optic nerve malformations.

Genetic analysis indicates that haploinsufficiency of gene causes classical aniridia phenotype, in which all eye tissues are affected. Most mutations in aniridia cause premature translational termination one one of the alleles. As most aa residues are conserved, missense mutations may alter degree and specificity of DNA binding and transcriptional regulation by Pax6 to diff extent.

To examine possibly functional changes by detected mutations, perform CAT assay in mouse embryonic carcinoma P19 cells.   A CAT-reporter construct carrying 6 copies of P6CON, consensus binding sequence of PAx6PD was used. Wt PAx6 strongly activated CAT-reporter gene expressoin. Transcriptional activation potential of Pax6 was affected by all mutations. Mutations in HD (F258S) impaired PD-mediated transcriptional activation. Consistent with functional interplay of PD and HD.

To examine effect of PAx6 and PAx2 proteins on activities of Pax2 and Pax6 promoters, use a CAT-reporter contsrucgt carrying a Pax6 promoter region or Pax2 promoter region. When increasing amoung of Pax6 or Pax2 expression construct was cotransfected into P19 cells with constant cmount of PAx2 or Pax6 reporter construct, each CAT activity decreased in dose-dependent manner. Indicates Pax6 represses PAx2 expression and vice versa.


Figure 2. Effects of PAX6 on PAX2 (a) and of PAX2 on PAX6 (c). CAT activities were measured in P19 cells after cotransfection of effecter and reporter constructs. Total volume of DNA was adjusted with an empty vector, pBluescript. Cell extracts were prepared after 48 h and assayed for CAT activities by use of a FAST CAT Green Reagent (Molecular Probes). The CAT activity was quantified by measurement with a phospho-fluor-imager (Molecular Dynamics) and illustrated in a fold-activation, compared with the condition with the vector alone. The levels of PAX2were suppressed with increasing amounts of wild type of PAX6, and vice versa. b, Transactivating potential of PAX6 mutants. 0.1 μg of effecter construct and 1 μg of reporter constructs were cotransfected in P19 cells. Transcription level from P6CON was disturbed significantly by P68S, Q205X, F258S, and S292I mutants and slightly by S363P, Q378R, M381V, and T391A mutants. d, Effects of PAX6 mutants on PAX2 expression. One μg of effecter construct and 1 μg of reporter constructs were cotransfected in P19 cells. The decreasing level of PAX6 was significantly disturbed with the P68S, Q205X, S292I, and M381V mutants. Each photograph of CAT assay under the bar graph is representative of at least three independent experiments.













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