Albert Erives

Whole-Genome Analysis of Dorsal-Ventral Patterning in the Drosophila Embryo

The maternal Dorsal regulatory gradient initiates the differentiation of several tissues in the early Drosophila embryo. Whole-genome microarray assays identified as many as 40 new Dorsal target genes, which encode a broad spectrum of cell signaling proteins and transcription factors. Evidence is presented that a tissue-specific form of the NF-Y transcription complex is essential for the activation of gene expression in the mesoderm. Tissue-specific enhancers were identified for new Dorsal target genes, and bioinformatics methods identified conserved cis-regulatory elements for coordinately regulated genes that respond to similar thresholds of the Dorsal gradient. The new Dorsal target genes and enhancers represent one of the most extensive gene networks known for any developmental process.

A regulatory code for neurogenic gene expression in the Drosophila embryo

Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permitted the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. We discuss the prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression.

Evolution Acts on Enhancer Organization to Fine-Tune Gradient Threshold Readouts

The elucidation of principles governing evolution of gene regulatory sequence is critical to the study of metazoan diversification. We are therefore exploring the structure and organizational constraints of regulatory sequences by studying functionally equivalent cis-regulatory modules (CRMs) that have been evolving in parallel across several loci. Such an independent dataset allows a multi-locus study that is not hampered by nonfunctional or constrained homology. The neurogenic ectoderm enhancers (NEEs) of Drosophila melanogaster are one such class of coordinately regulated CRMs. The NEEs share a common organization of binding sites and as a set would be useful to study the relationship between CRM organization and CRM activity across evolving lineages. We used the D. melanogaster transgenic system to screen for functional adaptations in the NEEs from divergent drosophilid species. We show that the individual NEE modules across a genome in any one lineage have independently evolved adaptations to compensate for lineage-specific developmental and/or genomic changes. Specifically, we show that both the site composition and the site organization of NEEs have been finely tuned by distinct, lineage-specific selection pressures in each of the three divergent species that we have examined: D. melanogaster, D. pseudoobscura, and D. virilis. Furthermore, by precisely altering the organization of NEEs with different morphogen gradient threshold readouts, we show that CRM organizational evolution is sufficient for explaining changes in enhancer activity. Thus, evolution can act on CRM organization to fine-tune morphogen gradient threshold readouts over a wide dynamic range. Our study demonstrates that equivalence classes of CRMs are powerful tools for detecting lineage-specific adaptations by gene regulatory sequences.

Evolution of the holozoan ribosome biogenesis regulon

BackgroundThe ribosome biogenesis (RiBi) genes encode a highly-conserved eukaryotic set of nucleolar proteins involved in rRNA transcription, assembly, processing, and export from the nucleus. While the mode of regulation of this suite of genes has been studied in the yeast, Saccharomyces cerevisiae, how this gene set is coordinately regulated in the larger and more complex metazoan genomes is not understood.ResultsHere we present genome-wide analyses indicating that a distinct mode of RiBi regulation co-evolved with the E(CG)-binding, Myc:Max bHLH heterodimer complex in a stem-holozoan, the ancestor of both Metazoa and Choanoflagellata, the protozoan group most closely related to animals. These results show that this mode of regulation, characterized by an E(CG)-bearing core-promoter, is specific to almost all of the known genes involved in ribosome biogenesis in these genomes. Interestingly, this holozoan RiBi promoter signature is absent in nematode genomes, which have not only secondarily lost Myc but are marked by invariant cell lineages typically producing small body plans of 1000 somatic cells. Furthermore, a detailed analysis of 10 fungal genomes shows that this holozoan signature in RiBi genes is not found in hemiascomycete fungi, which evolved their own unique regulatory signature for the RiBi regulon.ConclusionThese results indicate that a Myc regulon, which is activated in proliferating cells during normal development as well as during tumor progression, has primordial roots in the evolution of an inducible growth regime in a protozoan ancestor of animals. Furthermore, by comparing divergent bHLH repertoires, we conclude that regulation by Myc but not by other bHLH genes is responsible for the evolutionary maintenance of E(CG) sites across the RiBi suite of genes.

Temporal expression patterns of 39 Brachyury-downstream genes associated with notochord formation in the Ciona intestinalis embryo.

Expression of the Brachyury (Ci‐Bra) gene of the ascidian Ciona intestinalis is initiated at the 64‐cell stage. Gene expression is restricted to notochord precursor cells, and Ci‐Bra plays a key role in notochord differentiation. In a previous study, nearly 50 cDNA clones for potential Ci‐Bra‐downstream genes that are expressed in notochord cells were isolated. The present determination, by whole‐mount in situ hybridization, of the temporal expression patterns of 19 notochord‐specific and 20 notochord‐predominant genes demonstrated that the timings of initiation of the expression of various genes was not identical. The expression of several genes was initiated as early as the gastrula stage. However, the expression of most of the notochord‐specific genes commenced at the neural plate stage. Partial nucleotide sequence data of these clones suggest that genes expressed earlier encode potential transcriptional factors and/or nuclear proteins, while those expressed later encode proteins implicated in cell adhesion, signal transduction, regulation of the cytoskeleton, and components of the extracellular matrix. These gene activities may be associated with changes in cell shape and adhesion during the intercalation and extension of the notochord cells.

Dynamic evolution of precise regulatory encodings creates the clustered site signature of enhancers

Concentration gradients of morphogenic proteins pattern the embryonic axes of Drosophila by activating different genes at different concentrations. The neurogenic ectoderm enhancers (NEEs) activate different genes at different threshold levels of the Dorsal (Dl) morphogen, which patterns the dorsal/ventral axis. NEEs share a unique arrangement of highly constrained DNA-binding sites for Dl, Twist (Twi), Snail (Sna) and Suppressor of Hairless (Su(H)), and encode the threshold variable in the precise length of DNA that separates one well-defined Dl element from a Twi element. However, NEEs also possess dense clusters of variant Dl sites. Here, we show that these increasingly variant sites are eclipsed relic elements, which were superseded by more recently evolved threshold encodings. Given the divergence in egg size during Drosophila lineage evolution, the observed characteristic clusters of divergent sites indicate a history of frequent selection for changes in threshold responses to the Dl morphogen gradient and confirm the NEE structure/function model.

A closer look at the eve stripe 2 enhancers of Drosophila and Themira.

Gene regulatory sequences have been investigated and/or proposed to be important targets of natural selection during animal evolution [1]–[14]. However, much controversy has been generated by the contention that they are not likely to be as important as functional protein-coding evolution given the low number of such examples established to date [15],[16]. However, an important obstacle in identifying such sequences is our lack of understanding of the organizational basis for such sequences. Such an understanding could allow the rapid identification and annotation of gene regulatory functions in sequenced genomes. Gene regulatory sequences function by displaying clusters of sites for DNA sequence-specific binding factors. Such clusters are called cis-regulatory modules (CRMs), of which the transcriptional enhancers constitute a large and important class. The degree to which the constituent binding elements of enhancers are necessarily organized by position, orientation, and relative spacing in order to function will dictate the constraints governing enhancer evolution. Thus, the internal functional organization of enhancers is important for understanding the mode and tempo of gene regulatory evolution as well as for deciphering and annotating genomic sequences. Arguably, no other metazoan cis-regulatory module has yet been as genetically and biochemically defined as the even-skipped (eve) stripe 2 enhancer module of Drosophila melanogaster [17]–[23]. For this reason, this module has been intensely studied from a phylogenetic perspective amongst drosophilids [24]–[27]. These phylogenetic studies of the eve stripe 2 enhancer have now been extended into Themira, a sepsid fly [28]. This latest study is used to make a central claim that a lack of sequence conservation between the eve stripe 2 enhancers of Drosophila melanogaster and Themira putris suggests that “complex animal regulatory sequences can tolerate nearly complete rearrangement of their transcription factor binding sites”. Thus, this study is being interpreted to reach conclusions addressing an important ongoing debate on the degree of functional organization of enhancers [29]. The results of this debate therefore impact the much larger discussion on the genetic loci of evolution [15],[16]. Both Drosophila and Themira are acalyptrate flies and last shared a common ancestor at least ∼110 Mya, and so it is suggested that this distance is long enough for the sequences to be completely scrambled in divergent organisms still sharing a similar embryonic patterning system. While the sepsid study presents an informative taxonomic collection of an evolving enhancer, this study falls short in critically testing the claim that enhancer organization is not important. Moreover, here we report that we find extensive homology in what is claimed to be an exemplar of scrambled enhancer sequences. As explained below, these ordered blocks of homology encompass well-known activator and repressor binding sites. Thus, the organization of Acalyptratae eve stripe 2 enhancers has not diverged enough to rule out organized assembly of higher-order enhancesome complexes at these sequences. Extensive Homology in the eve Stripe 2 Enhancers of Drosophila and Themira We first began by graphing the Themira and Drosophila stripe 2 enhancer sequences on two-dimensional sequence alignment plots (Figure 1). Such a dot plot or graphic matrix shows all regions of similarity between two sequences [30]. Such an alignment is helpful for visualizing possible insertions, deletions, rearrangements, inversions, repeats, and overall homology, without being constrained by global alignments. We also computed the same dot plot using the reverse complement of one of the sequences (Figure 1B and 1E). In addition to showing similar sequences that happen to occur in the opposite orientation, graphing the reverse complement serves as an internal negative control for conservation of serial blocks of sequence. Here, we report that when we graph the eve stripe 2 enhancers in parallel orientations, we see large blocks of alignment spanning ∼600 bp, almost the entire length of the enhancer (Figure 1A). These blocks are larger and more numerous compared to the number and types of alignable blocks achieved when we align them in anti-parallel orientation, i.e., when we plot against the reverse complement of one of the sequences (compare Figure 1A and 1D versus 1B and 1E, or see score distributions in 1C and 1F, respectively). We made such plots for two different thresholds that correspond to an ∼14 bp length of alignment that would encompass most binding sites (Figure 1A–1C) as well as a more extensive ∼20 bp length of alignment (Figure 1D–1F). At the more stringent level, most of the alignments in the anti-parallel direction are lost (Figure 1E). However, a clear identity line of ordered blocks of conservation is visible in the parallel alignment (Figure 1D). Thus, there exists ordered blocks of highly conserved sequence of a length consistent with multiple binding sites spanning the length of the enhancer. Figure 1 Two-dimensional dot plots of the eve stripe 2 enhancers of Drosophila and Themira. The Drosophila/Themira study of an embryonic enhancer of the anterior posterior (A/P) axis could have been better informed by considering the Drosophila/Anopheles study of an embryonic enhancer of the dorsal/ventral axis (D/V) [31]. This study analyzed homologous vnd neuroectodermal enhancers from both Drosophila and the mosquito Anopheles, which last shared a common ancestor at least ∼250 Mya (Figure 2A). This study shows that core cis-elements are organized in a similar architectural plan (Figure 2B). Moreover, this conserved organization was present in non-homologous neuroectodermal enhancers that had evolved in parallel at other loci [31],[32]. However, the Drosophila and Anopheles vnd enhancers are so scrambled that it is difficult to find any evidence of serial sequence homology unlike the Drosophila/Themira pair (Figure 2C and 2D). This is consistent with the additional ∼140 My of divergence between Acalyptratae and mosquitoes on top of the ∼110 My of divergence between the Drosophila and Themira (Figure 2A). Figure 2 Evolutionary scrambling at the Drosophila and Anopheles vnd neuroectoderm enhancers (NEEs). The lesson in the mosquito example that should have informed the sepsid eve stripe 2 study is that the absence of extensive sequence homology is not indicative of the absence of conserved organization of binding sites. Therefore, a simple claim that an enhancer is scrambled is insufficient grounds to rule out functional organization of sites. However, in this particular case, the sepsid enhancer is actually more conserved than the Anopheles enhancer relative to each of their Drosophila orthologs (compare graphs and score in Figures 1 and ​and2).2). Below we show that these blocks of alignment in Acalyptratae sequences correspond to known transcription factor binding sites.

New insights into the roles of Xin repeat-containing proteins in cardiac development, function, and disease.

Since the discovery of Xin repeat-containing proteins in 1996, the importance of Xin proteins in muscle development, function, regeneration, and disease has been continuously implicated. Most Xin proteins are localized to myotendinous junctions of the skeletal muscle and also to intercalated discs (ICDs) of the heart. The Xin gene is only found in vertebrates, which are characterized by a true chambered heart. This suggests that the evolutionary origin of the Xin gene may have played a key role in vertebrate origins. Diverse vertebrates including mammals possess two paralogous genes, Xinα (or Xirp1) and Xinβ (or Xirp2), and this review focuses on the role of their encoded proteins in cardiac muscles. Complete loss of mouse Xinβ (mXinβ) results in the failure of forming ICD, severe growth retardation, and early postnatal lethality. Deletion of mouse Xinα (mXinα) leads to late-onset cardiomyopathy with conduction defects. Molecular studies have identified three classes of mXinα-interacting proteins: catenins, actin regulators/modulators, and ion-channel subunits. Thus, mXinα acts as a scaffolding protein modulating the N-cadherin-mediated adhesion and ion-channel surface expression. Xin expression is significantly upregulated in early stages of stressed hearts, whereas Xin expression is downregulated in failing hearts from various human cardiomyopathies. Thus, mutations in these Xin loci may lead to diverse cardiomyopathies and heart failure.

Metabolic and Chaperone Gene Loss Marks the Origin of Animals: Evidence for Hsp104 and Hsp78 Chaperones Sharing Mitochondrial Enzymes as Clients

The evolution of animals involved acquisition of an emergent gene repertoire for gastrulation. Whether loss of genes also co-evolved with this developmental reprogramming has not yet been addressed. Here, we identify twenty-four genetic functions that are retained in fungi and choanoflagellates but undetectable in animals. These lost genes encode: (i) sixteen distinct biosynthetic functions; (ii) the two ancestral eukaryotic ClpB disaggregases, Hsp78 and Hsp104, which function in the mitochondria and cytosol, respectively; and (iii) six other assorted functions. We present computational and experimental data that are consistent with a joint function for the differentially localized ClpB disaggregases, and with the possibility of a shared client/chaperone relationship between the mitochondrial Fe/S homoaconitase encoded by the lost LYS4 gene and the two ClpBs. Our analyses lead to the hypothesis that the evolution of gastrulation-based multicellularity in animals led to efficient extraction of nutrients from dietary sources, loss of natural selection for maintenance of energetically expensive biosynthetic pathways, and subsequent loss of their attendant ClpB chaperones.

Correction: Recessive coding and regulatory mutations in FBLIM1 underlie the pathogenesis of chronic recurrent multifocal osteomyelitis (CRMO)

[This corrects the article DOI: 10.1371/journal.pone.0169687.].