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Identification of retinal homeobox (rax) gene-dependent genes by a microarray approach: The DNA endoglycosylase neil3 is a major downstream component of the rax genetic pathway.

ABSTRACT: BACKGROUND:The retinal homeobox (rx/rax) gene is a transcription factor expressed in the developing eye field that is necessary for normal eye development. rax is necessary for retinal specification and stem cell development. The genetic program of early retinal development, including rax expression, can be induced in naïve ectoderm by activation of insulin-like growth factor (IGF) signaling. We have undertaken a microarray-based approach to identify rax-dependent IGF-induced genes. RESULTS:We identified 21 IGF-induced genes that exhibit at least a two-fold decrease in expression when rax expression is knocked down. Ten of these genes were expressed in the developing eye, eight were expressed in the ciliary marginal zone of the mature tadpole retina, and four could significantly rescue the rax knockdown phenotype. One of these, the nei endonuclease VIII-like 3 (neil3) gene, rescued the rax knockdown phenotype to a remarkable degree. We found that neil3 is necessary for normal retinal lamination and retinal neuron differentiation. CONCLUSIONS:We have identified neil3 as a component of the rax genetic pathway necessary for normal retinal progenitor cell development. neil3 is involved in the base excision DNA repair pathway, suggesting that this pathway is essential for normal rax-dependent progenitor cell development in the mature retina. Developmental Dynamics 247:1199-1210, 2018. © 2018 Wiley Periodicals, Inc.

SUBMITTER: Pan Y

PROVIDER: S-EPMC6246801 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Identification of retinal homeobox (rax) gene-dependent genes by a microarray approach: The DNA endoglycosylase neil3 is a major downstream component of the rax genetic pathway.

Pan Yi Y Kelly Lisa E LE El-Hodiri Heithem M HM

Developmental dynamics : an official publication of the American Association of Anatomists 20181109 11

<h4>Background</h4>The retinal homeobox (rx/rax) gene is a transcription factor expressed in the developing eye field that is necessary for normal eye development. rax is necessary for retinal specification and stem cell development. The genetic program of early retinal development, including rax expression, can be induced in naïve ectoderm by activation of insulin-like growth factor (IGF) signaling. We have undertaken a microarray-based approach to identify rax-dependent IGF-induced genes.<h4>R ...[more]

PMID: 30311321

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Project description:PurposeThe evolutionarily conserved retinal homeobox (Rax) transcription factor is essential for normal eye development in all vertebrates. Despite Rax's biologic significance, the molecular mechanisms underlying Rax molecular function as a transcriptional regulator are poorly defined. The rax gene encodes a conserved octapeptide motif (OP) near the N-terminus and several conserved regions in the C-terminus of unknown function, including the orthopedia, aristaless, rax (OAR) domain and the RX domain. The purpose of this study is to investigate the contribution of these conserved domains in Rax function.MethodsN-and C-terminal deletion and point mutations were generated in Xenopus laevis rax.L (previously known as Rx1A) using PCR-based methods. We examined the ability of mutated Rax to transactivate a reporter gene consisting of a portion of a rax target gene promoter (from the Xenopus rhodopsin gene) fused to a firefly luciferase coding region and transfected into human embryonic kidney 293T (HEK293T) cells. Portions of the Rax C-terminal region were also assayed for transactivation activity in the context of a heterologous DNA binding domain with an appropriate reporter gene.ResultsFull-length Rax weakly activated the reporter. Deletion of the Rax C-terminus increased Rax activity, suggesting that the C-terminus functions to repress Rax activity. Further deletion eventually resulted in a decrease in activity, suggesting that the C-terminal region also can function to enhance Rax activity. Deletion or mutation of the OP motif resulted in a slight decrease in Rax activity. Mutation or deletion of the N-terminal OP motif resulted in a mild decrease in activity and dampened the activity levels of the C-terminal deletions. Further, fusion of the C-terminus of Rax to a heterologous DNA binding domain enhanced transactivation.ConclusionsThe present data indicate that the C-terminus of Rax can function to repress or activate transcription in a context-dependent manner. These data support our hypothesis that the highly conserved OAR domain, in combination with other regulatory elements in the Rax C-terminus, coordinates Rax activity, perhaps through functional interaction with the N-terminal OP motif. Taken together, these data provide insight into the structural features that regulate Rax activity.

Project description:Due to the extensive use of chloroacetanilide herbicides over the past 60 years, bacteria have evolved catabolic pathways to mineralize these compounds. In the upstream catabolic pathway, chloroacetanilide herbicides are transformed into the two common metabolites 2-methyl-6-ethylaniline (MEA) and 2,6-diethylaniline (DEA) through N-dealkylation and amide hydrolysis. The pathway downstream of MEA is initiated by the hydroxylation of aromatic rings, followed by its conversion to a substrate for ring cleavage after several steps. Most of the key genes in the pathway have been identified. However, the genes involved in the initial hydroxylation step of MEA are still unknown. As a special aniline derivative, MEA cannot be transformed by the aniline dioxygenases that have been characterized. Sphingobium baderi DE-13 can completely degrade MEA and use it as a sole carbon source for growth. In this work, an MEA degradation-deficient mutant of S. baderi DE-13 was isolated. MEA catabolism genes were predicted through comparative genomic analysis. The results of genetic complementation and heterologous expression demonstrated that the products of meaX and meaY are responsible for the initial step of MEA degradation in S. baderi DE-13. MeaXY is a two-component flavoprotein monooxygenase system that catalyzes the hydroxylation of MEA and DEA using NADH and flavin mononucleotide (FMN) as cofactors. Nuclear magnetic resonance (NMR) analysis confirmed that MeaXY hydroxylates MEA and DEA at the para-position. Transcription of meaX was enhanced remarkably upon induction of MEA or DEA in S. baderi DE-13. Additionally, meaX and meaY were highly conserved among other MEA-degrading sphingomonads. This study fills a gap in our knowledge of the biochemical pathway that carries out mineralization of chloroacetanilide herbicides in sphingomonads.IMPORTANCE Much attention has been paid to the environmental fate of chloroacetanilide herbicides used for the past 60 years. Microbial degradation is considered an important mechanism in the degradation of these compounds. Bacterial degradation of chloroacetanilide herbicides has been investigated in many recent studies. Pure cultures or consortia able to mineralize these herbicides have been obtained. The catabolic pathway has been proposed, and most key genes involved have been identified. However, the genes responsible for the initiation step (from MEA to hydroxylated MEA or from DEA to hydroxylated DEA) of the downstream pathway have not been reported. The present study demonstrates that a two-component flavin-dependent monooxygenase system, MeaXY, catalyzes the para-hydroxylation of MEA or DEA in sphingomonads. Therefore, this work finds a missing link in the biochemical pathway that carries out the mineralization of chloroacetanilide herbicides in sphingomonads. Additionally, the results expand our understanding of the degradation of a special kind of aniline derivative.

Dataset Information

Identification of retinal homeobox (rax) gene-dependent genes by a microarray approach: The DNA endoglycosylase neil3 is a major downstream component of the rax genetic pathway.

Publications

Identification of retinal homeobox (rax) gene-dependent genes by a microarray approach: The DNA endoglycosylase neil3 is a major downstream component of the rax genetic pathway.

Similar Datasets

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