A survey of ancient conserved non-coding elements in the PAX6 locus reveals a landscape of interdigitated cis-regulatory archipelagos

Biological differences between cell types and developmental processes are characterised by differences in gene expression profiles. Gene-distal enhancers are key components of the regulatory networks that specify the tissue-specific expression patterns driving embryonic development and cell fate decisions, and variations in their sequences are a major contributor to genetic disease and disease susceptibility. Despite advances in the methods for discovery of putative cis-regulatory sequences, characterisation of their spatio-temporal enhancer activities in a mammalian model system remains a major bottle-neck. We employed a strategy that combines gnathostome sequence conservation with transgenic mouse and zebrafish reporter assays to survey the genomic locus of the developmental control gene PAX6 for the presence of novel cis-regulatory elements. Sequence comparison between human and the cartilaginous elephant shark (Callorhinchus milii) revealed several ancient gnathostome conserved non-coding elements (agCNEs) dispersed widely throughout the PAX6 locus, extending the range of the known PAX6 cis-regulatory landscape to contain the full upstream PAX6-RCN1 intergenic region. Our data indicates that ancient conserved regulatory sequences can be tested effectively in transgenic zebrafish even when not conserved in zebrafish themselves. The strategy also allows efficient dissection of compound regulatory regions previously assessed in transgenic mice. Remarkable overlap in expression patterns driven by sets of agCNEs indicates that PAX6 resides in a landscape of multiple tissue-specific regulatory archipelagos.     

Developmental effector gene regulation: Multiplexed strategies for functional analysis

The staggering complexity of the genome controls for developmental processes is revealed through massively parallel cis-regulatory analysis using new methods of perturbation and readout. The choice of combinations of these new methods is tailored to the system, question and resources at hand. Our focus is on issues that include the necessity or sufficiency of given cis-regulatory modules, cis-regulatory function in the normal spatial genomic context, and easily accessible high throughput and multiplexed analysis methods. In the sea urchin embryonic model, recombineered BACs offer new opportunities for consecutive modes of cis-regulatory analyses that answer these requirements, as we here demonstrate on a diverse suite of previously unstudied sea urchin effector genes expressed in skeletogenic cells. Positively active cis-regulatory modules were located in single Nanostring experiments per BAC containing the gene of interest, by application of our previously reported “barcode” tag vectors of which>?100 can be analyzed at one time. Computational analysis of DNA sequences that drive expression, based on the known skeletogenic regulatory state, then permitted effective identification of functional target site clusters. Deletion of these sub-regions from the parent BACs revealed module necessity, as simultaneous tests of the same regions in short constructs revealed sufficiency. Predicted functional inputs were then confirmed by site mutations, all generated and tested in multiplex formats. There emerged the simple conclusion that each effector gene utilizes a small subset of inputs from the skeletogenic GRN. These inputs may function to only adjust expression levels or in some cases necessary for expression. Since we know the GRN architecture upstream of the effector genes, we could then conceptually isolate and compare the wiring of the effector gene driver sub-circuits and identify the inputs whose removal abolish expression.     

Precise cis-regulatory control of spatial and temporal expression of the alx-1 gene in the skeletogenic lineage of s. purpuratus

Deployment of the gene-regulatory network (GRN) responsible for skeletogenesis in the embryo of the sea urchin Strongylocentrotus purpuratus is restricted to the large micromere lineage by a double negative regulatory gate. The gate consists of a GRN subcircuit composed of the pmar1 and hesC genes, which encode repressors and are wired in tandem, plus a set of target regulatory genes under hesC control. The skeletogenic cell state is specified initially by micromere-specific expression of these regulatory genes, viz. alx1, ets1, tbrain and tel, plus the gene encoding the Notch ligand Delta. Here we use a recently developed high throughput methodology for experimental cis-regulatory analysis to elucidate the genomic regulatory system controlling alx1 expression in time and embryonic space. The results entirely confirm the double negative gate control system at the cis-regulatory level, including definition of the functional HesC target sites, and add the crucial new information that the drivers of alx1 expression are initially Ets1, and then Alx1 itself plus Ets1. Cis-regulatory analysis demonstrates that these inputs quantitatively account for the magnitude of alx1 expression. Furthermore, the Alx1 gene product not only performs an auto-regulatory role, promoting a fast rise in alx1 expression, but also, when at high levels, it behaves as an auto-repressor. A synthetic experiment indicates that this behavior is probably due to dimerization. In summary, the results we report provide the sequence level basis for control of alx1 spatial expression by the double negative gate GRN architecture, and explain the rising, then falling temporal expression profile of the alx1 gene in terms of its auto-regulatory genetic wiring.     

Cis-regulatory characterization of sequence conservation surrounding the Hox4 genes

Hox genes are key regulators of anterior-posterior axis patterning and have a major role in hindbrain development. The zebrafish Hox4 paralogs have strong overlapping activities in hindbrain rhombomeres 7 and 8, in the spinal cord and in the pharyngeal arches. With the aim to predict enhancers that act on the hoxa4a, hoxb4a, hoxc4a and hoxd4a genes, we used sequence conservation around the Hox4 genes to analyze all fish:human conserved non-coding sequences by reporter assays in stable zebrafish transgenesis. Thirty-four elements were functionally tested in GFP reporter gene constructs and more than 100 F1 lines were analyzed to establish a correlation between sequence conservation and cis-regulatory function, constituting a catalog of Hox4 CNEs. Sixteen tissue-specific enhancers could be identified. Multiple alignments of the CNEs revealed paralogous cis-regulatory sequences, however, the CNE sequence similarities were found not to correlate with tissue specificity. To identify ancestral enhancers that direct Hox4 gene activity, genome sequence alignments of mammals, teleosts, horn shark and the cephalochordate amphioxus, which is the most basal extant chordate possessing a single prototypical Hox cluster, were performed. Three elements were identified and two of them exhibited regulatory activity in transgenic zebrafish, however revealing no specificity. Our data show that the approach to identify cis-regulatory sequences by genome sequence alignments and subsequent testing in zebrafish transgenesis can be used to define enhancers within the Hox clusters and that these have significantly diverged in their function during evolution.     

RNA-ID,a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay

We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.     

Molecular domestication – more than a sporadic episode in evolution

Transposable elements are short but complex pieces of DNA or RNA containing a streamlined minimal-genome with the capacity for its selfish replication in a foreign genomic environment. Cis-regulatory sections within the elements orchestrate tempo and mode of TE expression. Proteins encoded by TEs mainly direct their own propagation within the genome by recruitment of host-encoded factors. On the other hand, TE-encoded proteins harbor a very attractive repertoire of functional abilities for a cell. These proteins mediate excision, replication and integration of defined DNA fragments. Furthermore, some of these proteins are able to manipulate important host factors by altering their original function. Thus, if the host genome succeeds in domesticating such TE-encoded proteins by taming their ‘anarchistic behavior,’ such an event can be considered as an important evolutionary innovation for its own benefit. In fact, the domestication of TE-derived cis-regulatory modules and protein coding sections took place repeatedly in the course of genome evolution. We will present prominent cases that impressively demonstrate the beneficial impact of TEs on host biology over evolutionary time. Furthermore, we will propose that molecular domestication might be considered as a resumption of the same evolutionary process that drove the transition from ‘primitive genomes’ to ‘modern’ ones at the early dawn of life, that is, the adaptive integration of a short piece of autonomous DNA into a complex regulatory network. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparative genomics using <Emphasis Type="Italic">Fugu</Emphasis> reveals insights into regulatory subfunctionalization

Background  

A major mechanism for the preservation of gene duplicates in the genome is thought to be mediated via loss or modification of cis-regulatory subfunctions between paralogs following duplication (a process known as regulatory subfunctionalization). Despite a number of gene expression studies that support this mechanism, no comprehensive analysis of regulatory subfunctionalization has been undertaken at the level of the distal cis-regulatory modules involved. We have exploited fish-mammal genomic alignments to identify and compare more than 800 conserved non-coding elements (CNEs) that associate with genes that have undergone fish-specific duplication and retention.     

Conservation of upstream regulators of scute on the notum of cyclorraphous Diptera

Bristles on the notum of many cyclorraphous flies are arranged into species-specific stereotyped patterns. Differences in the spatial expression of the proneural gene scute correlate with the positions of bristles in those species looked at so far. However, the examination of a number of genes encoding trans-regulatory factors, such as pannier, stripe, u-shaped, caupolican and wingless, indicates that they are expressed in conserved domains on the prospective notum. This suggests that the function of a trans-regulatory network of genes is relatively unchanged in derived Diptera, and that many differences are likely to be due to changes in cis-regulatory sequences of scute. In contrast, in Anopheles gambiae, a basal species with no stereotyped bristle pattern, the expression patterns of pannier and wingless are not conserved, and expression of AgASH, the Anopheles proneural gene, does not correlate in a similar manner with the bristle pattern. We discuss the possibility that independently acting cis-regulatory sequences at the scute locus may have arisen in the lineage giving rise to cyclorraphous flies.