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Introduction: The articulating ends of limb bones have precise morphology and asymmetry that ensures proper joint function. Growth differentiation factor 5 (Gdf5) is a secreted morphogen involved in cartilage and bone development that contributes to the architecture of developing joints. Dysregulation of Gdf5 results in joint dysmorphogenesis often leading to progressive joint degeneration or osteoarthritis (OA). The transcription factors and cis-regulatory modules (CRMs) that regulate Gdf5 expression are not well characterized. We previously identified a Gdf5-associated regulatory region (GARR) that contains predicted binding sites for Lmx1b, Osr2, Fox, and the Sox transcription factors. These transcription factors are recognized factors involved in joint morphogenesis and skeletal development. Methods: We used in situ hybridization to Gdf5, Col2A1, and the transcription factors of interest in developing chicken limbs to determine potential overlap in expression. We further analyzed scRNA-seq data derived from limbs and knees in published mouse and chicken datasets, identifying cells with coexpression of Gdf5 and the transcription factors of interest. We also performed site-directed mutatgenesis of the predicted transcription factor binding sites in a GARR-reporter construct and determined any change in activity using targeted regional electroporation (TREP) in micromass and embryonic chicken wing bioassays. Results: Gdf5 expression overlapped the expression of these transcription factors during joint development both by in situ hybridization (ISH) and scRNA-seq analyses. Within the GARR CRM, mutation of two binding sites common to Fox and Sox transcripstion factors reduced enhancer activity to background levels in micromass cultures and in ovo embryonic chicken wing bioassays, whereas mutation of two Sox-only binding sites caused a significant increase in activity. These results indicate that the Fox/Sox binding sites are required for activity, while the Sox-only sites are involved in repression of activity. Mutation of Lmx1b binding sites in GARR caused an overall reduction in enhancer activity in vitro and a dorsal reduction in ovo. Despite a recognized role for Osr2 in joint development, disruption of the predicted Osr2 site did not alter GARR activity. Conclusion: Taken together, our data indicates that GARR integrates positive, repressive, and asymmetrical inputs to fine-tune the expression of Gdf5 during elbow joint development.

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Long non-coding RNAs (lncRNAs), a class of poorly conserved transcripts without protein-encoding ability, are widely involved in plant organogenesis and stress responses by mediating the transmission and expression of genetic information at the transcriptional, posttranscriptional, and epigenetic levels. Here, we cloned and characterized a novel lncRNA molecule through sequence alignment, Sanger sequencing, transient expression in protoplasts, and genetic transformation in poplar. lncWOX11a is a 215 bp transcript located on poplar chromosome 13, ~50 kbp upstream of PeWOX11a on the reverse strand, and the lncRNA may fold into a series of complex stem-loop structures. Despite the small open reading frame (sORF) of 51 bp within lncWOX11a, bioinformatics analysis and protoplast transfection revealed that lncWOX11a has no protein-coding ability. The overexpression of lncWOX11a led to a decrease in the quantity of adventitious roots on the cuttings of transgenic poplars. Further, cis-regulatory module prediction and CRISPR/Cas9 knockout experiments with poplar protoplasts demonstrated that lncWOX11a acts as a negative regulator of adventitious rooting by downregulating the WUSCHEL-related homeobox gene WOX11, which is supposed to activate adventitious root development in plants. Collectively, our findings imply that lncWOX11a is essential for modulating the formation and development of adventitious roots.

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How complex morphological patterns form is an intriguing question in developmental biology. However, the mechanisms that generate complex patterns remain largely unknown. Here, we sought to identify the genetic mechanisms that regulate the tan (t) gene in a multi-spotted pigmentation pattern on the abdomen and wings of Drosophila guttifera. Previously, we showed that yellow (y) gene expression completely prefigures the abdominal and wing pigment patterns of this species. In the current study, we demonstrate that the t gene is co-expressed with the y gene in nearly identical patterns, both transcripts foreshadowing the adult abdominal and wing melanin spot patterns. We identified cis-regulatory modules (CRMs) of t, one of which drives reporter expression in six longitudinal rows of spots on the developing pupal abdomen, while the second CRM activates the reporter gene in a spotted wing pattern. Comparing the abdominal spot CRMs of y and t, we found a similar composition of putative transcription factor binding sites that are thought to regulate the complex expression patterns of both terminal pigmentation genes y and t. In contrast, the y and t wing spots appear to be regulated by distinct upstream factors. Our results suggest that the D. guttifera abdominal and wing melanin spot patterns have been established through the co-regulation of y and t, shedding light on how complex morphological traits may be regulated through the parallel coordination of downstream target genes.

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Metazoa gene expression is controlled by modular DNA segments called cis-regulatory modules (CRMs). CRMs can convey promoter/enhancer/insulator roles, generating additional regulation layers in transcription. Experiments for understanding CRM roles are low-throughput and costly. Large-scale CRM function investigation still depends on computational methods. However, existing in silico tools only recognize enhancers or promoters exclusively, thus accumulating errors when considering CRM promoter/enhancer/insulator roles altogether. Currently, no algorithm can concurrently consider these CRM roles. In this research, we developed the CRM Function Annotator (CFA) model. CFA provides complete CRM transcriptional role labeling based on epigenetic profiling interpretation. We demonstrated that CFA achieves high performance (test macro auROC/auPRC = 94.1%/90.3%) and outperforms existing tools in promoter/enhancer/insulator identification. CFA is also inspected to recognize explainable epigenetic codes consistent with previous findings when labeling CRM roles. By considering the higher-order combinations of the epigenetic codes, CFA significantly reduces false-positive rates in CRM transcriptional role annotation. CFA is available at https://github.com/cobisLab/CFA/.

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We provide here an updated description of the REDfly (Regulatory Element Database for Fly) database of transcriptional regulatory elements, a unique resource that provides regulatory annotation for the genome of Drosophila and other insects. The genomic sequences regulating insect gene expression-transcriptional cis-regulatory modules (CRMs, e.g., "enhancers") and transcription factor binding sites (TFBSs)-are not currently curated by any other major database resources. However, knowledge of such sequences is important, as CRMs play critical roles with respect to disease as well as normal development, phenotypic variation, and evolution. Characterized CRMs also provide useful tools for both basic and applied research, including developing methods for insect control. REDfly, which is the most detailed existing platform for metazoan regulatory-element annotation, includes over 40,000 experimentally verified CRMs and TFBSs along with their DNA sequences, their associated genes, and the expression patterns they direct. Here, we briefly describe REDfly's contents and data model, with an emphasis on the new features implemented since 2020. We then provide an illustrated walk-through of several common REDfly search use cases.

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KEY MESSAGE: In SlHDC-A promoter, SlHDC-A core-ES is an essential region for fruit-specific expression and interacts with GATA, HSF and AP1. Triplication of essential region was proposed as a minimal fruit-specific promoter. In plant biotechnology, fruit-specific promoter is an important tool for the improvement and utilization of tomato fruit. To expand our understanding on fruit-specific expression, it is necessary to determine the promoter region involved in fruit-specific transcriptional activity and transcriptional regulations of the promoter. In previous study, we isolated a fruit-specific SlHDC-A core promoter specifically expressed during tomato ripening stages. In this study, we identified SlHDC-A promoter region (SlHDC-A core-ES) that is essential for fruit-specific expression of the SlHDC-A. To understand the molecular mechanisms of fruit-specific expression of the SlHDC-A promoter, we first identified the putative transcription factor binding elements in the SlHDC-A core promoter region and corresponding putative transcription factors which are highly expressed during fruit maturation. Yeast one hybrid analysis confirmed that GATA, HSF, and AP1 interact with the SlHDC-A core-ES promoter region. Further transactivation analysis revealed that expression of the three transcription factors significantly activated expression of a reporter gene driven by SlHDC-A core-ES promoter. These results suggest that GATA, HSF, and AP1 are involved in the fruit-specific expression of SlHDC-A promoter. Furthermore, the synthetic promoter composed of three tandem repeats of SlHDC-A core-ES showed relatively higher activity than the constitutive 35S promoter in the transgenic tomato fruits at the orange stage. Taken together, we propose a new synthetic promoter that is specifically expressed during fruit ripening stage.

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Adeno-associated viruses (AAVs) targeting specific cell types are powerful tools for studying distinct cell types in the central nervous system (CNS). Cis-regulatory modules (CRMs), e.g., enhancers, are highly cell-type-specific and can be integrated into AAVs to render cell type specificity. Chromatin accessibility has been commonly used to nominate CRMs, which have then been incorporated into AAVs and tested for cell type specificity in the CNS. However, chromatin accessibility data alone cannot accurately annotate active CRMs, as many chromatin-accessible CRMs are not active and fail to drive gene expression in vivo. Using available large-scale datasets on chromatin accessibility, such as those published by the ENCODE project, here we explored strategies to increase efficiency in identifying active CRMs for AAV-based cell-type-specific labeling and manipulation. We found that prescreening of chromatin-accessible putative CRMs based on the density of cell-type-specific transcription factor binding sites (TFBSs) can significantly increase efficiency in identifying active CRMs. In addition, generation of synthetic CRMs by stitching chromatin-accessible regions flanking cell-type-specific genes can render cell type specificity in many cases. Using these straightforward strategies, we generated AAVs that can target the extensively studied interneuron and glial cell types in the retina and brain. Both strategies utilize available genomic datasets and can be employed to generate AAVs targeting specific cell types in CNS without conducting comprehensive screening and sequencing experiments, making a step forward in cell-type-specific research.

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The vertebrate retina is generated by retinal progenitor cells (RPCs), which produce >100 cell types. Although some RPCs produce many cell types, other RPCs produce restricted types of daughter cells, such as a cone photoreceptor and a horizontal cell (HC). We used genome-wide assays of chromatin structure to compare the profiles of a restricted cone/HC RPC and those of other RPCs in chicks. These data nominated regions of regulatory activity, which were tested in tissue, leading to the identification of many cis-regulatory modules (CRMs) active in cone/HC RPCs and developing cones. Two transcription factors, Otx2 and Oc1, were found to bind to many of these CRMs, including those near genes important for cone development and function, and their binding sites were required for activity. We also found that Otx2 has a predicted autoregulatory CRM. These results suggest that Otx2, Oc1 and possibly other Onecut proteins have a broad role in coordinating cone development and function. The many newly discovered CRMs for cones are potentially useful reagents for gene therapy of cone diseases.

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Transcription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is necessary. We analyzed how the expression of the homeobox TF, orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during development in the mouse retina. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs, respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages. We conclude that retinal cells use a cohort of TFs with different expression patterns and multiple CRMs with different chromatin configurations to regulate the expression of Otx2 precisely.

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The notochord is a structure required for support and patterning of all chordate embryos, from sea squirts to humans. An increasing amount of information on notochord development and on the molecular strategies that ensure its proper morphogenesis has been gleaned through studies in the sea squirt Ciona. This invertebrate chordate offers a fortunate combination of experimental advantages, ranging from translucent, fast-developing embryos to a compact genome and impressive biomolecular resources. These assets have enabled the rapid identification of numerous notochord genes and cis-regulatory regions, and provide a rather unique opportunity to reconstruct the gene regulatory network that controls the formation of this developmental and evolutionary chordate landmark. This chapter summarizes the morphogenetic milestones that punctuate notochord formation in Ciona, their molecular effectors, and the current knowledge of the gene regulatory network that ensures the accurate spatial and temporal orchestration of these processes.

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Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal-ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.

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The LEAFY COTYLEDON1 (LEC1) transcription factor is a central regulator of seed development, because it controls diverse biological programs during seed development, such as embryo morphogenesis, photosynthesis, and seed maturation. To understand how LEC1 regulates different gene sets during development, we explored the possibility that LEC1 acts in combination with other transcription factors. We identified and compared genes that are directly transcriptionally regulated by ABA-RESPONSIVE ELEMENT BINDING PROTEIN3 (AREB3), BASIC LEUCINE ZIPPER67 (bZIP67), and ABA INSENSITIVE3 (ABI3) with those regulated by LEC1. We showed that LEC1 operates with specific sets of transcription factors to regulate different gene sets and, therefore, distinct developmental processes. Thus, LEC1 controls diverse processes through its combinatorial interactions with other transcription factors. DNA binding sites for the transcription factors are closely clustered in genomic regions upstream of target genes, defining cis-regulatory modules that are enriched for DNA sequence motifs that resemble sequences known to be bound by these transcription factors. Moreover, cis-regulatory modules for genes regulated by distinct transcription factor combinations are enriched for different sets of DNA motifs. Expression assays with embryo cells indicate that the enriched DNA motifs are functional cis elements that regulate transcription. Together, the results suggest that combinatorial interactions between LEC1 and other transcription factors are mediated by cis-regulatory modules containing clustered cis elements and by physical interactions that are documented to occur between the transcription factors.

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Gene regulatory regions contain short and degenerated DNA binding sites recognized by transcription factors (TFBS). When TFBS harbor SNPs, the DNA binding site may be affected, thereby altering the transcriptional regulation of the target genes. Such regulatory SNPs have been implicated as causal variants in Genome-Wide Association Study (GWAS) studies. In this study, we describe improved versions of the programs Variation-tools designed to predict regulatory variants, and present four case studies to illustrate their usage and applications. In brief, Variation-tools facilitate i) obtaining variation information, ii) interconversion of variation file formats, iii) retrieval of sequences surrounding variants, and iv) calculating the change on predicted transcription factor affinity scores between alleles, using motif scanning approaches. Notably, the tools support the analysis of haplotypes. The tools are included within the well-maintained suite Regulatory Sequence Analysis Tools (RSAT, http://rsat.eu), and accessible through a web interface that currently enables analysis of five metazoa and ten plant genomes. Variation-tools can also be used in command-line with any locally-installed Ensembl genome. Users can input personal collections of variants and motifs, providing flexibility in the analysis.

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The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.

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Programs of gene transcription are controlled by cis-acting DNA elements, including enhancers, silencers, and promoters. Local accessibility of chromatin has proven to be a highly informative structural feature for identifying such regulatory elements, which tend to be relatively open due to their interactions with proteins. Recently, ATAC-seq (assay for transposase-accessible chromatin using sequencing) has emerged as one of the most powerful approaches for genome-wide chromatin accessibility profiling. This method assesses DNA accessibility using hyperactive Tn5 transposase, which simultaneously cuts DNA and inserts sequencing adaptors, preferentially in regions of open chromatin. ATAC-seq is a relatively simple procedure which can be applied to only a few thousand cells. It is well-suited to developing embryos of sea urchins and other echinoderms, which are a prominent experimental model for understanding the genomic control of animal development. In this chapter, we present a protocol for applying ATAC-seq to embryonic cells of sea urchins.

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Transcriptional enhancers play a major role in regulating metazoan gene expression. Recent developments in genomics and next-generation sequencing have accelerated and revitalized the study of this important class of sequence elements. Increased interest and attention, however, has also led to troubling trends in the enhancer literature. In this Opinion, I describe some of these issues and show how they arise from shifting and nonuniform enhancer definitions, and genome-era biases. I discuss how they can lead to interpretative errors and an unduly narrow focus on certain aspects of enhancer biology to the potential exclusion of others.

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During development diverse transcription factor inputs are integrated by cis-regulatory modules (CRMs) to yield cell-specific gene expression. Defining how CRMs recruit the appropriate combinations of factors to either activate or repress gene expression remains a challenge. In this study, we compare and contrast the ability of two CRMs within the Drosophila embryo to recruit functional Hox transcription factor complexes. The DCRE CRM recruits Ultrabithorax (Ubx) and Abdominal-A (Abd-A) Hox complexes that include the Extradenticle (Exd) and Homothorax (Hth) transcription factors to repress the Distal-less leg selector gene, whereas the RhoA CRM selectively recruits Abd-A/Exd/Hth complexes to activate rhomboid and stimulate Epidermal Growth Factor secretion in sensory cell precursors. By swapping binding sites between these elements, we found that the RhoA Exd/Hth/Hox site configuration that mediates Abd-A specific activation can convey transcriptional repression by both Ubx and Abd-A when placed into the DCRE. We further show that the orientation and spacing of Hox sites relative to additional binding sites within the RhoA and DCRE is critical to mediate cell- and segment-specific output. These results indicate that the configuration of Exd, Hth, and Hox site within RhoA is neither Abd-A specific nor activation specific. Instead Hox specific output is largely dependent upon the presence of appropriately spaced and oriented binding sites for additional TF inputs. Taken together, these studies provide insight into the cis-regulatory logic used to generate cell-specific outputs via recruiting Hox transcription factor complexes.

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In a previous bioinformatics analysis we identified 10 conserved Drosophila melanogaster sequences that reside upstream from protein coding genes (CGs). Here we characterize one of these genomic regions, which constitutes a Drosophila melanogaster cis-regulatory module (CRM) that we denominate TT-CRM. The TT-CRM is 646 bp long and is located in one of the introns of CG32239 and resides about 3,500 bp upstream of CG13711 and about 620 bp upstream of CG12493. Analysis of 646 bp-lacZ lines revealed that TT-CRM drives gene expression not only to the larval, prepupal, and pupal tracheal system but also to the adult dorsal longitudinal muscles. The patterns of mRNA expression of the transgene and of the CGs that lie in the vicinity of TT-CRM were investigated both in dissected trachea and in adult thoraces. Through RT-qPCR we observed that in the tracheal system the pattern of expression of 646 bp-lacZ is similar to the pattern of expression of CG32239 and CG13711, whereas in the thoracic muscles 646 bp-lacZ expression accompanies the expression of CG12493. Together, these results suggest new functions for two previously characterized D. melanogaster genes and also contribute to the initial characterization of a novel CRM that drives a dynamic pattern of expression throughout development.

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Ascidian embryos have been employed as model systems for studies of developmental biology for well over a century, owing to their desirable blend of experimental advantages, which include their rapid development, traceable cell lineage, and evolutionarily conserved morphogenetic movements. Two decades ago, the development of a streamlined electroporation method drastically reduced the time and cost of transgenic experiments, and, along with the elucidation of the complete genomic sequences of several ascidian species, propelled these simple chordates to the forefront of the model organisms available for studies of regulation of gene expression. Numerous ascidian sequences with tissue-specific enhancer activity were isolated and rapidly characterized through systematic in vivo experiments that would require several weeks in most other model systems. These cis-regulatory sequences include a large collection of notochord enhancers, which have been used to visualize notochord development in vivo, to generate mutant phenotypes, and to knock down genes of interest. Moreover, their detailed characterization has allowed the reconstruction of different branches of the notochord gene regulatory network. This chapter describes how the use of transgenic techniques has rendered the ascidian Ciona a competitive model organism for studies of notochord development, evolution, and gene regulation.

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The DNA puff BhC4-1 gene of Bradysia hygida (Diptera, Sciaridae) is amplified and expressed in the salivary glands at the end of the last larval instar. Even though there are no BhC4-1 orthologs in Drosophila melanogaster, the mechanisms that regulate BhC4-1 gene expression in B. hygida are for the most part conserved in D. melanogaster. The BhC4-1 promoter contains a 129bp (-186/-58) cis-regulatory module (CRM) that drives developmentally regulated expression in transgenic salivary glands at the onset of metamorphosis. Both in the sciarid and in transgenic D. melanogaster, BhC4-1 gene expression is induced by the increase in ecdysone titers that triggers metamorphosis. Genetic interaction experiments revealed that in the absence of the Eip74EF-PA early gene isoform BhC4-1-lacZ levels of expression in the salivary gland are severely reduced. Here we show that the overexpression of the Eip74EF-PA transcription factor is sufficient to anticipate BhC4-1-lacZ expression in transgenic D. melanogaster. Through yeast one-hybrid assays we confirm that the Eip74EF-PA transcription factor directly binds to the 129 bp sciarid CRM. Together, these results contribute to the characterization of an insect CRM and indicate that the ecdysone gene regulatory network that promotes metamorphosis is conserved between D. melanogaster and the sciarid B. hygida.

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