[Junk DNA is out of fashion]. / Chroniques génomiques - Le Junk DNA n'est plus ce qu'il était.

A systematic search for non-conventional open reading frames in human DNA reveals a large number of small ORFs encoding peptides generally smaller than 100 amino-acids. These ORFs are transcribed and translated into small proteins, which are demonstrated to have functional significance by bulk CRISPR inactivation. Evidence is also found for bicistronic mRNAs including such a small ORF upstream of a canonical coding sequence. These findings add a new facet to our understanding of biological processes.

Regulation of MFGE8 by the intergenic coronary artery disease locus on 15q26.1.

BACKGROUND AND AIMS: A recently identified locus for coronary artery disease (CAD) tagged by rs8042271 is in a region of tight linkage disequilibrium (LD) between 2 genes (MFGE8, ABHD2) previously linked to atherosclerosis. Here we have explored the regulatory framework of this region to identify its functional relationship to CAD. METHODS: The CAD Associated Region between MFGE8 and ABHD2 (CARMA) was investigated by bioinformatic approaches and transcriptional reporter assays to prioritize target genes and identify putative causal variants. Findings were integrated with publicly available gene expression datasets. MFGE8 silencing was performed in cell models relevant to CAD. RESULTS: The regulatory potential of CARMA is disseminated sparsely over the entire region. CARMA contains multiple eQTL that regulate MFGE8 in coronary artery and coronary artery smooth muscle cell (CoSMC). SNPs that predict the expression of MFGE8 in artery are concordantly associated with higher risk of CAD (pval = 0.0014). Targeting CARMA by CRISPR/Cas9 in a cellular model increased MFGE8 expression. MFGE8 silencing was found to reduce CoSMC and monocyte (THP-1) but not endothelial cell proliferation. CONCLUSIONS: These findings support a mechanistic link between a GWAS identified CAD risk locus and atherosclerosis. The intergenic locus CARMA regulates MFGE8 in a haplotype dependent manner. Individuals genetically susceptible to increased MFGE8 expression exhibit greater CAD risk. Suppressing MFGE8 expression reduced SMC and THP-1 proliferation. These data support an atherogenic contribution of CARMA/MFGE8 that may be linked to cell proliferation and/or improved survival of CAD relevant cell types.

Contribution of noncoding pathogenic variants to RPGRIP1-mediated inherited retinal degeneration.

PURPOSE: With the advent of gene therapies for inherited retinal degenerations (IRDs), genetic diagnostics will have an increasing role in clinical decision-making. Yet the genetic cause of disease cannot be identified using exon-based sequencing for a significant portion of patients. We hypothesized that noncoding pathogenic variants contribute significantly to the genetic causality of IRDs and evaluated patients with single coding pathogenic variants in RPGRIP1 to test this hypothesis. METHODS: IRD families underwent targeted panel sequencing. Unsolved cases were explored by exome and genome sequencing looking for additional pathogenic variants. Candidate pathogenic variants were then validated by Sanger sequencing, quantitative polymerase chain reaction, and in vitro splicing assays in two cell lines analyzed through amplicon sequencing. RESULTS: Among 1722 families, 3 had biallelic loss-of-function pathogenic variants in RPGRIP1 while 7 had a single disruptive coding pathogenic variants. Exome and genome sequencing revealed potential noncoding pathogenic variants in these 7 families. In 6, the noncoding pathogenic variants were shown to lead to loss of function in vitro. CONCLUSION: Noncoding pathogenic variants were identified in 6 of 7 families with single coding pathogenic variants in RPGRIP1. The results suggest that noncoding pathogenic variants contribute significantly to the genetic causality of IRDs and RPGRIP1-mediated IRDs are more common than previously thought.

The intergenic region of the maize defensin-like protein genes Def1 and Def2 functions as an embryo-specific asymmetric bidirectional promoter.

Bidirectional promoters are identified in diverse organisms with widely varied genome sizes, including bacteria, yeast, mammals, and plants. However, little research has been done on any individual endogenous bidirectional promoter from plants. Here, we describe a promoter positioned in the intergenic region of two defensin-like protein genes, Def1 and Def2 in maize (Zea mays). We examined the expression profiles of Def1 and Def2 in 14 maize tissues by qRT-PCR, and the results showed that this gene pair was expressed abundantly and specifically in seeds. When fused to either green fluorescent protein (GFP) or ß-glucuronidase (GUS) reporter genes, P ZmBD1 , P ZmDef1 , and P ZmDef2 were active and reproduced the expression patterns of both Def1 and Def2 genes in transformed immature maize embryos, as well as in developing seeds of transgenic maize. Comparative analysis revealed that PZmBD1 shared most of the expression characteristics of the two polar promoters, but displayed more stringent embryo specificity, delayed expression initiation, and asymmetric promoter activity. Moreover, a truncated promoter study revealed that the core promoters only exhibit basic bidirectional activity, while interacting with necessary cis-elements, which leads to polarity and different strengths. The sophisticated interaction or counteraction between the core promoter and cis-elements may potentially regulate bidirectional promoters.

Functions of noncoding sequences in mammalian genomes.

Most of the mammalian genome consists of nucleotide sequences not coding for proteins. Exons of genes make up only 3% of the human genome, while the significance of most other sequences remains unknown. Recent genome studies with high-throughput methods demonstrate that the so-called noncoding part of the genome may perform important functions. This hypothesis is supported by three groups of experimental data: 1) approximately 10% of the sequences, most of which are located in noncoding parts of the genome, is evolutionarily conserved and thus can be of functional importance; 2) up to 99% of the mammalian genome is being transcribed forming short and long noncoding RNAs in addition to common mRNA; and 3) mutations in noncoding parts of the genome can be accompanied by progression of pathological states of the organism. In the light of these data, in the review we consider the functional role of numerous known sequences of noncoding parts of the genome including introns, DNA methylation regions, enhancers and locus control regions, insulators, S/MAR sequences, pseudogenes, and genes of noncoding RNAs, as well as transposons and simple repeats of centromeric and telomeric regions of chromosomes. The assumption is made that the intergenic noncoding sequences without definite/clear functions can be involved in spatial organization of genetic loci in interphase nuclei.

Functional characterization of SNPs in CHRNA3/B4 intergenic region associated with drug behaviors.

The cluster of human neuronal nicotinic receptor genes (CHRNA5/A3/B4) (15q25.1) has been associated with a variety of smoking and drug-related behaviors, as well as risk for lung cancer. CHRNA3/B4 intergenic single nucleotide polymorphisms (SNPs) rs1948 and rs8023462 have been associated with early initiation of alcohol and tobacco use, and rs6495309 has been associated with nicotine dependence and risk for lung cancer. An in vitro luciferase expression assay was used to determine whether these SNPs and surrounding sequences contribute to differences in gene expression using cell lines either expressing proteins characteristic of neuronal tissue or derived from lung cancers. Electrophoretic mobility shift assays (EMSAs) were performed to investigate whether nuclear proteins from these cell lines bind SNP alleles differentially. Results from expression assays were dependent on cell culture type and haplotype. EMSAs indicated that rs8023462 and rs6495309 bind nuclear proteins in an allele-specific way. Additionally, GATA transcription factors appeared to bind rs8023462 only when the minor/risk allele was present. Much work has been done to describe the rat Chrnb4/a3 intergenic region, but few studies have examined the human intergenic region effects on expression; therefore, these studies greatly aid human genetic research as it relates to observed nicotine phenotypes, lung cancer risk and potential underlying genetic mechanisms. Data from these experiments support the hypothesis that SNPs associated with human addiction-related phenotypes and lung cancer risk can affect gene expression, and are potential therapeutic targets. Additionally, this is the first evidence that rs8023462 interacts with GATA transcription factors to influence gene expression.

A meta-analysis of the genomic and transcriptomic composition of complex life.

It is now clear that animal genomes are predominantly non-protein-coding, and that these sequences encode a wide array of RNA transcripts and other regulatory elements that are fundamental to the development of complex life. We have previously argued that the proportion of an animal genome that is non-protein-coding DNA (ncDNA) correlates well with its apparent biological complexity. Here we extend on that work and, using data from a total of 1,627 prokaryotic and 153 eukaryotic complete and annotated genomes, show that the proportion of ncDNA per haploid genome is significantly positively correlated with a previously published proxy of biological complexity, the number of distinct cell types. This is in contrast to the amount of the genome that encodes proteins, which we show is essentially unchanged across Metazoa. Furthermore, using a total of 179 RNA-seq data sets from nematode (47), fruit fly (72), zebrafish (20) and human (42), we show, consistent with other recent reports, that the vast majority of ncDNA in animals is transcribed. This includes more than 60 human loci previously considered "gene deserts," many of which are expressed tissue-specifically and associated with previously reported GWAS SNPs. These results suggest that ncDNA, and the ncRNAs encoded within it, may be intimately involved in the evolution, maintenance and development of complex life.

Regulatory element copy number differences shape primate expression profiles.

Gene expression differences are shaped by selective pressures and contribute to phenotypic differences between species. We identified 964 copy number differences (CNDs) of conserved sequences across three primate species and examined their potential effects on gene expression profiles. Samples with copy number different genes had significantly different expression than samples with neutral copy number. Genes encoding regulatory molecules differed in copy number and were associated with significant expression differences. Additionally, we identified 127 CNDs that were processed pseudogenes and some of which were expressed. Furthermore, there were copy number-different regulatory regions such as ultraconserved elements and long intergenic noncoding RNAs with the potential to affect expression. We postulate that CNDs of these conserved sequences fine-tune developmental pathways by altering the levels of RNA.

Identification of a novel type of spacer element required for imprinting in fission yeast.

Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during lagging-strand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fully-processed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers.

Heterogeneous diversity of spacers within CRISPR (clustered regularly interspaced short palindromic repeats).

Clustered regularly interspaced short palindromic repeats (CRISPR) in bacterial and archaeal DNA have recently been shown to be a new type of antiviral immune system in these organisms. We here study the diversity of spacers in CRISPR under selective pressure. We propose a population dynamics model that explains the biological observation that the leader-proximal end of CRISPR is more diversified and the leader-distal end of CRISPR is more conserved. This result is shown to be in agreement with recent experiments. Our results show that the CRISPR spacer structure is influenced by and provides a record of the viral challenges that bacteria face.

Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts.

Metazoan genes are encrypted with at least two superimposed codes: the genetic code to specify the primary structure of proteins and the splicing code to expand their proteomic output via alternative splicing. Here, we define the specificity of a central regulator of pre-mRNA splicing, the conserved, essential splicing factor SFRS1. Cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) identified 23,632 binding sites for SFRS1 in the transcriptome of cultured human embryonic kidney cells. SFRS1 was found to engage many different classes of functionally distinct transcripts including mRNA, miRNA, snoRNAs, ncRNAs, and conserved intergenic transcripts of unknown function. The majority of these diverse transcripts share a purine-rich consensus motif corresponding to the canonical SFRS1 binding site. The consensus site was not only enriched in exons cross-linked to SFRS1 in vivo, but was also enriched in close proximity to splice sites. mRNAs encoding RNA processing factors were significantly overrepresented, suggesting that SFRS1 may broadly influence the post-transcriptional control of gene expression in vivo. Finally, a search for the SFRS1 consensus motif within the Human Gene Mutation Database identified 181 mutations in 82 different genes that disrupt predicted SFRS1 binding sites. This comprehensive analysis substantially expands the known roles of human SR proteins in the regulation of a diverse array of RNA transcripts.

Identification of a promoter motif involved in Curtovirus sense-gene expression in transgenic Arabidopsis.

Expression of the seven open reading frames (ORFs) of single-stranded DNA Curtoviruses such as Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV) is driven by a bi-directional promoter. To investigate this bi-directional promoter activity with respect to viral late gene expression, transgenic Arabidopsis plants expressing a GUS reporter gene under the control of either the BCTV or BSCTV bi-directional promoter were constructed. Transgenic plants harboring constructs showed higher expression levels when the promoter of the less virulent BCTV was used than when the promoter of the more virulent BSCTV was used. In transgenic seedlings, the reporter gene constructs were expressed primarily in actively dividing tissues such as root tips and apical meristems. As the transgenic plants matured, reporter gene expression diminished but viral infection of mature transgenic plants restored reporter gene expression, particularly in transgenic plants containing BCTV virion-sense gene promoter constructs. A 30 base pair conserved late element (CLE) motif was identified that was present three times in tandem in the BCTV promoter and once in that of BSCTV. Progressive deletion of these repeats from the BCTV promoter resulted in decreased reporter gene expression, but BSCTV promoters in which one or two extra copies of this motif were inserted did not exhibit increased late gene promoter activity. These results demonstrate that Curtovirus late gene expression by virion-sense promoters depends on the developmental stage of the host plant as well as on the number of CLE motifs present in the promoter.

BAC transgenic analysis reveals enhancers sufficient for Hoxa13 and neighborhood gene expression in mouse embryonic distal limbs and genital bud.

We previously demonstrated that a approximately 1 Mb domain of genes upstream of and including Hoxa13 is co-expressed in the developing mouse limbs and genitalia. A highly conserved non-coding sequence, mmA13CNS, was shown to be insufficient in transgenic mice to direct precise Hoxa13-like expression in the limb buds or genital bud, although some LacZ expression from the transgene was reproducibly found in these tissues. In this report, we used beta-globin minimal promoter LacZ recombinant BAC transgenes encompassing mmA13CNS to identify a single critical region involved in mouse Hoxa13-like embryonic genital bud expression. By analyzing the expression patterns of these overlapping BAC clones in transgenic mice, we show that at least two sequences remote to the HoxA cluster are required collectively to drive Hoxa13-like expression in developing distal limbs. Given that the paralogous posterior HoxD and neighboring genes have been shown to be under the influence of long-range distal limb and genital bud enhancers, we hypothesize that both long-range enhancers have one ancestral origin, which diverged in both sequence and function after the HoxA/D cluster duplication.

A discrete class of intergenic DNA dictates meiotic DNA break hotspots in fission yeast.

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) made by Spo11 (Rec12 in fission yeast), which becomes covalently linked to the DSB ends. Like recombination events, DSBs occur at hotspots in the genome, but the genetic factors responsible for most hotspots have remained elusive. Here we describe in fission yeast the genome-wide distribution of meiosis-specific Rec12-DNA linkages, which closely parallel DSBs measured by conventional Southern blot hybridization. Prominent DSB hotspots are located approximately 65 kb apart, separated by intervals with little or no detectable breakage. Most hotspots lie within exceptionally large intergenic regions. Thus, the chromosomal architecture responsible for hotspots in fission yeast is markedly different from that of budding yeast, in which DSB hotspots are much more closely spaced and, in many regions of the genome, occur at each promoter. Our analysis in fission yeast reveals a clearly identifiable chromosomal feature that can predict the majority of recombination hotspots across a whole genome and provides a basis for searching for the chromosomal features that dictate hotspots of meiotic recombination in other organisms, including humans.

The HBS1L-MYB intergenic region on chromosome 6q23.3 influences erythrocyte, platelet, and monocyte counts in humans.

Common sequence variants situated between the HBS1L and MYB genes on chromosome 6q23.3 (HMIP) influence the proportion of F cells (erythrocytes that carry measurable amounts of fetal hemoglobin). Since the physiological processes underlying the F-cell variability are thought to be linked to kinetics of erythrocyte maturation and differentiation, we have investigated the influence of the HMIP locus on other hematologic parameters. Here we show a significant impact of HMIP variability on several types of peripheral blood cells: erythrocyte, platelet, and monocyte counts as well as erythrocyte volume and hemoglobin content in healthy individuals of European ancestry. These results support the notion that changes of F-cell abundance can be an indicator of more general shifts in hematopoietic patterns in humans.

Identification of long intergenic region sequences involved in maize streak virus replication.

The main cis-acting control regions for replication of the single-stranded DNA genome of maize streak virus (MSV) are believed to reside within an approximately 310 nt long intergenic region (LIR). However, neither the minimum LIR sequence required nor the sequence determinants of replication specificity have been determined experimentally. There are iterated sequences, or iterons, both within the conserved inverted-repeat sequences with the potential to form a stem-loop structure at the origin of virion-strand replication, and upstream of the rep gene TATA box (the rep-proximal iteron or RPI). Based on experimental analyses of similar iterons in viruses from other geminivirus genera and their proximity to known Rep-binding sites in the distantly related mastrevirus wheat dwarf virus, it has been hypothesized that the iterons may be Rep-binding and/or -recognition sequences. Here, a series of LIR deletion mutants was used to define the upper bounds of the LIR sequence required for replication. After identifying MSV strains and distinct mastreviruses with incompatible replication-specificity determinants (RSDs), LIR chimaeras were used to map the primary MSV RSD to a 67 nt sequence containing the RPI. Although the results generally support the prevailing hypothesis that MSV iterons are functional analogues of those found in other geminivirus genera, it is demonstrated that neither the inverted-repeat nor RPI sequences are absolute determinants of replication specificity. Moreover, widely divergent mastreviruses can trans-replicate one another. These results also suggest that sequences in the 67 nt region surrounding the RPI interact in a sequence-specific manner with those of the inverted repeat.

Genetic evidence suggests that the intergenic region between pstA and pstB plays a role in the regulation of rpoS translation during phosphate limitation.

In addition to the Pho regulon, phosphate starvation also stimulates the accumulation of RpoS. Several deletion mutations within the pstSCAB-phoU operon were tested for the accumulation of RpoS during exponential growth. Our data suggest that the processed 3' end of the pstA message stimulates translation of rpoS.

An iterated sequence in the genome of Banana bunchy top virus is essential for efficient replication.

Banana bunchy top virus (BBTV) has a multi-component genome of circular, single-stranded DNA. BBTV replicates via a rolling-circle mechanism, probably involving sequence-specific interaction of the replication initiation protein (Rep) with iterated sequences (iterons) within the viral genome. Three putative iterons (designated F1, F2 and R), with the sequence GGGAC, have been identified in the intergenic region of each BBTV component. To investigate their role in replication, each of the iterons was mutated, singularly and in tandem, in a BBTV DNA-N 1.1mer and the ability of these molecules to be replicated by the BBTV 'master' Rep was evaluated in banana cells using transient biolistic assays. All iteron mutants were replicated less efficiently than the native DNA-N. Mutation of the F1 and R iterons caused a 42 and 62 % reduction in DNA-N replication, respectively, whereas mutation of the F2 and combined F1F2 iteron virtually abolished DNA-N replication.