Overlapping genes in natural and engineered genomes.

Modern genome-scale methods that identify new genes, such as proteogenomics and ribosome profiling, have revealed, to the surprise of many, that overlap in genes, open reading frames and even coding sequences is widespread and functionally integrated into prokaryotic, eukaryotic and viral genomes. In parallel, the constraints that overlapping regions place on genome sequences and their evolution can be harnessed in bioengineering to build more robust synthetic strains and constructs. With a focus on overlapping protein-coding and RNA-coding genes, this Review examines their discovery, topology and biogenesis in the context of their genome biology. We highlight exciting new uses for sequence overlap to control translation, compress synthetic genetic constructs, and protect against mutation.

Gene Birth Contributes to Structural Disorder Encoded by Overlapping Genes.

The same nucleotide sequence can encode two protein products in different reading frames. Overlapping gene regions encode higher levels of intrinsic structural disorder (ISD) than nonoverlapping genes (39% vs. 25% in our viral dataset). This might be because of the intrinsic properties of the genetic code, because one member per pair was recently born de novo in a process that favors high ISD, or because high ISD relieves increased evolutionary constraint imposed by dual-coding. Here, we quantify the relative contributions of these three alternative hypotheses. We estimate that the recency of de novo gene birth explains [Formula: see text] or more of the elevation in ISD in overlapping regions of viral genes. While the two reading frames within a same-strand overlapping gene pair have markedly different ISD tendencies that must be controlled for, their effects cancel out to make no net contribution to ISD. The remaining elevation of ISD in the older members of overlapping gene pairs, presumed due to the need to alleviate evolutionary constraint, was already present prior to the origin of the overlap. Same-strand overlapping gene birth events can occur in two different frames, favoring high ISD either in the ancestral gene or in the novel gene; surprisingly, most de novo gene birth events contained completely within the body of an ancestral gene favor high ISD in the ancestral gene (23 phylogenetically independent events vs. 1). This can be explained by mutation bias favoring the frame with more start codons and fewer stop codons.

Overlapping function of Lmx1a and Lmx1b in anterior hindbrain roof plate formation and cerebellar growth.

The roof plate is an organizing center in the dorsal CNS that controls specification and differentiation of adjacent neurons through secretion of the BMP and WNT signaling molecules. Lmx1a, a member of the LIM-homeodomain (LIM-HD) transcription factor family, is expressed in the roof plate and its progenitors at all axial levels of the CNS and is necessary and sufficient for roof plate formation in the spinal cord. In the anterior CNS, however, a residual roof plate develops in the absence of Lmx1a. Lmx1b, another member of the LIM-HD transcription factor family which is highly related to Lmx1a, is expressed in the roof plate in the anterior CNS. Although Lmx1b-null mice do not show a substantial deficiency in hindbrain roof plate formation, Lmx1a/Lmx1b compound-null mutants fail to generate hindbrain roof plate. This observation indicates that both genes act in concert to direct normal hindbrain roof plate formation. Since the requirement of Lmx1b function for normal isthmic organizer at the mid-hindbrain boundary complicates analysis of a distinct dorsal patterning role of this gene, we also used a conditional knock-out strategy to specifically delete dorsal midline Lmx1b expression. Phenotypic analysis of single and compound conditional mutants confirmed overlapping roles for Lmx1 genes in regulating hindbrain roof plate formation and growth and also revealed roles in regulating adjacent cerebellar morphogenesis. Our data provides the first evidence of overlapping function of the Lmx1 genes during embryonic CNS development.

Preferential protection of protein interaction network hubs in yeast: evolved functionality of genetic redundancy.

The widely observed dispensability of duplicate genes is typically interpreted to suggest that a proportion of the duplicate pairs are at least partially redundant in their functions, thus allowing for compensatory affects. However, because redundancy is expected to be evolutionarily short lived, there is currently debate on both the proportion of redundant duplicates and their functional importance. Here, we examined these compensatory interactions by relying on a genome wide data analysis, followed by experiments and literature mining in yeast. Our data, thus, strongly suggest that compensated duplicates are not randomly distributed within the protein interaction network but are rather strategically allocated to the most highly connected proteins. This design is appealing because it suggests that many of the potentially vulnerable nodes that would otherwise be highly sensitive to mutations are often protected by redundancy. Furthermore, divergence analyses show that this association between redundancy and protein connectivity becomes even more significant among the ancient duplicates, suggesting that these functional overlaps have undergone purifying selection. Our results suggest an intriguing conclusion-although redundancy is typically transient on evolutionary time scales, it tends to be preserved among some of the central proteins in the cellular interaction network.

Overlapping expression of FoxA and Zic confers responsiveness to FGF signaling to specify notochord in ascidian embryos.

Differences in cell responsiveness to an inductive signal contribute to the emergence of a variety of tissue types during animal development. In ascidian embryos, the Fibroblast Growth Factor (FGF) signal secreted from endoderm cells induces several different tissue types, such as notochord, mesenchyme and brain, at different positions in the embryo at the 32-cell stage. We show here in Halocynthia roretzi that FoxA and Zic are required for notochord formation in cells that receive the FGF signal. We also show that these transcription factors, only when both are supplied, are able to induce ectopic expression of the brachyury gene, a notochord-specific marker, in cells of all the three germ layers in an FGF-dependent manner. These results suggest that FoxA and Zic confer notochord-specific responsiveness to FGF signaling. Further analyses including knockdown and over-expression experiments showed that combinatorial inputs from maternally supplied and zigotically activated factors lead to overlapping expression of FoxA and Zic in the presumptive notochord cells, which eventually activate the expression of the brachyury gene in cooperation with FGF signaling. Our data illustrate how a complex gene network specifies the notochord at its specific position within the embryo.

P2 growth restriction on an rpoC mutant is suppressed by alleles of the Rz1 homolog lysC.

Escherichia coli strain 397c carries a temperature-sensitive mutation, rpoC397, that removes the last 50 amino acids of the RNA polymerase beta' subunit and is nonpermissive for plating of bacteriophage P2. P2 gor mutants productively infect 397c and define a new gene, lysC, encoded by a reading frame that extensively overlaps the P2 lysis accessory gene, lysB. The unusual location of lysC with respect to lysB is reminiscent of the Rz/Rz1 lysis gene pair of phage lambda. Indeed, coexpression of lysB and lysC complemented the growth defect of lambda Rz/Rz1 null mutants, indicating that the LysB/C pair is similar to Rz/Rz1 in both gene arrangement and function. Cells carrying the rpoC397 mutation exhibited an early onset of P2-induced lysis, which was suppressed by the gor mutation in lysC. We propose that changes in host gene expression resulting from the rpoC397 mutation result in changes in the composition of the bacterial cell wall, making the cell more susceptible to P2-mediated lysis and preventing accumulation of progeny phage sufficient for plaque formation.

Discovering novel cis-regulatory motifs using functional networks.

We combined functional information such as protein-protein interactions or metabolic networks with genome information in Saccharomyces cerevisiae to predict cis-regulatory motifs in the upstream region of genes. We developed a new scoring metric combining these two information sources and used this metric in motif discovery. To estimate the statistical significance of this metric, we used brute-force randomization, which shows a consistent well-behaved trend. In contrast, real data showed complex nonrandom behavior. With conservative parameters we were able to find 42 degenerate motifs (that touch 40% of yeast genes) based on 647 original patterns, five of which are well known. Some of these motifs also show limited spatial position in the promoter, indicative of a true motif. We also tested the metric on other known motifs and show that this metric is a good discriminator of real motifs. As well as a pragmatic motif discovery method, with many applications beyond this work, these results also show that interacting proteins are often coordinated at the level of transcription, even in the absence of obvious coregulation in gene expression data sets.

Properties of overlapping EREs: synergistic activation of transcription and cooperative binding of ER.

We have designed a novel estrogen-responsive unit, overERE, which consists of two overlapping ERE separated by 5 bp (center-to-center). In gel retardation assays, this sequence forms a low-mobility complex that migrates like an estrogen receptor tetramer. The receptor-overERE complex was specific and was supershifted by anti-ER H222 antibodies. Dose response studies showed that the formation of the receptor tetramer-overERE complex was cooperative. Truncated receptors were used to assess the contribution of the receptor domains. Deletion of the E domain of the ER prevented the formation of an ER-tetramer complex, which reflects a novel function of this receptor domain. In transfection experiments, 17-beta-estradiol activated transcription from an overERE-containing promoter 4-6 times better than from an ERE-containing promoter. This synergistic effect was observed using either the natural hormone (17-beta-estradiol) or xenoestrogens (phenol red, chlordane). We conclude that two overlapping estrogen-responsive elements can elicit synergistic induction of transcription.