Transcription factors (TFs) recognize short sequence motifs that are present in millions of copies in large eukaryotic genomes. TFsmust distinguish their target binding sites from a vast genomic ...excess of spurious motif occurrences; however, it is unclear whether functional sites are distinguished from nonfunctional motifs by local primary sequence features or by the larger genomic context in which motifs reside. We used a massively parallel enhancer assay in living mouse retinas to compare 1,300 sequences bound in the genome by the photoreceptor transcription factor Cone-rod homeobox (Crx), to 3,000 control sequences. We found that very short sequences bound in the genome by Crx activated transcription at high levels, whereas unbound genomic regions with equal numbers of Crx motifs did not activate above background levels, even when liberated from their larger genomic context. High local GC content strongly distinguishes bound motifs from unbound motifs across the entire genome. Our results show that the cis -regulatory potential of TF-bound DNA is determined largely by highly local sequence features and not by genomic context.
Cis -regulatory elements (CREs) control gene expression by recruiting transcription factors (TFs) and other DNA binding proteins. We aim to understand how individual nucleotides contribute to the ...function of CREs. Here we introduce CRE analysis by sequencing (CRE-seq), a high-throughput method for producing and testing large numbers of reporter genes in mammalian cells. We used CRE-seq to assay >1,000 single and double nucleotide mutations in a 52-bp CRE in the Rhodopsin promoter that drives strong and specific expression in mammalian photoreceptors. We find that this particular CRE is remarkably complex. The majority (86%) of single nucleotide substitutions in this sequence exert significant effects on regulatory activity. Although changes in the affinity of known TF binding sites explain some of these expression changes, we present evidence for complex phenomena, including binding site turnover and TF competition. Analysis of double mutants revealed complex, nucleotide-specific interactions between residues in different TF binding sites. We conclude that some mammalian CREs are finely tuned by evolution and function through complex, nonadditive interactions between bound TFs. CRE-seq will be an important tool to uncover the rules that govern these interactions.
Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation ...of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell-derived organoids.
Transcription factors often activate and repress different target genes in the same cell. How activation and repression are encoded by different arrangements of transcription factor binding sites in ...cis-regulatory elements is poorly understood. We investigated how sites for the transcription factor CRX encode both activation and repression in photoreceptors by assaying thousands of genomic and synthetic cis-regulatory elements in wild-type and Crx−/− retinas. We found that sequences with high affinity for CRX repress transcription, whereas sequences with lower affinity activate. This rule is modified by a cooperative interaction between CRX sites and sites for the transcription factor NRL, which overrides the repressive effect of high affinity for CRX. Our results show how simple rearrangements of transcription factor binding sites encode qualitatively different responses to a single transcription factor and explain how CRX plays multiple cis-regulatory roles in the same cell.
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•CRX acts directly as both a repressor and activator in rod photoreceptors•Activation and repression encoded by different affinity for CRX•A binding site for the transcription factor NRL overrides repression•Designed cis-regulatory elements recapitulate the behavior of genomic CRX targets
Transcription factors often play different regulatory roles in the same cell. White et al. show how transcriptional activation and repression are encoded in regulatory DNA by the number and affinity of binding sites for the transcription factor CRX, enabling CRX to act as both repressor and activator in rod photoreceptors.
Rod photoreceptors are specialized neurons that mediate vision in dim light and are the predominant photoreceptor type in nocturnal mammals. The rods of nocturnal mammals are unique among vertebrate ...cell types in having an 'inverted' nuclear architecture, with a dense mass of heterochromatin in the center of the nucleus rather than dispersed clumps at the periphery. To test if this unique nuclear architecture is correlated with a unique epigenomic landscape, we performed ATAC-seq on mouse rods and their most closely related cell type, cone photoreceptors. We find that thousands of loci are selectively closed in rods relative to cones as well as >60 additional cell types. Furthermore, we find that the open chromatin profile of photoreceptors lacking the rod master regulator Nrl is nearly indistinguishable from that of native cones, indicating that Nrl is required for selective chromatin closure in rods. Finally, we identified distinct enrichments of transcription factor binding sites in rods and cones, revealing key differences in the cis-regulatory grammar of these cell types. Taken together, these data provide insight into the development and maintenance of photoreceptor identity, and highlight rods as an attractive system for studying the relationship between nuclear organization and local changes in gene regulation.
The effects of transcription factor binding sites (TFBSs) on the activity of a cis-regulatory element (CRE) depend on the local sequence context. In rod photoreceptors, binding sites for the ...transcription factor (TF) Cone-rod homeobox (CRX) occur in both enhancers and silencers, but the sequence context that determines whether CRX binding sites contribute to activation or repression of transcription is not understood. To investigate the context-dependent activity of CRX sites, we fit neural network-based models to the activities of synthetic CREs composed of photoreceptor TFBSs. The models revealed that CRX binding sites consistently make positive, independent contributions to CRE activity, while negative homotypic interactions between sites cause CREs composed of multiple CRX sites to function as silencers. The effects of negative homotypic interactions can be overcome by the presence of other TFBSs that either interact cooperatively with CRX sites or make independent positive contributions to activity. The context-dependent activity of CRX sites is thus determined by the balance between positive heterotypic interactions, independent contributions of TFBSs, and negative homotypic interactions. Our findings explain observed patterns of activity among genomic CRX-bound enhancers and silencers, and suggest that enhancers may require diverse TFBSs to overcome negative homotypic interactions between TFBSs.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Approximately 98% of mammalian DNA is noncoding, yet we understand relatively little about the function of this enigmatic portion of the genome. The cis-regulatory elements that control gene ...expression reside in noncoding regions and can be identified by mapping the binding sites of tissue-specific transcription factors. Cone-rod homeobox (CRX) is a key transcription factor in photoreceptor differentiation and survival, but its in vivo targets are largely unknown. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) on CRX to identify 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 data set 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. In addition, this study pinpoints thousands of noncoding regions of relevance to both Mendelian and complex retinal disease.
The photoreceptor cells of the retina are subject to a greater number of genetic diseases than any other cell type in the human body. The majority of more than 120 cloned human blindness genes are ...highly expressed in photoreceptors. In order to establish an integrative framework in which to understand these diseases, we have undertaken an experimental and computational analysis of the network controlled by the mammalian photoreceptor transcription factors, Crx, Nrl, and Nr2e3. Using microarray and in situ hybridization datasets we have produced a model of this network which contains over 600 genes, including numerous retinal disease loci as well as previously uncharacterized photoreceptor transcription factors. To elucidate the connectivity of this network, we devised a computational algorithm to identify the photoreceptor-specific cis-regulatory elements (CREs) mediating the interactions between these transcription factors and their target genes. In vivo validation of our computational predictions resulted in the discovery of 19 novel photoreceptor-specific CREs near retinal disease genes. Examination of these CREs permitted the definition of a simple cis-regulatory grammar rule associated with high-level expression. To test the generality of this rule, we used an expanded form of it as a selection filter to evolve photoreceptor CREs from random DNA sequences in silico. When fused to fluorescent reporters, these evolved CREs drove strong, photoreceptor-specific expression in vivo. This study represents the first systematic identification and in vivo validation of CREs in a mammalian neuronal cell type and lays the groundwork for a systems biology of photoreceptor transcriptional regulation.
Massively parallel reporter gene assays are key tools in regulatory genomics but cannot be used to identify cell-type-specific regulatory elements without performing assays serially across different ...cell types. To address this problem, we developed a single-cell massively parallel reporter assay (scMPRA) to measure the activity of libraries of cis-regulatory sequences (CRSs) across multiple cell types simultaneously. We assayed a library of core promoters in a mixture of HEK293 and K562 cells and showed that scMPRA is a reproducible, highly parallel, single-cell reporter gene assay that detects cell-type-specific cis-regulatory activity. We then measured a library of promoter variants across multiple cell types in live mouse retinas and showed that subtle genetic variants can produce cell-type-specific effects on cis-regulatory activity. We anticipate that scMPRA will be widely applicable for studying the role of CRSs across diverse cell types.
A prime goal of regenerative medicine is to direct cell fates in a therapeutically useful manner. Retinitis pigmentosa is one of the most common degenerative diseases of the eye and is associated ...with early rod photoreceptor death followed by secondary cone degeneration. We hypothesized that converting adult rods into cones, via knockdown of the rod photoreceptor determinant Nrl, could make the cells resistant to the effects of mutations in rodspecific genes, thereby preventing secondary cone loss. To test this idea, we engineered a tamoxifen-inducible allele of Nrl to acutely inactivate the gene in adult rods. This manipulation resulted in reprogramming of rods into cells with a variety of cone-like molecular, histologie, and functional properties. Moreover, reprogramming of adult rods achieved cellular and functional rescue of retinal degeneration in a mouse model of retinitis pigmentosa. These findings suggest that elimination of Nrl in adult rods may represent a unique therapy for retinal degeneration.
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