Epigenetics and cell death: DNA hypermethylation in programmed retinal cell death.

BackgroundVertebrate genomes undergo epigenetic reprogramming during development and disease. Emerging evidence suggests that DNA methylation plays a key role in cell fate determination in the retina. Despite extensive studies of the programmed cell death that occurs during retinal development and degeneration, little is known about how DNA methylation might regulate neuronal cell death in the retina.MethodsThe developing chicken retina and the rd1 and rhodopsin-GFP mouse models of retinal degeneration were used to investigate programmed cell death during retinal development and degeneration. Changes in DNA methylation were determined by immunohistochemistry using antibodies against 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC).ResultsPunctate patterns of hypermethylation paralleled patterns of caspase3-dependent apoptotic cell death previously reported to occur during development in the chicken retina. Degenerating rd1 mouse retinas, at time points corresponding to the peak of rod cell death, showed elevated signals for 5mC and 5hmC in photoreceptors throughout the retina, with the most intense staining observed in the peripheral retina. Hypermethylation of photoreceptors in rd1 mice was associated with TUNEL and PAR staining and appeared to be cCaspase3-independent. After peak rod degeneration, during the period of cone death, occasional hypermethylation was observed in the outer nuclear layer.ConclusionThe finding that cell-specific increases of 5mC and 5hmC immunostaining are associated with the death of retinal neurons during both development and degeneration suggests that changes in DNA methylation may play a role in modulating gene expression during the process of retinal degeneration. During retinal development, hypermethylation of retinal neurons associates with classical caspase-dependent apoptosis as well as caspase-3 independent cell death, while hypermethylation in the rd1 mouse photoreceptors is primarily associated with caspase-3 independent programmed cell death. These findings suggest a previously unrecognized role for epigenetic mechanisms in the onset and/or progression of programed cell death in the retina

Cornea organoids from human induced pluripotent stem cells.

The cornea is the transparent outermost surface of the eye, consisting of a stratified epithelium, a collagenous stroma and an innermost single-cell layered endothelium and providing 2/3 of the refractive power of the eye. Multiple diseases of the cornea arise from genetic defects where the ultimate phenotype can be influenced by cross talk between the cell types and the extracellular matrix. Cell culture modeling of diseases can benefit from cornea organoids that include multiple corneal cell types and extracellular matrices. Here we present human iPS cell-derived organoids through sequential rounds of differentiation programs. These organoids share features of the developing cornea, harboring three distinct cell types with expression of key epithelial, stromal and endothelial cell markers. Cornea organoid cultures provide a powerful 3D model system for investigating corneal developmental processes and their disruptions in diseased conditions

Identification of tissue-specific cis-regulatory modules based on interactions between transcription factors

<p>Abstract</p> <p>Background</p> <p>Evolutionary conservation has been used successfully to help identify cis-acting DNA regions that are important in regulating tissue-specific gene expression. Motivated by increasing evidence that some DNA regulatory regions are not evolutionary conserved, we have developed an approach for cis-regulatory region identification that does not rely upon evolutionary sequence conservation.</p> <p>Results</p> <p>The conservation-independent approach is based on an empirical potential energy between interacting transcription factors (TFs). In this analysis, the potential energy is defined as a function of the number of TF interactions in a genomic region and the strength of the interactions. By identifying sets of interacting TFs, the analysis locates regions enriched with the binding sites of these interacting TFs. We applied this approach to 30 human tissues and identified 6232 putative cis-regulatory modules (CRMs) regulating 2130 tissue-specific genes. Interestingly, some genes appear to be regulated by different CRMs in different tissues. Known regulatory regions are highly enriched in our predicted CRMs. In addition, DNase I hypersensitive sites, which tend to be associated with active regulatory regions, significantly overlap with the predicted CRMs, but not with more conserved regions. We also find that conserved and non-conserved CRMs regulate distinct gene groups. Conserved CRMs control more essential genes and genes involved in fundamental cellular activities such as transcription. In contrast, non-conserved CRMs, in general, regulate more non-essential genes, such as genes related to neural activity.</p> <p>Conclusion</p> <p>These results demonstrate that identifying relevant sets of binding motifs can help in the mapping of DNA regulatory regions, and suggest that non-conserved CRMs play an important role in gene regulation.</p

Zinc-finger domains of the transcriptional repressor KLF15 bind multiple sites in rhodopsin and IRBP promoters including the CRS-1 and G-rich repressor elements

BACKGROUND: In the retina, many of the genes that encode components of the visual transduction cascade and retinoid recycling are exclusively expressed in photoreceptor cells and show highly stereotyped temporal and spatial expression patterns. Multiple transcriptional activators of photoreceptor-specific genes have been identified, but little is known about negative regulation of gene expression in the retina. We recently identified KLF15, a member of the Sp/Krüppel-like Factor family of zinc-finger containing transcription factors, as an in vitro repressor of the promoters of the photoreceptor-specific genes rhodopsin and IRBP/Rbp3. To gain further insight into the mechanism of KLF15-mediated regulation of gene expression, we have characterized the binding characteristics and specificity of KLF15's DNA binding domains and defined the KLF15 binding sites in the rhodopsin and IRBP promoters. RESULTS: In EMSA and DNAseI footprinting assays, a KLF15-GST fusion protein containing the C-terminal zinc-finger domains (123 amino acids) showed zinc-dependent and sequence-specific binding to a 9 bp consensus sequence containing a core CG/TCCCC. Both the bovine rhodopsin and IRBP promoters contained multiple KLF15 binding sites that included the previously identified CRS-1 and G-rich repressor elements. KLF15 binding sites were highly conserved between the bovine, human, chimp and dog rhodopsin promoters, but less conserved in rodents. KLF15 reduced luciferase expression by bRho130-luc (containing 4 KLF15 sites) and repressed promoter activation by CRX (cone rod homeobox) and/or NRL (neural retina leucine zipper), although the magnitude of the reduction was smaller than previously reported for a longer bRho225-luc (containing 6 KFL15 sites). CONCLUSION: KLF15 binds to multiple 9 bp consensus sites in the Rhodospin and IRBP promoters including the CRS-1 and G-rich repressor elements. Based on the known expression pattern of KLF15 in non-photoreceptor cells, we hypothesize an in vivo role for KLF15 in repressing photoreceptor-specific gene expression in the inner retina

TiGER: A database for tissue-specific gene expression and regulation

<p>Abstract</p> <p>Background</p> <p>Understanding how genes are expressed and regulated in different tissues is a fundamental and challenging question. However, most of currently available biological databases do not focus on tissue-specific gene regulation.</p> <p>Results</p> <p>The recent development of computational methods for tissue-specific combinational gene regulation, based on transcription factor binding sites, enables us to perform a large-scale analysis of tissue-specific gene regulation in human tissues. The results are stored in a web database called TiGER (Tissue-specific Gene Expression and Regulation). The database contains three types of data including tissue-specific gene expression profiles, combinatorial gene regulations, and cis-regulatory module (CRM) detections. At present the database contains expression profiles for 19,526 UniGene genes, combinatorial regulations for 7,341 transcription factor pairs and 6,232 putative CRMs for 2,130 RefSeq genes.</p> <p>Conclusion</p> <p>We have developed and made publicly available a database, TiGER, which summarizes and provides large scale data sets for tissue-specific gene expression and regulation in a variety of human tissues. This resource is available at <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>.</p

Analysis of regulatory network topology reveals functionally distinct classes of microRNAs

MicroRNAs (miRNAs) negatively regulate the expression of target genes at the post-transcriptional level. Little is known about the crosstalk between miRNAs and transcription factors (TFs). Here we provide data suggesting that the interaction patterns between TFs and miRNAs can influence the biological functions of miRNAs. From this global survey, we find that a regulated feedback loop, in which two TFs regulate each other and one miRNA regulates both of the factors, is the most significantly overrepresented network motif. Mathematical modeling shows that the miRNA in this motif stabilizes the feedback loop to resist environmental perturbation, providing one mechanism to explain the robustness of developmental programs that is contributed by miRNAs. Furthermore, on the basis of a network motif profile analysis, we demonstrate the existence of two classes of miRNAs with distinct network topological properties. The first class of miRNAs is regulated by a large number of TFs, whereas the second is regulated by only a few TFs. The differential expression level of the two classes of miRNAs in embryonic developmental stages versus adult tissues suggests that the two classes may have fundamentally different biological functions. Our results demonstrate that the TFs and miRNAs extensively interact with each other and the biological functions of miRNAs may be wired in the regulatory network topology

Genome-wide prediction and characterization of interactions between transcription factors in Saccharomyces cerevisiae

Combinatorial regulation by transcription factor complexes is an important feature of eukaryotic gene regulation. Here, we propose a new method for identification of interactions between transcription factors (TFs) that relies on the relationship of their binding sites, and we test it using Saccharomyces cerevisiae as a model system. The algorithm predicts interacting TF pairs based on the co-occurrence of their binding motifs and the distance between the motifs in promoter sequences. This allows investigation of interactions between TFs without known binding motifs or expression data. With this approach, 300 significant interactions involving 77 TFs were identified. These included more than 70% of the known protein–protein interactions. Approximately half of the detected interacting motif pairs showed strong preferences for particular distances and orientations in the promoter sequences. These one dimensional features may reflect constraints on allowable spatial arrangements for protein–protein interactions. Evidence for biological relevance of the observed characteristic distances is provided by the finding that target genes with the same characteristic distances show significantly higher co-expression than those without preferred distances. Furthermore, the observed interactions were dynamic: most of the TF pairs were not constitutively active, but rather showed variable activity depending on the physiological condition of the cells. Interestingly, some TF pairs active in multiple conditions showed preferences for different distances and orientations depending on the condition. Our prediction and characterization of TF interactions may help to understand the transcriptional regulatory networks in eukaryotic systems

Photoreceptor Outer Segment-like Structures in Long-Term 3D Retinas from Human Pluripotent Stem Cells.

The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro

Identification of regulatory targets of tissue-specific transcription factors: application to retina-specific gene regulation

Identification of tissue-specific gene regulatory networks can yield insights into the molecular basis of a tissue's development, function and pathology. Here, we present a computational approach designed to identify potential regulatory target genes of photoreceptor cell-specific transcription factors (TFs). The approach is based on the hypothesis that genes related to the retina in terms of expression, disease and/or function are more likely to be the targets of retina-specific TFs than other genes. A list of genes that are preferentially expressed in retina was obtained by integrating expressed sequence tag, SAGE and microarray datasets. The regulatory targets of retina-specific TFs are enriched in this set of retina-related genes. A Bayesian approach was employed to integrate information about binding site location relative to a gene's transcription start site. Our method was applied to three retina-specific TFs, CRX, NRL and NR2E3, and a number of potential targets were predicted. To experimentally assess the validity of the bioinformatic predictions, mobility shift, transient transfection and chromatin immunoprecipitation assays were performed with five predicted CRX targets, and the results were suggestive of CRX regulation in 5/5, 3/5 and 4/5 cases, respectively. Together, these experiments strongly suggest that RP1, GUCY2D, ABCA4 are novel targets of CRX