Inducible CRISPRa screen identifies putative enhancers

Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates. However, the identification of enhancers in native cellular contexts still remains a challenge. Here, we develop an inducible CRISPR activation (CRISPRa) system by transgenic expression of doxycycline (Dox)-inducible dCas9-VPR in mouse embryonic stem cells (iVPR ESC). With this line, a simple introduction of specific guide RNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible, reversible, and Dox concentration-dependent manner. Taking advantage of this system, we induce tiled CRISPRa across genomic regions (105 kilobases) surrounding T (Brachyury), one of the key mesodermal development regulator genes. Moreover, we identify several CRISPRa-responsive elements with chromatin features of putative enhancers, including a region the homologous sequence in which humans harbors a body height risk variant. Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation. Therefore, our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers.     

Single-molecule footprinting identifies context-dependent regulation of enhancers by DNA methylation

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A systematic approach to identify functional motifs within vertebrate developmental enhancers

Uncovering the cis-regulatory logic of developmental enhancers is critical to understanding the role of non-coding DNA in development. However, it is cumbersome to identify functional motifs within enhancers, and thus few vertebrate enhancers have their core functional motifs revealed. Here we report a combined experimental and computational approach for discovering regulatory motifs in developmental enhancers. Making use of the zebrafish gene expression database, we computationally identified conserved non-coding elements (CNEs) likely to have a desired tissue-specificity based on the expression of nearby genes. Through a high throughput and robust enhancer assay, we tested the activity of ∼ 100 such CNEs and efficiently uncovered developmental enhancers with desired spatial and temporal expression patterns in the zebrafish brain. Application of de novo motif prediction algorithms on a group of forebrain enhancers identified five top-ranked motifs, all of which were experimentally validated as critical for forebrain enhancer activity. These results demonstrate a systematic approach to discover important regulatory motifs in vertebrate developmental enhancers. Moreover, this dataset provides a useful resource for further dissection of vertebrate brain development and function.     

Distribution of CT-rich tracts is conserved in vertebrate chromosomes

The distribution of d(CT)-rich pyrimidine tracts in the karyotypes of a variety of vertebrates was studied by in situ hybridization. The probe for these studies was a 56 bp homopyrimidine/homopurine sequence obtained from a mouse genomic library constructed with DNA prepared from a restriction enzyme digestion of metaphase chromosomes. Single-stranded DNA nuclease digestions and two-dimensional gel analysis of topoisomers of this sequence indicated that it is capable of adopting a triplex conformation in vitro. In situ hybridization with this probe to the karyotypes of ten different vertebrate species revealed a highly conserved chromosomal distribution of d(CT)-rich tracts. These tracts are found throughout the chromosomal arms and in some karyotypes they are clustered, producing a banding pattern. However, at the resolution of the light microscope these tracts appeared to be absent from the centromeric regions of all chromosomes examined except those of chicken. The non-random distribution of these tracts to the chromosomal arm regions implies an organizational or functional role for this repeat class. It is unlikely that the 56 bp sequence type contributed to the formation of the triplex DNA structure previously detected in centromeric domains of mouse.     

Regulation of Six1 expression by evolutionarily conserved enhancers in tetrapods

The Six1 homeobox gene plays critical roles in vertebrate organogenesis. Mice deficient for Six1 show severe defects in organs such as skeletal muscle, kidney, thymus, sensory organs and ganglia derived from cranial placodes, and mutations in human SIX1 cause branchio-oto-renal syndrome, an autosomal dominant developmental disorder characterized by hearing loss and branchial defects. The present study was designed to identify enhancers responsible for the dynamic expression pattern of Six1 during mouse embryogenesis. The results showed distinct enhancer activities of seven conserved non-coding sequences (CNSs) retained in tetrapod Six1 loci. The activities were detected in all cranial placodes (excluding the lens placode), dorsal root ganglia, somites, nephrogenic cord, notochord and cranial mesoderm. The major Six1-expression domains during development were covered by the sum of activities of these enhancers, together with the previously identified enhancer for the pre-placodal region and foregut endoderm. Thus, the eight CNSs identified in a series of our study represent major evolutionarily conserved enhancers responsible for the expression of Six1 in tetrapods. The results also confirmed that chick electroporation is a robust means to decipher regulatory information stored in vertebrate genomes. Mutational analysis of the most conserved placode-specific enhancer, Six1-21, indicated that the enhancer integrates a variety of inputs from Sox, Pax, Fox, Six, Wnt/Lef1 and basic helix-loop-helix proteins. Positive autoregulation of Six1 is achieved through the regulation of Six protein-binding sites. The identified Six1 enhancers provide valuable tools to understand the mechanism of Six1 regulation and to manipulate gene expression in the developing embryo, particularly in the sensory organs.     

Comparative analysis of vertebrate <Emphasis Type="Italic">Shh</Emphasis> genes identifies novel conserved non-coding sequence

Abstract The puffer fish Takifugu rubripes (Fugu), with its compact genome, is an ideal model organism for comparative genomics. Sonic hedgehog (Shh) is a key protein in the patterning of differentiating cells during embryonic development. We have sequenced the Fugu Shh gene and compared it with the mammalian and zebrafish orthologs, identifying a number of novel conserved, non-coding sequences upstream of exon one and within the two introns. Additional conserved sequences serve to delineate activator regions and enhancers previously characterized through functional analysis. Control elements can thus be rapidly and effectively predicted by comparative methodology in its own right as well as complementing other, functional methods. This work demonstrates the value of using Fugu in comparative genomics, which has allowed identification of new putative regulatory elements, as well as corroborating enhancers identified by the more traditional deletion mapping method.     

Analysis of conserved domains identifies a unique structural feature of a chloroplast heat shock protein

Summary A low molecular weight heat shock protein which localizes to chloroplasts has been identified in several plant species. This protein belongs to a eukaryotic superfamily of small HSPs, all of which contain a conserved carboxyl-terminal domain. To investigate further the structure of this HSP, we isolated and sequenced cDNA clones for the chloroplast LMW HSPs from Petunia hybrida and Arabidopsis thaliana. The cloning of chloroplast HSPs from these two species enabled us to compare the amino acid sequences of this protein from plant species (petunia, Arabidopsis, pea, soybean and maize) that represent evolutionarily divergent taxonomic subclasses. Three conserved regions were identified, which are designated as regions I, II and III. Regions I and II are also shared by cytoplasmic LMW HSPs and therefore are likely to have functional roles common to all eukaryotic LMW HSPs. In contrast, consensus region III is not found in other LMW HSPs. Secondary structure analysis predicts that this region forms an amphipathic -helix with high conservation of methionine residues on the hydrophobic face and 100% conservation of residues on the hydrophilic face. This structure is similar to three helices, termed methionine bristles, which are found in a methionine-rich domain of a 54 kDa protein component of signal recognition particle (SRP54). The conservation of regions I and II among LMW cytoplasmic and chloroplast HSPs suggests that these HSPs perform related functions in different cellular compartments. However, identification of the methionine bristle domain suggests that chloroplast HSPs also have unique functions or substrates within the special environment of the chloroplast or other plastids.Abbreviations HS heat shock - HSP heat shock protein - LMW low molecular weight     

Evolutionary growth process of highly conserved sequences in vertebrate genomes

Genome sequence comparison between evolutionarily distant species revealed ultraconserved elements (UCEs) among mammals under strong purifying selection. Most of them were also conserved among vertebrates. Because they tend to be located in the flanking regions of developmental genes, they would have fundamental roles in creating vertebrate body plans. However, the evolutionary origin and selection mechanism of these UCEs remain unclear. Here we report that UCEs arose in primitive vertebrates, and gradually grew in vertebrate evolution. We searched for UCEs in two teleost fishes, Tetraodon nigroviridis and Oryzias latipes, and found 554 UCEs with 100% identity over 100 bps. Comparison of teleost and mammalian UCEs revealed 43 pairs of common, jawed-vertebrate UCEs (jUCE) with high sequence identities, ranging from 83.1% to 99.2%. Ten of them retain lower similarities to the Petromyzon marinus genome, and the substitution rates of four non-exonic jUCEs were reduced after the teleost-mammal divergence, suggesting that robust conservation had been acquired in the jawed vertebrate lineage. Our results indicate that prototypical UCEs originated before the divergence of jawed and jawless vertebrates and have been frozen as perfect conserved sequences in the jawed vertebrate lineage. In addition, our comparative sequence analyses of UCEs and neighboring regions resulted in a discovery of lineage-specific conserved sequences. They were added progressively to prototypical UCEs, suggesting step-wise acquisition of novel regulatory roles. Our results indicate that conserved non-coding elements (CNEs) consist of blocks with distinct evolutionary history, each having been frozen since different evolutionary era along the vertebrate lineage.