Non-coding DNA conservation across species has been often used as a predictor for transcriptional enhancer activity. However, only a few systematic analyses of the function of these highly conserved ...non-coding regions (HCNRs) have been performed. Here we use zebrafish transgenic assays to perform a systematic study of 113 HCNRs from human chromosome 16. By comparing transient and stable transgenesis, we show that the first method is highly inefficient, leading to 40% of false positives and 20% of false negatives. When analyzed in stable transgenic lines, a great majority of HCNRs were active in the central nervous system, although some of them drove expression in other organs such as the eye and the excretory system. Finally, by testing a fraction of the HCNRs lacking enhancer activity for in vivo insulator activity, we find that 20% of them may contain enhancer-blocking function. Altogether our data indicate that HCNRs may contain different types of cis-regulatory activity, including enhancer, insulators as well as other not yet discovered functions.
Cis-regulatory elements (CREs) and transcription factors (TFs) associated with them determine temporal and spatial domains of gene expression. Therefore, identification of these CREs and TFs is ...crucial to elucidating transcriptional programs across taxa. With chromatin accessibility facilitating transcription factor access to DNA, the identification of regions of open chromatin sheds light both on the function of the regulatory elements and their evolution, thus allowing the recognition of potential CREs. Buenrostro and colleagues have developed a novel method for exploring chromatin accessibility: assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), which can be used for the purpose of identifying putative CREs. This method was shown to have considerable advantages when compared to traditional methods such as sequence conservation analyses or functional assays. Here we present the adaptation of the ATAC-seq method to echinoderm species and discuss how it can be used for CRE discovery.
The Regulators of Complement Activation (RCA) gene cluster comprises several tandemly arranged genes with shared functions within the immune system. RCA members, such as complement receptor 2 (
), ...are well-established susceptibility genes in complex autoimmune diseases. Altered expression of RCA genes has been demonstrated at both the functional and genetic level, but the mechanisms underlying their regulation are not fully characterised. We aimed to investigate the structural organisation of the RCA gene cluster to identify key regulatory elements that influence the expression of
and other genes in this immunomodulatory region. Using 4C, we captured extensive CTCF-mediated chromatin looping across the RCA gene cluster in B cells and showed these were organised into two topologically associated domains (TADs). Interestingly, an inter-TAD boundary was located within the
gene at a well-characterised segmental duplication. Additionally, we mapped numerous gene-gene and gene-enhancer interactions across the region, revealing extensive co-regulation. Importantly, we identified an intergenic enhancer and functionally demonstrated this element upregulates two RCA members (
and
) in B cells. We have uncovered novel, long-range mechanisms whereby autoimmune disease susceptibility may be influenced by genetic variants, thus highlighting the important contribution of chromatin topology to gene regulation and complex genetic disease.
Ascidian species of the Phallusia and Ciona genera are distantly related, their last common ancestor dating several hundred million years ago. Although their genome sequences have extensively ...diverged since this radiation, Phallusia and Ciona species share almost identical early morphogenesis and stereotyped cell lineages.
Here, we explored the evolution of transcriptional control between P. mammillata and C. robusta. We combined genome-wide mapping of open chromatin regions in both species with a comparative analysis of the regulatory sequences of a test set of 10 pairs of orthologous early regulatory genes with conserved expression patterns.
We find that ascidian chromatin accessibility landscapes obey similar rules as in other metazoa. Open-chromatin regions are short, highly conserved within each genus and cluster around regulatory genes. The dynamics of chromatin accessibility and closest-gene expression are strongly correlated during early embryogenesis. Open-chromatin regions are highly enriched in cis-regulatory elements: 73% of 49 open chromatin regions around our test genes behaved as either distal enhancers or proximal enhancer/promoters following electroporation in Phallusia eggs. Analysis of this datasets suggests a pervasive use in ascidians of “shadow” enhancers with partially overlapping activities. Cross-species electroporations point to a deep conservation of both the trans-regulatory logic between these distantly-related ascidians and the cis-regulatory activities of individual enhancers. Finally, we found that the relative order and approximate distance to the transcription start site of open chromatin regions can be conserved between Ciona and Phallusia species despite extensive sequence divergence, a property that can be used to identify orthologous enhancers, whose regulatory activity can partially diverge.
•Open chromatin regions are highly enriched for cis-regulatory elements in ascidian early embryos.•Ascidian Clusters of Open Regulatory Elements (COREs) are found next to regulatory genes.•Regulatory gene expression, the trans-regulatory code, is conserved between distantly-related Ciona and Phallusia ascidians.•Ascidians show pervasive use of “shadow” enhancers.•The position of homologous enhancers can be conserved, despite extensive genome divergence.
Despite their evolutionary, developmental and functional importance, the origin of vertebrate paired appendages remains uncertain. In mice, a single enhancer termed ZRS is solely responsible for Shh ...expression in limbs. Here, zebrafish and mouse transgenic assays trace the functional equivalence of ZRS across the gnathostome phylogeny. CRISPR/Cas9-mediated deletion of the medaka (Oryzias latipes) ZRS and enhancer assays identify the existence of ZRS shadow enhancers in both teleost and human genomes. Deletion of both ZRS and shadow ZRS abolishes shh expression and completely truncates pectoral fin formation. Strikingly, deletion of ZRS results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and median fins and that paired fins likely emerged by the co-option of developmental programs established in the median fins of stem gnathostomes. Shh function was later reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness.
In this study, we investigated the gene regulatory network that governs formation of the Zona limitans intrathalamica (ZLI), a signaling center that secretes Sonic Hedgehog (Shh) to control the ...growth and regionalization of the caudal forebrain. Using loss- and gain-of-function, explants and grafting experiments in amphibians, we demonstrate that barhl2 acts downstream of otx2 and together with the iroquois (irx)-3 gene in establishment of the ZLI compartment initiated by Shh influence. We find that the presumptive (pre)-ZLI domain expresses barhl2, otx2 and irx3, whereas the thalamus territory caudally bordering the pre-ZLI expresses barhl2, otx2 and irx1/2 and early on irx3. We demonstrate that Barhl2 activity is required for determination of the ZLI and thalamus fates and that within the p2 alar plate the ratio of Irx3 to Irx1/2 contributes to ZLI specification and size determination. We show that when continuously exposed to Shh, neuroepithelial cells coexpressing barhl2, otx2 and irx3 acquire two characteristics of the ZLI compartment-the competence to express shh and the ability to segregate from anterior neural plate cells. In contrast, neuroepithelial cells expressing barhl2, otx2 and irx1/2, are not competent to express shh. Noteworthy in explants, under Shh influence, ZLI-like cells segregate from thalamic-like cells. Our study establishes that Barhl2 activity plays a key role in p2 alar plate patterning, specifically ZLI formation, and provides new insights on establishment of the signaling center of the caudal forebrain.
During eye development, retinal progenitors are drawn from a multipotent, proliferative cell population. In Drosophila the maintenance of this cell population requires the function of the ...TALE-homeodomain transcription factor Hth, although its mechanisms of action are still unknown. Here we investigate whether members of the Meis gene family, the vertebrate homologs of hth, are also involved in early stages of eye development in the zebrafish. We show that meis1 is initially expressed throughout the eye primordium. Later, meis1 becomes repressed as neurogenesis is initiated, and its expression is confined to the ciliary margin, where the retinal stem population resides. Knocking down meis1 function through morpholino injection causes a delay in the G1-to-S phase transition of the eye cells, and results in severely reduced eyes. This role in cell cycle control is mediated by meis1 regulating cyclin D1 and c-myc transcription. The forced maintenance of meis1 expression in cell clones is incompatible with the normal differentiation of the meis1-expressing cells, which in turn tend to reside in undifferentiated regions of the retinal neuroepithelium, such as the ciliary margin. Together, these results implicate meis1 as a positive cell cycle regulator in early retinal cells, and provide evidence of an evolutionary conserved function for Hth/Meis genes in the maintenance of the proliferative, multipotent cell state during early eye development.
Abstract
We investigated how the two rounds of whole-genome duplication that occurred at the base of the vertebrate lineage have impacted ancient microsyntenic associations involving developmental ...regulators (known as genomic regulatory blocks, GRBs). We showed that the majority of GRBs identified in the last common ancestor of chordates have been maintained as a single copy in humans. We found evidence that dismantling of the duplicated GRB copies occurred early in vertebrate evolution often through the differential retention of the regulatory gene but loss of the bystander gene’s exonic sequences. Despite the large evolutionary scale, the presence of duplicated highly conserved noncoding regions provided unambiguous proof for this scenario for multiple ancient GRBs. Remarkably, the dismantling of ancient GRB duplicates has contributed to the creation of large gene deserts associated with regulatory genes in vertebrates, providing a potentially widespread mechanism for the origin of these enigmatic genomic traits.
The
Iroquois (
Irx) genes encode homeoproteins conserved during evolution. Vertebrate genomes contain six
Irx genes organized in two clusters,
IrxA (which harbors
Irx1,
Irx2 and
Irx4) and
IrxB (which ...harbors
Irx3,
Irx5 and
Irx6). To determine the precise role of these genes during development and their putative redundancies, we conducted a comparative expression analysis and a comprehensive loss-of-function study of all the early expressed
Irx genes (
Irx1–5) using specific morpholinos in
Xenopus. We found that the five
Irx genes display largely overlapping expression patterns and contribute to neural patterning. All
Irx genes are required for proper formation of posterior forebrain, midbrain, hindbrain and, to a lesser an extent, spinal cord. Nevertheless,
Irx1 and
Irx3 seem to have a predominant role during regionalization of the neural plate. In addition, we find that the common anterior limit of
Irx gene expression, which will correspond to the future border between the prethalamus and thalamus, is defined by mutual repression between Fezf and Irx proteins. This mutual repression is likely direct. Finally, we show that Arx, another anteriorly expressed repressor, also contribute to delineate the anterior border of
Irx expression.
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