Ann C. Morris

CREB Regulates MHC Class II Expression in a CIITA-Dependent Manner

The X2 box of MHC class II promoters is homologous to TRE/CRE elements and is required for expression of MHC class II genes. The X2 box-specific DNA binding activity, X2BP, was purified to homogeneity, sequenced, and identified as CREB. Transient transactivation experiments showed that CREB can cooperate with CIITA to enhance activation of transcription from MHC class II promoters in a dose-dependent manner. Binding of CREB to the class II promoter in vivo was demonstrated by a chromatin immunoprecipitation assay. Additionally, ICER, a dominant inhibitor of CREB function, was found to repress class II expression. These results demonstrate that CREB binds to the X2 box in vivo and cooperates with CIITA to direct MHC class II expression.

Kinetics of a Gamma Interferon Response: Expression and Assembly of CIITA Promoter IV and Inhibition by Methylation

ABSTRACT Chromatin immunoprecipitation assays were employed to assess the kinetics of transcription factor assembly and histone modifications that occur during gamma interferon (IFN-γ) induction of CIITA gene expression. CIITA is the master regulator of major histocompatibility complex class II transcription. Promoter IV (PIV), the major IFN-γ responsive promoter for CIITA expression, requires both STAT1 and IFN regulatory factor 1 (IRF-1) for induction by IFN-γ. STAT1 binding to PIV was detected first and was accompanied by a modest acetylation of histones H3 and H4 that were associated with the region. Despite these changes, which occurred within 30 min of IFN-γ treatment, CIITA mRNA was not detected until IRF-1 protein was synthesized and bound to its site, a process that required >120 min. In contrast to these events, fetal trophoblast-like cell lines, which are refractory to CIITA induction by IFN-γ, failed to assemble the above factors or modify their chromatin, suggesting that accessibility to the promoter is blocked. Bisulfite sequencing of PIV showed strong hypermethylation of PIV, providing a link between methylation, chromatin structure, and factor binding. Together, this analysis provides a kinetic view of the activation of the CIITA gene in response to IFN-γ and shows that regulatory factor assembly, chromatin modification, and gene expression proceed in discrete steps.

Methylation of Class II trans -Activator Promoter IV: A Novel Mechanism of MHC Class II Gene Control

Inhibition of class II trans-activator (CIITA) expression prevents embryonic trophoblast cells from up-regulating MHC class II genes in response to IFN-γ. This is thought to be one mechanism of maternal tolerance to the fetal allograft. The CIITA gene is regulated by four distinct promoters; promoter III directs constitutive (B cell) expression, and promoter IV regulates IFN-γ-inducible expression. Using in vivo genomic footprinting, promoter-reporter analysis, Southern blot analysis, and RT-PCR, we have examined the cause of CIITA silencing in a trophoblast-derived cell line. We report here that methylation of promoter IV DNA at CpG sites in Jar cells prevents promoter occupancy and IFN-γ-inducible transcription. The inhibition of CpG methylation in Jar cells by treatment with 5-aza-2′-deoxycytidine restores IFN-γ inducibility to CIITA. This is the first description of an epigenetic mechanism involved in regulation of CIITA and MHC class II gene expression.

Sp1 Binding Is Critical for Promoter Assembly and Activation of the MCP-1 Gene by Tumor Necrosis Factor

The monocyte chemoattractant protein-1 gene (MCP-1) is induced by the inflammatory cytokine tumor necrosis factor through the coordinate assembly of an NF-κB-dependent distal regulatory region and a proximal region that has been suggested to bind Sp1 as well as other factors. To provide a genetic correlation for Sp1 activity in this system, a cell line homozygous for a targeted truncation of the Sp1 gene was derived and examined. We found that the lack of Sp1 binding activity resulted in the inability of both the distal and proximal regions to assemble in vivo even though the binding of NF-κB to distal region DNA was unaffected in vitro. We also found that Sp1 and NF-κB were the minimal mammalian transcription factors required for efficient activity when transfected into Drosophila Schneider cells. Additionally, Sp3 was able to compensate for Sp1 in the Drosophila tissue cell system but not in the Sp1−/− cell line suggesting that Sp1 usage is site-specific and is likely to depend on the context of the binding site. Together, these data provide genetic and biochemical proof for Sp1 in regulating the MCP-1 gene.

Genetic Dissection Reveals Two Separate Pathways for Rod and Cone Regeneration in the Teleost Retina

Development of therapies to treat visual system dystrophies resulting from the degeneration of rod and cone photoreceptors may directly benefit from studies of animal models, such as the zebrafish, that display continuous retinal neurogenesis and the capacity for injury‐induced regeneration. Previous studies of retinal regeneration in fish have been conducted on adult animals and have relied on methods that cause acute damage to both rods and cones, as well as other retinal cell types. We report here the use of a genetic approach to study progenitor cell responses to photoreceptor degeneration in the larval and adult zebrafish retina. We have compared the responses to selective rod or cone degeneration using, respectively, the XOPS‐mCFP transgenic line and zebrafish with a null mutation in the pde6c gene. Notably, rod degeneration induces increased proliferation of progenitors in the outer nuclear layer (ONL) and is not associated with proliferation or reactive gliosis in the inner nuclear layer (INL). Molecular characterization of the rod progenitor cells demonstrated that they are committed to the rod photoreceptor fate while they are still mitotic. In contrast, cone degeneration induces both Müller cell proliferation and reactive gliosis, with little change in proliferation in the ONL. We found that in both lines, proliferative responses to photoreceptor degeneration can be observed as 7 days post fertilization (dpf). These two genetic models therefore offer new opportunities for investigating the molecular mechanisms of selective degeneration and regeneration of rods and cones.

MHC class II gene silencing in trophoblast cells is caused by inhibition of CIITA expression

PROBLEM: Major histocompatibility complex (MHC) class II molecule expression is specifically suppressed on fetal trophoblasts, even in response to interferon (IFN)‐γ, a potent inducer of MHC class II genes. The suppression of class II induction has been suggested to play a role in preventing rejection of the fetal allograft. The mechanism of this suppression is unknown.

Tbx2b is required for ultraviolet photoreceptor cell specification during zebrafish retinal development

The vertebrate rod and cone photoreceptors are highly specialized sensory neurons that transduce light into the chemical and electrical signals of the nervous system. Although the physiological properties of cones and rods are well known, only a handful of genes have been identified that regulate the specification of photoreceptor subtypes. Taking advantage of the mosaic organization of photoreceptors in zebrafish, we report the isolation of a mutation resulting in a unique change in photoreceptor cell fate. Mutation of the lots-of-rods (lor) locus results in a near one-for-one transformation of UV-cone precursors into rods. The transformed cells exhibit morphological characteristics and a gene-expression pattern typical of rods, but differentiate in a temporal and spatial pattern consistent with UV-cone development. In mutant larvae and adults, the highly ordered photoreceptor mosaic is maintained and degeneration is not observed, suggesting that lor functions after the specification of the other photoreceptor subtypes. In genetic chimeras, lor functions cell-autonomously in the specification of photoreceptor cell fate. Linkage analysis and genetic-complementation testing indicate that lor is an allele of tbx2b/fby (from beyond). fby was identified by a pineal complex phenotype, and carries a nonsense mutation in the T-box domain of the tbx2b transcription factor. Homozygous fby mutant larvae and lor/fby transheterozygotes also display the lots-of-rods phenotype. Based upon these data, we propose a previously undescribed function for tbx2b in photoreceptor cell precursors, to promote the UV cone fate by repressing the rod differentiation pathway.

Studying rod photoreceptor development in zebrafish.

The zebrafish has rapidly become a favored model vertebrate organism, well suited for studies of developmental processes using large-scale genetic screens. In particular, zebrafish morphological and behavioral genetic screens have led to the identification of genes important for development of the retinal photoreceptors. This may help clarify the genetic mechanisms underlying human photoreceptor development and dysfunction in retinal diseases. In this review, we present the advantages of zebrafish as a vertebrate model organism, summarize retinal and photoreceptor cell development in zebrafish, with emphasis on the rod photoreceptors, and describe zebrafish visual behaviors that can be used for genetic screens. We then describe some of the photoreceptor cell mutants that have been isolated in morphological and behavioral screens and discuss the limitations of current screening methods for uncovering mutations that specifically affect rod function. Finally, we present some alternative strategies to target the rod developmental pathway in zebrafish.

Microarray Analysis of XOPS-mCFP Zebrafish Retina Identifies Genes Associated with Rod Photoreceptor Degeneration and Regeneration

PURPOSE XOPS-mCFP transgenic zebrafish experience a continual cycle of rod photoreceptor development and degeneration throughout life, making them a useful model for investigating the molecular determinants of rod photoreceptor regeneration. The purpose of this study was to compare the gene expression profiles of wild-type and XOPS-mCFP retinas and identify genes that may contribute to the regeneration of the rods. METHODS Adult wild-type and XOPS-mCFP retinal mRNA was subjected to microarray analysis. Pathway analysis was used to identify biologically relevant processes that were significantly represented in the dataset. Expression changes were verified by RT-PCR. Selected genes were further examined during retinal development and in adult retinas by in situ hybridization and immunohistochemistry and in a transgenic fluorescent reporter line. RESULTS More than 600 genes displayed significant expression changes in XOPS-mCFP retinas compared with expression in wild-type controls. Many of the downregulated genes were associated with phototransduction, whereas upregulated genes were associated with several biological functions, including cell cycle, DNA replication and repair, and cell development and death. RT-PCR analysis of a subset of these genes confirmed the microarray RESULTS Three transcription factors (sox11b, insm1a, and c-myb), displaying increased expression in XOPS-mCFP retinas, were also expressed throughout retinal development and in the persistently neurogenic ciliary marginal zone. CONCLUSIONS This study identified numerous gene expression changes in response to rod degeneration in zebrafish and further suggests a role for the transcriptional regulators sox11b, insm1a, and c-myb in both retinal development and rod photoreceptor regeneration.

Sox11 Is Required to Maintain Proper Levels of Hedgehog Signaling during Vertebrate Ocular Morphogenesis

Ocular coloboma is a sight-threatening malformation caused by failure of the choroid fissure to close during morphogenesis of the eye, and is frequently associated with additional anomalies, including microphthalmia and cataracts. Although Hedgehog signaling is known to play a critical role in choroid fissure closure, genetic regulation of this pathway remains poorly understood. Here, we show that the transcription factor Sox11 is required to maintain specific levels of Hedgehog signaling during ocular development. Sox11-deficient zebrafish embryos displayed delayed and abnormal lens formation, coloboma, and a specific reduction in rod photoreceptors, all of which could be rescued by treatment with the Hedgehog pathway inhibitor cyclopamine. We further demonstrate that the elevated Hedgehog signaling in Sox11-deficient zebrafish was caused by a large increase in shha transcription; indeed, suppressing Shha expression rescued the ocular phenotypes of sox11 morphants. Conversely, over-expression of sox11 induced cyclopia, a phenotype consistent with reduced levels of Sonic hedgehog. We screened DNA samples from 79 patients with microphthalmia, anophthalmia, or coloboma (MAC) and identified two novel heterozygous SOX11 variants in individuals with coloboma. In contrast to wild type human SOX11 mRNA, mRNA containing either variant failed to rescue the lens and coloboma phenotypes of Sox11-deficient zebrafish, and both exhibited significantly reduced transactivation ability in a luciferase reporter assay. Moreover, decreased gene dosage from a segmental deletion encompassing the SOX11 locus resulted in microphthalmia and related ocular phenotypes. Therefore, our study reveals a novel role for Sox11 in controlling Hedgehog signaling, and suggests that SOX11 variants contribute to pediatric eye disorders.