Cis-regulation in the Mammalian Rod Photoreceptor PDF Download
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Author: Jamie C. Kwasnieski
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 123
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Book Description
Transcription factors regulate the expression level of target genes by binding to cis-regulatory elements (CREs) present in gene promoters. The goal of my thesis research is to define the sequence components of CREs that determine transcriptional output. In order to accomplish this goal, I developed a method to measure the regulatory activity of thousands of CREs in a single experiment. In this method I insert unique barcodes in the 3'UTR of a reporter gene and multiplex expression measurements with RNA sequencing. Using this technique in explanted retinas, I determined the impact of single nucleotide variants in a mammalian promoter by measuring expression controlled by all single nucleotide variants of the Rhodopsin proximal promoter. I found that nearly all (86%) sequence variants drive significantly different activity than the wild-type promoter and that the mechanism of most variants can be interpreted as altered transcription factor binding. In addition, we found that the largest changes in expression resulted from variants located in characterized transcription factor binding site sequences. Next, I explored how combinations of binding sites drive particular levels of gene expression by utilizing a synthetic biology approach. I generated synthetic CREs composed of various combinations of binding sites found in the Rhodopsin promoter and measured the expression driven by these sequences. In this study I found that synthetic CREs containing binding sites for transcriptional activators yielded diverse expression outputs, including both activation and repression of a minimal promoter. Together, these experiments demonstrate that interactions between binding sites and dual regulation of a single binding site can produce diverse gene expression patterns. I conclude that simple cis-regulatory elements can produce complex expression outputs due to interactions between transcriptional activators and detailed quantitative models will be necessary to predict expression from these sequences.
Author: Jamie C. Kwasnieski
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 123
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Book Description
Transcription factors regulate the expression level of target genes by binding to cis-regulatory elements (CREs) present in gene promoters. The goal of my thesis research is to define the sequence components of CREs that determine transcriptional output. In order to accomplish this goal, I developed a method to measure the regulatory activity of thousands of CREs in a single experiment. In this method I insert unique barcodes in the 3'UTR of a reporter gene and multiplex expression measurements with RNA sequencing. Using this technique in explanted retinas, I determined the impact of single nucleotide variants in a mammalian promoter by measuring expression controlled by all single nucleotide variants of the Rhodopsin proximal promoter. I found that nearly all (86%) sequence variants drive significantly different activity than the wild-type promoter and that the mechanism of most variants can be interpreted as altered transcription factor binding. In addition, we found that the largest changes in expression resulted from variants located in characterized transcription factor binding site sequences. Next, I explored how combinations of binding sites drive particular levels of gene expression by utilizing a synthetic biology approach. I generated synthetic CREs composed of various combinations of binding sites found in the Rhodopsin promoter and measured the expression driven by these sequences. In this study I found that synthetic CREs containing binding sites for transcriptional activators yielded diverse expression outputs, including both activation and repression of a minimal promoter. Together, these experiments demonstrate that interactions between binding sites and dual regulation of a single binding site can produce diverse gene expression patterns. I conclude that simple cis-regulatory elements can produce complex expression outputs due to interactions between transcriptional activators and detailed quantitative models will be necessary to predict expression from these sequences.
Author: Karen Ann Lawrence
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 132
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Book Description
Crx is the principal transcription factor of the photoreceptor transcriptional network and is a key regulator of photoreceptor differentiation and survival. Mice mutant for Crx lack functional photoreceptors and are blind at birth. Mutations in human CRX are associated with several retinal diseases including autosomal dominant cone-rod dystrophy 2 and Leber's congenital amaurosis, a severe form of blindness in newborns. Given the importance of Crx in photoreceptor development and maintenance, genome-wide mapping of Crx binding sites was used to facilitate identification of photoreceptor cis-regulatory elements. ChIP-seq against Crx identified thousands of cis-regulatory regions around photoreceptor genes in adult mouse retina. Crx directly regulates downstream photoreceptor transcription factors and their target genes via a network of spatially distributed regulatory elements around each locus. Crx-bound regions act in a synergistic fashion to activate transcription and contain multiple Crx binding sites which interact in a spacing- and orientation-dependent manner to fine-tune transcript levels. Crx ChIP-seq was also performed on Nrl-/- retinas which represent an enriched source of cone photoreceptors. Comparison with the wild-type ChIP-seq dataset identified numerous rod- and cone-specific Crx-bound regions as well as many shared elements. Thus, Crx combinatorially orchestrates the transcriptional networks of both rods and cones by coordinating the expression of photoreceptor genes including most retinal disease genes. This study pinpoints thousands of non-coding regions of relevance to both Mendelian and complex retinal disease and allows prioritization of human retinal disease genes which has already resulted in identification of FAM161A and MAK, novel disease genes implicated in retinitis pigmentosa.
Author: Helga Kolb
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Author: Andrew Everett Oliver Hughes
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 152
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Book Description
Photoreceptors are light-sensitive neurons that mediate vision, and they are the most commonly affected cell type in genetic forms of blindness. In mice, there are two basic types of photoreceptors, rods and cones, which mediate vision in dim and bright environments, respectively. The transcription factors (TFs) that control rod and cone development have been studied in detail, but the cis-regulatory elements (CREs) through which these TFs act are less well understood. To comprehensively identify photoreceptor CREs in mice and to understand their relationship with gene expression, we performed open chromatin (ATAC-seq) and transcriptome (RNA-seq) profiling of FACS-purified rods and cones. We find that rods have significantly fewer regions of open chromatin than cones (as well as >60 additional cell types and tissues), and we demonstrate that this uniquely closed chromatin architecture depends on the rod master regulator Nrl. Finally, we find that regions of rod- and cone-specific open chromatin are enriched for distinct sets of TF binding sites, providing insight into the cis-regulatory grammar of these cell types.We also sought to understand how the regulatory activity of rod and cone open chromatin regions is encoded in DNA sequence. Cone-rod homeobox (CRX) is a paired-like homeodomain TF and master regulator of both rod and cone development, and CRX binding sites are by far the most enriched TF binding sites in photoreceptor CREs. The in vitro DNA binding preferences of CRX have been extensively characterized, but how well in vitro models of TF binding site affinity predict in vivo regulatory activity is not known. In addition, paired-class homeodomain TFs bind DNA as both monomers and dimers, but whether monomeric and dimeric CRX binding sites have distinct regulatory activities is not known. To address these questions, we used a massively parallel reporter assay to quantify the activity of thousands native and mutant CRX binding sites in explanted mouse retinas. These data reveal that dimeric CRX binding sites encode stronger enhancers than monomeric CRX binding sites. Moreover, the activity of half-sites within dimeric CRX binding sites is cooperative and spacing-dependent. In addition, saturating mutagenesis of 195 CRX binding sites reveals that, while TF binding site affinity and activity are moderately correlated across mutations within individual CREs, they are poorly correlated across mutations from distinct CREs. Accordingly, we show that accounting for baseline CRE activity improves the prediction of the effects of mutations in regulatory DNA from sequence-based models. Taken together, these data demonstrate that the activity of CRX binding sites depends on multiple layers of sequence context, providing insight into photoreceptor gene regulation and illustrating functional principles of homeodomain TF binding sites.
Author: Yunlu Xue
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 128
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Book Description
The visual perception of vertebrates begins in rod and cone photoreceptors. Both photoreceptors require visual pigments to detect light. At the first step of light detection, a chromophore molecule (i.e. 11-cis retinal), which is conjugated to the visual pigment in photoreceptor outer segment, absorbs a photon. Photoisomerization of the chromophore activates the visual pigment, triggers the phototransduction cascade, and produces electrical signals. After photoisomerization, the chromophore is ultimately converted to all-trans retinol, which must be recycled to regenerate the visual pigment. This visual pigment regeneration process is called the visual cycle. It is the rate-limiting step of the photoreceptor dark adaptation after extensive light activation. The chromophore is recycled through retinal pigment epithelium (RPE) cells. In addition, cones can access a second visual cycle through the retinal Müller cells. This second visual cycle is cone-specific and fast-operating. However, it is unknown how important this retina visual cycle is to mammalian cone function and dark adaptation. To address this question, we studied whether this pathway could be impaired by deleting one of its components, the cellular retinaldehyde binding protein (CRALBP), and how this impairment would affect cone function and survival. We found that the deletion of CRALBP in mice led to impaired retina visual cycle and cone overall dark adaption, causing chronic chromophore deprivation, which desensitized M-cones, mislocalized M-opsin, and decreased M-cone numbers. We discovered that only rescuing the retina, but not RPE visual cycle, could partially restore the cone function. Considering the changes in ambient luminance, chromophore consumption is vastly different at day compared to at night. It is not clear whether the efficiency of the RPE visual cycle is modulated to reflect this chromophore consumption difference. To explore this question, we conducted rod dark adaptation experiments at subjective day, subjective night and objective day using electroretinography (ERG) on both melatonin-proficient and melatonin-deficient mouse strains. We observed that in melatonin-proficient mice the RPE visual cycle during the day is slightly down-regulated by the circadian clock and dramatically down-regulated by light exposure. We did not observe any such differences in melatonin-deficient strains, suggesting that this daytime down-regulation is melatonin-dependent. Cones, but not rods can oxidize the 11-cis retinol produced by the retina visual cycle. However, the 11-cis retinol dehydrogenase (RDH) driving this reaction in cones has not been unidentified. To address this question, we examined how knocking out RDH10, an 11-cis RDH candidate, selectively in cones or in the retina affects the retina visual cycle. We did not observe any alteration in cone function and the retina visual cycle, suggesting that RDH10 is not necessary for the retina visual cycle. In addition, the transgenic RDH10 rods did not accelerate rod dark adaptation in vivo, suggesting that RDH10 is not sufficient for rods to access the retina visual cycle. The identity of the cone 11-cis RDH(s) is still unclear. In summary, we first reported that the retina visual cycle supports cone function and dark adaptation. CRALBP plays a crucial role in retina visual cycle, whereas RDH10 appears not to be involved in this pathway. The RPE visual cycle is down-regulated to decrease the chromophore turnover for saturated rods during the day. These findings strongly support the existence of a functional retina visual cycle and provide hints for future study on the evolution of this pathway.
Author: G. Berlucchi
Publisher: Springer Science & Business Media
ISBN: 3642654959
Category : Medical
Languages : en
Pages : 758
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Author: Gordon L. Walls
Publisher: Alpha Edition
ISBN: 9789354009846
Category : History
Languages : en
Pages : 806
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Book Description
This book has been considered by academicians and scholars of great significance and value to literature. This forms a part of the knowledge base for future generations. So that the book is never forgotten we have represented this book in a print format as the same form as it was originally first published. Hence any marks or annotations seen are left intentionally to preserve its true nature.
Author: Tapas Kumar Kundu
Publisher: Springer Science & Business Media
ISBN: 9400745257
Category : Medical
Languages : en
Pages : 698
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Book Description
Epigenetics fine-tunes the life processes dictated by DNA sequences, but also kick-starts pathophysiological processes including diabetes, AIDS and cancer. This volume tracks the latest research on epigenetics, including work on new-generation therapeutics.
Author: Ernesto Carafoli
Publisher: Springer Science & Business Media
ISBN: 1402061919
Category : Medical
Languages : en
Pages : 591
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Book Description
Authors highlight several promising discoveries in the field of calcium signaling that provide new information about both genetic and acquired pathologies. Their discussions will give you new insights into the underlying causes of congenital and acquired diseases and point the way to new, even more promising research and therapies.
Author: Takahisa Furukawa
Publisher: Springer
ISBN: 9784431563358
Category : Medical
Languages : en
Pages : 0
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Book Description
This book provides a series of comprehensive views on various important aspects of vertebrate photoreceptors. The vertebrate retina is a tissue that provides unique experimental advantages to neuroscientists. Photoreceptor neurons are abundant in this tissue and they are readily identifiable and easily isolated. These features make them an outstanding model for studying neuronal mechanisms of signal transduction, adaptation, synaptic transmission, development, differentiation, diseases and regeneration. Thanks to recent advances in genetic analysis, it also is possible to link biochemical and physiological investigations to understand the molecular mechanisms of vertebrate photoreceptors within a functioning retina in a living animal. Photoreceptors are the most deeply studied sensory receptor cells, but readers will find that many important questions remain. We still do not know how photoreceptors, visual pigments and their signaling pathways evolved, how they were generated and how they are maintained. This book will make clear what is known and what is not known. The chapters are selected from fields of studies that have contributed to a broad understanding of the birth, development, structure, function and death of photoreceptor neurons. The underlying common word in all of the chapters that is used to describe these mechanisms is “molecule”. Only with this word can we understand how these highly specific neurons function and survive. It is challenging for even the foremost researchers to cover all aspects of the subject. Understanding photoreceptors from several different points of view that share a molecular perspective will provide readers with a useful interdisciplinary perspective.
