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1.  Deep Vertebrate Roots for Mammalian Zinc Finger Transcription Factor Subfamilies 
Genome Biology and Evolution  2014;6(3):510-525.
While many vertebrate transcription factor (TF) families are conserved, the C2H2 zinc finger (ZNF) family stands out as a notable exception. In particular, novel ZNF gene types have arisen, duplicated, and diverged independently throughout evolution to yield many lineage-specific TF genes. This evolutionary dynamic not only raises many intriguing questions but also severely complicates identification of those ZNF genes that remain functionally conserved. To address this problem, we searched for vertebrate “DNA binding orthologs” by mining ZNF loci from eight sequenced genomes and then aligning the patterns of DNA-binding amino acids, or “fingerprints,” extracted from the encoded ZNF motifs. Using this approach, we found hundreds of lineage-specific genes in each species and also hundreds of orthologous groups. Most groups of orthologs displayed some degree of fingerprint divergence between species, but 174 groups showed fingerprint patterns that have been very rigidly conserved. Focusing on the dynamic KRAB-ZNF subfamily—including nearly 400 human genes thought to possess potent KRAB-mediated epigenetic silencing activities—we found only three genes conserved between mammals and nonmammalian groups. These three genes, members of an ancient familial cluster, encode an unusual KRAB domain that functions as a transcriptional activator. Evolutionary analysis confirms the ancient provenance of this activating KRAB and reveals the independent expansion of KRAB-ZNFs in every vertebrate lineage. Most human ZNF genes, from the most deeply conserved to the primate-specific genes, are highly expressed in immune and reproductive tissues, indicating that they have been enlisted to regulate evolutionarily divergent biological traits.
doi:10.1093/gbe/evu030
PMCID: PMC3971581  PMID: 24534434
zinc finger genes; transcription factor evolution; vertebrate gene families
2.  Zfp322a Regulates Mouse ES Cell Pluripotency and Enhances Reprogramming Efficiency 
PLoS Genetics  2014;10(2):e1004038.
Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a) is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotency maintenance of mouse ES cells. Here, we report that Zfp322a is required for mES cell identity since depletion of Zfp322a directs mES cells towards differentiation. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays revealed that Zfp322a binds to Pou5f1 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of mES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated mES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits mES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka's factors (OSKM). It can be even used in combination with Yamanaka's factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of mouse ES cells.
Author Summary
Embryonic stem (ES) cells are featured by their ability to self-renew and by their potential to differentiate into many different cell types. Recent studies have revealed that the unique properties of mouse ES cells are governed by a specific transcription regulatory network, including master regulators Oct4/Sox2/Nanog and other pluripotency factors. The importance of these factors was highlighted by the subsequent finding that combination of several transcription factors can reprogram differentiated fibroblasts back to pluripotent stem cells. Here, we report that Zfp322a is a novel factor which is required for mES cell identity. We revealed that Zfp322a can regulate the key pluripotency genes Pou5f1 and Nanog and functions as a repressor of MAPK/ERK pathway in mES cells, therefore preventing mES cell differentiation. Furthermore, we discovered that Zfp332a can promote the generation of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Our results reveal that Zfp322a is a novel essential transcription factor which not only regulates ES cell pluripotency but also enhances iPSC formation.
doi:10.1371/journal.pgen.1004038
PMCID: PMC3923668  PMID: 24550733
3.  Genetic Disruption of the Copulatory Plug in Mice Leads to Severely Reduced Fertility 
PLoS Genetics  2013;9(1):e1003185.
Seminal fluid proteins affect fertility at multiple stages in reproduction. In many species, a male's ejaculate coagulates to form a copulatory plug. Although taxonomically widespread, the molecular details of plug formation remain poorly understood, limiting our ability to manipulate the structure and understand its role in reproduction. Here I show that male mice knockouts for transglutaminase IV (Tgm4) fail to form a copulatory plug, demonstrating that this gene is necessary for plug formation and lending a powerful new genetic tool to begin characterizing plug function. Tgm4 knockout males show normal sperm count, sperm motility, and reproductive morphology. However, very little of their ejaculate migrates into the female's reproductive tract, suggesting the plug prevents ejaculate leakage. Poor ejaculate migration leads to a reduction in the proportion of oocytes fertilized. However, Tgm4 knockout males fertilized between 3–11 oocytes, which should be adequate for a normal litter. Nevertheless, females mated to Tgm4 knockout males for approximately 14 days were significantly less likely to give birth to a litter compared to females mated to wild-type males. Therefore, it appears that the plug also affects post-fertilization events such as implantation and/or gestation. This study shows that a gene influencing the viscosity of seminal fluid has a major influence on male fertility.
Author Summary
Male reproductive fitness is strongly affected by seminal fluid. In many animals, the male's ejaculate coagulates in the female's reproductive tract to form a structure known as the copulatory plug. Here, I show that male mice without a functional copy of the gene transglutaminase IV cannot form a plug and suffer severe fertility defects. In spite of normal reproductive morphology, less of the ejaculate migrates through the female's reproductive tract and Tgm4 knockout males sire significantly fewer litters than wild type. This study demonstrates that the copulatory plug and/or Tgm4 itself is necessary for normal fertility.
doi:10.1371/journal.pgen.1003185
PMCID: PMC3547826  PMID: 23341775
4.  Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1 
PLoS Genetics  2012;8(3):e1002577.
Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.
Author Summary
Osteoporosis is a disease caused by disruption of the balance between bone formation and resorption resulting in a net loss of bone mass. Although anti-resorptive agents are the current mainstay of osteoporosis therapy, novel strategies to promote bone formation are critically needed for more effective prevention and treatment of the disease. Notch signaling, an evolutionally conserved mechanism among multi-cellular organisms, was recently shown to control bone formation and therefore represents a potential target pathway for novel bone-promoting therapeutics. In this study we elucidate the intracellular signaling mechanism through which Notch controls bone formation, providing a molecular framework that may guide future drug development.
doi:10.1371/journal.pgen.1002577
PMCID: PMC3310726  PMID: 22457635
5.  Rapid Sequence and Expression Divergence Suggest Selection for Novel Function in Primate-Specific KRAB-ZNF Genes 
Molecular Biology and Evolution  2010;27(11):2606-2617.
Recent segmental duplications (SDs), arising from duplication events that occurred within the past 35–40 My, have provided a major resource for the evolution of proteins with primate-specific functions. KRAB zinc finger (KRAB-ZNF) transcription factor genes are overrepresented among genes contained within these recent human SDs. Here, we examine the structural and functional diversity of the 70 human KRAB-ZNF genes involved in the most recent primate SD events including genes that arose in the hominid lineage. Despite their recent advent, many parent–daughter KRAB-ZNF gene pairs display significant differences in zinc finger structure and sequence, expression, and splicing patterns, each of which could significantly alter the regulatory functions of the paralogous genes. Paralogs that emerged on the lineage to humans and chimpanzees have undergone more evolutionary changes per unit of time than genes already present in the common ancestor of rhesus macaques and great apes. Taken together, these data indicate that a substantial fraction of the recently evolved primate-specific KRAB-ZNF gene duplicates have acquired novel functions that may possibly define novel regulatory pathways and suggest an active ongoing selection for regulatory diversity in primates.
doi:10.1093/molbev/msq157
PMCID: PMC2981486  PMID: 20573777
KRAB-ZNF genes; recently evolved primate genes; transcription factor; primate evolution; paralog divergence
6.  MicroRNA Expression and Regulation in Human, Chimpanzee, and Macaque Brains 
PLoS Genetics  2011;7(10):e1002327.
Among other factors, changes in gene expression on the human evolutionary lineage have been suggested to play an important role in the establishment of human-specific phenotypes. However, the molecular mechanisms underlying these expression changes are largely unknown. Here, we have explored the role of microRNA (miRNA) in the regulation of gene expression divergence among adult humans, chimpanzees, and rhesus macaques, in two brain regions: prefrontal cortex and cerebellum. Using a combination of high-throughput sequencing, miRNA microarrays, and Q-PCR, we have shown that up to 11% of the 325 expressed miRNA diverged significantly between humans and chimpanzees and up to 31% between humans and macaques. Measuring mRNA and protein expression in human and chimpanzee brains, we found a significant inverse relationship between the miRNA and the target genes expression divergence, explaining 2%–4% of mRNA and 4%–6% of protein expression differences. Notably, miRNA showing human-specific expression localize in neurons and target genes that are involved in neural functions. Enrichment in neural functions, as well as miRNA–driven regulation on the human evolutionary lineage, was further confirmed by experimental validation of predicted miRNA targets in two neuroblastoma cell lines. Finally, we identified a signature of positive selection in the upstream region of one of the five miRNA with human-specific expression, miR-34c-5p. This suggests that miR-34c-5p expression change took place after the split of the human and the Neanderthal lineages and had adaptive significance. Taken together these results indicate that changes in miRNA expression might have contributed to evolution of human cognitive functions.
Author Summary
Humans are remarkably similar to apes and monkeys on the genome sequence level but remain remarkably distinct with respect to cognitive abilities. How could human cognition evolve within such a short evolutionary time? Among many hypotheses, evolution in expression of a few key regulators affecting hundreds of their target genes was proposed as one possible solution. Here, we tested this notion by studying expression divergence of a specific type of regulatory RNA, microRNA (miRNA), and its effect on gene expression profiles in brains of humans, chimpanzees, and rhesus macaques. Our results indicate that changes in miRNA expression have played a considerable role in the establishment of gene expression divergence between human brains and brains of non-human primates at both mRNA and protein expression levels. Furthermore, we find indications that some of the human-specific gene expression profiles caused by miRNA expression divergence might be associated with evolution of human-specific functions.
doi:10.1371/journal.pgen.1002327
PMCID: PMC3192836  PMID: 22022286
7.  Gain, Loss and Divergence in Primate Zinc-Finger Genes: A Rich Resource for Evolution of Gene Regulatory Differences between Species 
PLoS ONE  2011;6(6):e21553.
The molecular changes underlying major phenotypic differences between humans and other primates are not well understood, but alterations in gene regulation are likely to play a major role. Here we performed a thorough evolutionary analysis of the largest family of primate transcription factors, the Krüppel-type zinc finger (KZNF) gene family. We identified and curated gene and pseudogene models for KZNFs in three primate species, chimpanzee, orangutan and rhesus macaque, to allow for a comparison with the curated set of human KZNFs. We show that the recent evolutionary history of primate KZNFs has been complex, including many lineage-specific duplications and deletions. We found 213 species-specific KZNFs, among them 7 human-specific and 23 chimpanzee-specific genes. Two human-specific genes were validated experimentally. Ten genes have been lost in humans and 13 in chimpanzees, either through deletion or pseudogenization. We also identified 30 KZNF orthologs with human-specific and 42 with chimpanzee-specific sequence changes that are predicted to affect DNA binding properties of the proteins. Eleven of these genes show signatures of accelerated evolution, suggesting positive selection between humans and chimpanzees. During primate evolution the most extensive re-shaping of the KZNF repertoire, including most gene additions, pseudogenizations, and structural changes occurred within the subfamily homininae. Using zinc finger (ZNF) binding predictions, we suggest potential impact these changes have had on human gene regulatory networks. The large species differences in this family of TFs stands in stark contrast to the overall high conservation of primate genomes and potentially represents a potent driver of primate evolution.
doi:10.1371/journal.pone.0021553
PMCID: PMC3126818  PMID: 21738707
8.  Lineage-specific transcription factors and the evolution of gene regulatory networks 
Nature is replete with examples of diverse cell types, tissues and body plans, forming very different creatures from genomes with similar gene complements. However, while the genes and the structures of proteins they encode can be highly conserved, the production of those proteins in specific cell types and at specific developmental time points might differ considerably between species. A full understanding of the factors that orchestrate gene expression will be essential to fully understand evolutionary variety. Transcription factor (TF) proteins, which form gene regulatory networks (GRNs) to act in cooperative or competitive partnerships to regulate gene expression, are key components of these unique regulatory programs. Although many TFs are conserved in structure and function, certain classes of TFs display extensive levels of species diversity. In this review, we highlight families of TFs that have expanded through gene duplication events to create species-unique repertoires in different evolutionary lineages. We discuss how the hierarchical structures of GRNs allow for flexible small to large-scale phenotypic changes. We survey evidence that explains how newly evolved TFs may be integrated into an existing GRN and how molecular changes in TFs might impact the GRNs. Finally, we review examples of traits that evolved due to lineage-specific TFs and species differences in GRNs.
doi:10.1093/bfgp/elp056
PMCID: PMC3096533  PMID: 20081217
transcription factors; gene regulatory network; evolution; lineage-specific genes
9.  PPS, a Large Multidomain Protein, Functions with Sex-Lethal to Regulate Alternative Splicing in Drosophila 
PLoS Genetics  2010;6(3):e1000872.
Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL–mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance.
Author Summary
In Drosophila the sex-specific ON/OFF regulation of Sex-lethal (Sxl) is controlled by an autoregulatory splicing mechanism that depends on the SXL protein interacting with general splicing factors. Here we identify PPS as a novel component of the machinery required for Sxl splicing autoregulation by showing that the lack of pps function interferes with Sxl expression and that the PPS protein is physically linked to the Sxl pre–mRNA, the SXL protein and components of the general splicing machinery. PPS, however, stands apart from all other proteins known to control Sxl splicing because it is not a general splicing factor. Furthermore, PPS has a distinct pattern of accumulation along the Sxl transcription unit that suggests PPS is loaded onto the RNA at the promoter. Together with the observation that the PPS protein contains four signature motifs typically found in proteins that function in transcriptional regulation, our data suggest that linking transcription to splicing regulation is important for controlling Sxl expression. This idea is especially intriguing because it indicates that the coupling of transcription and splicing seen in vitro and in cell culture studies is likely to be pertinent to developmentally controlled patterns of gene expression in the living animal.
doi:10.1371/journal.pgen.1000872
PMCID: PMC2832672  PMID: 20221253
10.  VEZF1 Elements Mediate Protection from DNA Methylation 
PLoS Genetics  2010;6(1):e1000804.
There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state.
Author Summary
DNA sequences known as chromatin insulator or barrier elements are considered key components of genome organization as they can establish boundaries between transcriptionally permissive and repressive chromatin domains. Here we address the hypothesis that barrier elements in vertebrates can protect genes from transcriptional silencing that is marked by DNA methylation. We have found that the HS4 insulator element from the β-globin gene locus can protect a gene promoter from DNA methylation. Protection from DNA methylation is separable from other insulator activities and is mapped to three transcription factor binding sites occupied by the zinc finger protein VEZF1, a novel chromatin barrier protein. VEZF1 is a candidate factor for the protection of promoters from DNA methylation. We found that VEZF1-specific binding sites are sufficient to mediate demethylation and protection of the APRT gene promoter from DNA methylation. We propose that barrier elements in vertebrates must be capable of preventing DNA methylation in addition to blocking the propagation of silencing histone modifications, as either process is sufficient to direct the establishment of an inactive chromatin state.
doi:10.1371/journal.pgen.1000804
PMCID: PMC2795164  PMID: 20062523
11.  The Human Retinoblastoma Gene Is Imprinted 
PLoS Genetics  2009;5(12):e1000790.
Genomic imprinting is an epigenetic process leading to parent-of-origin–specific DNA methylation and gene expression. To date, ∼60 imprinted human genes are known. Based on genome-wide methylation analysis of a patient with multiple imprinting defects, we have identified a differentially methylated CpG island in intron 2 of the retinoblastoma (RB1) gene on chromosome 13. The CpG island is part of a 5′-truncated, processed pseudogene derived from the KIAA0649 gene on chromosome 9 and corresponds to two small CpG islands in the open reading frame of the ancestral gene. It is methylated on the maternal chromosome 13 and acts as a weak promoter for an alternative RB1 transcript on the paternal chromosome 13. In four other KIAA0649 pseudogene copies, which are located on chromosome 22, the two CpG islands have deteriorated and the CpG dinucleotides are fully methylated. By analysing allelic RB1 transcript levels in blood cells, as well as in hypermethylated and 5-aza-2′-deoxycytidine–treated lymphoblastoid cells, we have found that differential methylation of the CpG island skews RB1 gene expression in favor of the maternal allele. Thus, RB1 is imprinted in the same direction as CDKN1C, which operates upstream of RB1. The imprinting of two components of the same pathway indicates that there has been strong evolutionary selection for maternal inhibition of cell proliferation.
Author Summary
Genomic imprinting is an epigenetic process leading to parent-of-origin–specific DNA methylation and gene expression. Defects in this process lead to abnormal development, growth, or behavior. It is still unclear why and how imprinting evolved and how many human genes are imprinted. Based on genome-wide DNA methylation analysis in a patient with a generalized imprinting defect, we have found that the paradigmatic retinoblastoma 1 (RB1) gene on chromosome 13 is imprinted. Imprinting of RB1 is linked to the insertion of a DNA sequence derived by retrotransposition from a gene on chromosome 9. Part of the inserted DNA sequence has evolved into a differentially methylated alternative RB1 promoter. Differential methylation of this sequence skews expression of the RB1 gene in favour of the maternal allele. The direction of the imprint imposed on the RB1 gene is the same as of the maternally expressed CDKN1C gene, which operates upstream of RB1. The imprinting of two components of the same pathway indicates that there has been strong evolutionary selection for maternal inhibition of cell proliferation.
doi:10.1371/journal.pgen.1000790
PMCID: PMC2791201  PMID: 20041224
12.  A Common Variant Associated with Dyslexia Reduces Expression of the KIAA0319 Gene 
PLoS Genetics  2009;5(3):e1000436.
Numerous genetic association studies have implicated the KIAA0319 gene on human chromosome 6p22 in dyslexia susceptibility. The causative variant(s) remains unknown but may modulate gene expression, given that (1) a dyslexia-associated haplotype has been implicated in the reduced expression of KIAA0319, and (2) the strongest association has been found for the region spanning exon 1 of KIAA0319. Here, we test the hypothesis that variant(s) responsible for reduced KIAA0319 expression resides on the risk haplotype close to the gene's transcription start site. We identified seven single-nucleotide polymorphisms on the risk haplotype immediately upstream of KIAA0319 and determined that three of these are strongly associated with multiple reading-related traits. Using luciferase-expressing constructs containing the KIAA0319 upstream region, we characterized the minimal promoter and additional putative transcriptional regulator regions. This revealed that the minor allele of rs9461045, which shows the strongest association with dyslexia in our sample (max p-value = 0.0001), confers reduced luciferase expression in both neuronal and non-neuronal cell lines. Additionally, we found that the presence of this rs9461045 dyslexia-associated allele creates a nuclear protein-binding site, likely for the transcriptional silencer OCT-1. Knocking down OCT-1 expression in the neuronal cell line SHSY5Y using an siRNA restores KIAA0319 expression from the risk haplotype to nearly that seen from the non-risk haplotype. Our study thus pinpoints a common variant as altering the function of a dyslexia candidate gene and provides an illustrative example of the strategic approach needed to dissect the molecular basis of complex genetic traits.
Author Summary
Dyslexia, or reading disability, is a common disorder caused by both genetic and environmental factors. Genetic studies have implicated a number of genes as candidates for playing a role in dyslexia. We functionally characterized one such gene (KIAA0319) to identify variant(s) that might affect gene expression and contribute to the disorder. We discovered a variant residing outside of the protein-coding region of KIAA0319 that reduces expression of the gene. This variant creates a binding site for the transcription factor OCT-1. Previous studies have shown that OCT-1 binding to a specific DNA sequence upstream of a gene can reduce the expression of that gene. In this case, reduced KIAA0319 expression could lead to improper development of regions of the brain involved in reading ability. This is the first study to identify a functional variant implicated in dyslexia. More broadly, our study illustrates the steps that can be utilized for identifying mutations causing other complex genetic disorders.
doi:10.1371/journal.pgen.1000436
PMCID: PMC2653637  PMID: 19325871
13.  Deletion Hotspots in AMACR Promoter CpG Island Are cis-Regulatory Elements Controlling the Gene Expression in the Colon 
PLoS Genetics  2009;5(1):e1000334.
Alpha-methylacyl-coenzyme A racemase (AMACR) regulates peroxisomal β-oxidation of phytol-derived, branched-chain fatty acids from red meat and dairy products — suspected risk factors for colon carcinoma (CCa). AMACR was first found overexpressed in prostate cancer but not in benign glands and is now an established diagnostic marker for prostate cancer. Aberrant expression of AMACR was recently reported in Cca; however, little is known about how this gene is abnormally activated in cancer. By using a panel of immunostained-laser-capture-microdissected clinical samples comprising the entire colon adenoma–carcinoma sequence, we show that deregulation of AMACR during colon carcinogenesis involves two nonrandom events, resulting in the mutually exclusive existence of double-deletion at CG3 and CG10 and deletion of CG12-16 in a newly identified CpG island within the core promoter of AMACR. The double-deletion at CG3 and CG10 was found to be a somatic lesion. It existed in histologically normal colonic glands and tubular adenomas with low AMACR expression and was absent in villous adenomas and all CCas expressing variable levels of AMACR. In contrast, deletion of CG12-16 was shown to be a constitutional allele with a frequency of 43% in a general population. Its prevalence reached 89% in moderately differentiated CCas strongly expressing AMACR but only existed at 14% in poorly differentiated CCas expressing little or no AMACR. The DNA sequences housing these deletions were found to be putative cis-regulatory elements for Sp1 at CG3 and CG10, and ZNF202 at CG12-16. Chromatin immunoprecipitation, siRNA knockdown, gel shift assay, ectopic expression, and promoter analyses supported the regulation by Sp1 and ZNF202 of AMACR gene expression in an opposite manner. Our findings identified key in vivo events and novel transcription factors responsible for AMACR regulation in CCas and suggested these AMACR deletions may have diagnostic/prognostic value for colon carcinogenesis.
Author Summary
Men consuming high amounts of red meat and dairy products are at a higher risk of developing colon and prostate cancer. Alpha-methylacyl-coenzyme A racemase (AMACR) is an enzyme that helps to break down fat from these foods to produce energy. An increase in the utilization of energy from fat is a hallmark of many cancers including colon and prostate cancers. Indeed, the AMACR gene was first found to be abnormally active in prostate cancers, and its abnormal expression has become a diagnostic marker for the cancer. However, little is known about how AMACR becomes activated in cancer cells. Here, we show that AMACR is also highly expressed in certain stages of colon cancer, though not all stages. A close examination of the AMACR gene in a panel of normal and progressively malignant colon tissues reveals that deletions of specific sequences in the AMACR gene may trigger its abnormal expression during the evolution of colon cancer. We also identify unique proteins known as “transcription factors” that normally bind to these deleted sequences to maintain normal expression of the gene. Finally, we report a new deletion variant of the AMACR gene in the general population that may influence the course of colon carcinogenesis.
doi:10.1371/journal.pgen.1000334
PMCID: PMC2613032  PMID: 19148275
14.  YY1 as a controlling factor for the Peg3 and Gnas imprinted domains 
Genomics  2006;89(2):262-269.
Imprinting Control Regions (ICRs) often harbor tandem arrays of transcription factor binding sites, as demonstrated by the identification of multiple YY1 binding sites within the ICRs of Peg3, Nespas, and Xist/Tsix domains. In the current study, we have sought to characterize possible roles of YY1 in transcriptional control and epigenetic modification of these imprinted domains. RNA interference-based knockdown experiments in Neuro2A cells resulted in overall transcriptional up-regulation of most of the imprinted genes within the Peg3 domain and also, concomitantly, caused significant loss in the DNA methylation of Peg3-DMR (Differentially Methylated Regions). A similar overall and coordinated expression change was also observed for the imprinted genes of the Gnas domain: up-regulation of Nespas and down-regulation of Nesp and Gnasxl. YY1 knockdown also resulted in changes in the expression levels of Xist and Snrpn. These results support the idea that YY1 plays a major role, as a trans factor, for the control of these imprinted domains.
doi:10.1016/j.ygeno.2006.09.009
PMCID: PMC1828871  PMID: 17067777
genomic imprinting; ICRs; YY1
15.  The “Alternative” Choice of Constitutive Exons throughout Evolution 
PLoS Genetics  2007;3(11):e203.
Alternative cassette exons are known to originate from two processes—exonization of intronic sequences and exon shuffling. Herein, we suggest an additional mechanism by which constitutively spliced exons become alternative cassette exons during evolution. We compiled a dataset of orthologous exons from human and mouse that are constitutively spliced in one species but alternatively spliced in the other. Examination of these exons suggests that the common ancestors were constitutively spliced. We show that relaxation of the 5′ splice site during evolution is one of the molecular mechanisms by which exons shift from constitutive to alternative splicing. This shift is associated with the fixation of exonic splicing regulatory sequences (ESRs) that are essential for exon definition and control the inclusion level only after the transition to alternative splicing. The effect of each ESR on splicing and the combinatorial effects between two ESRs are conserved from fish to human. Our results uncover an evolutionary pathway that increases transcriptome diversity by shifting exons from constitutive to alternative splicing.
Author Summary
Alternative splicing is believed to play a major role in the creation of transcriptomic diversification leading to higher order of organismal complexity, especially in mammals. As much as 80% of human genes generate more than one type of mRNA by alternative splicing. Thus, alternative splicing can bridge the low number of protein coding genes (∼24,500) and the total number of proteins generated in the human proteome (∼90,000). The correlation between the higher order of phenotypic diversity and alternative splicing was recently demonstrated and thus the origin of alternative splicing is of great interest. There are currently two models regarding the origin of alternatively spliced exons—exonization of intronic sequences and exon shuffling. According to these two mechanisms, a protein-coding gene was first established and only then a new alternative exon appeared within it or was added to the gene. Our current study provides evidences for a new mechanism indicating that during evolution constitutively spliced exons became alternatively spliced. Large-scale bioinformatic analyses reveal the magnitude of this process and experimental validation systems provide insights into its mechanisms.
doi:10.1371/journal.pgen.0030203
PMCID: PMC2077895  PMID: 18020709
16.  The ets-Related Transcription Factor GABP Directs Bidirectional Transcription 
PLoS Genetics  2007;3(11):e208.
Approximately 10% of genes in the human genome are distributed such that their transcription start sites are located less than 1 kb apart on opposite strands. These divergent gene pairs have a single intergenic segment of DNA, which in some cases appears to share regulatory elements, but it is unclear whether these regions represent functional bidirectional promoters or two overlapping promoters. A recent study showed that divergent promoters are enriched for consensus binding sequences of a small group of transcription factors, including the ubiquitous ets-family transcription factor GA-binding protein (GABP). Here we show that GABP binds to more than 80% of divergent promoters in at least one cell type. Furthermore, we demonstrate that GABP binding is correlated and associated with bidirectional transcriptional activity in a luciferase transfection assay. In addition, we find that the addition of a strict consensus GABP site into a set of promoters that normally function in only one direction significantly increases activity in the opposite direction in 67% of cases. Our findings demonstrate that GABP regulates the majority of divergent promoters and suggest that bidirectional transcriptional activity is mediated through GABP binding and transactivation at both divergent and nondivergent promoters.
Author Summary
Surveys of the locations of genes in the human genome have revealed that a surprising number of genes, greater than 10%, have transcription start sites within 1 kb of one another on opposite strands. These divergent gene pairs, sometimes referred to as bidirectional genes, are common in organisms such as bacteria and yeast, but it is unknown why such an arrangement exists in large, mammalian genomes. Recently, it has become apparent that the promoters of these divergent genes are regulated by a subset of transcription factors, and we have focused on one of these, GA-binding protein (GABP). We find that it regulates a large number of human genes, including the majority of divergent genes, and that its binding is associated with, correlated with, and sufficient for bidirectional transcriptional activity. Although clearly GABP is a major regulator of divergent genes, which carry out a variety of roles critical for the function and survival of the cell, these data also propose novel roles for GABP as a transcription factor. For example, the ability of GABP to promote bidirectional transcription may prove to be biologically relevant in generating many of the transcripts that have been observed outside of protein coding genes.
doi:10.1371/journal.pgen.0030208
PMCID: PMC2077898  PMID: 18020712
17.  The “Alternative” Choice of Constitutive Exons throughout Evolution 
PLoS Genetics  2007;3(11):e203.
Alternative cassette exons are known to originate from two processes—exonization of intronic sequences and exon shuffling. Herein, we suggest an additional mechanism by which constitutively spliced exons become alternative cassette exons during evolution. We compiled a dataset of orthologous exons from human and mouse that are constitutively spliced in one species but alternatively spliced in the other. Examination of these exons suggests that the common ancestors were constitutively spliced. We show that relaxation of the 5′ splice site during evolution is one of the molecular mechanisms by which exons shift from constitutive to alternative splicing. This shift is associated with the fixation of exonic splicing regulatory sequences (ESRs) that are essential for exon definition and control the inclusion level only after the transition to alternative splicing. The effect of each ESR on splicing and the combinatorial effects between two ESRs are conserved from fish to human. Our results uncover an evolutionary pathway that increases transcriptome diversity by shifting exons from constitutive to alternative splicing.
Author Summary
Alternative splicing is believed to play a major role in the creation of transcriptomic diversification leading to higher order of organismal complexity, especially in mammals. As much as 80% of human genes generate more than one type of mRNA by alternative splicing. Thus, alternative splicing can bridge the low number of protein coding genes (∼24,500) and the total number of proteins generated in the human proteome (∼90,000). The correlation between the higher order of phenotypic diversity and alternative splicing was recently demonstrated and thus the origin of alternative splicing is of great interest. There are currently two models regarding the origin of alternatively spliced exons—exonization of intronic sequences and exon shuffling. According to these two mechanisms, a protein-coding gene was first established and only then a new alternative exon appeared within it or was added to the gene. Our current study provides evidences for a new mechanism indicating that during evolution constitutively spliced exons became alternatively spliced. Large-scale bioinformatic analyses reveal the magnitude of this process and experimental validation systems provide insights into its mechanisms.
doi:10.1371/journal.pgen.0030203
PMCID: PMC2077895  PMID: 18020709
18.  The ets-Related Transcription Factor GABP Directs Bidirectional Transcription 
PLoS Genetics  2007;3(11):e208.
Approximately 10% of genes in the human genome are distributed such that their transcription start sites are located less than 1 kb apart on opposite strands. These divergent gene pairs have a single intergenic segment of DNA, which in some cases appears to share regulatory elements, but it is unclear whether these regions represent functional bidirectional promoters or two overlapping promoters. A recent study showed that divergent promoters are enriched for consensus binding sequences of a small group of transcription factors, including the ubiquitous ets-family transcription factor GA-binding protein (GABP). Here we show that GABP binds to more than 80% of divergent promoters in at least one cell type. Furthermore, we demonstrate that GABP binding is correlated and associated with bidirectional transcriptional activity in a luciferase transfection assay. In addition, we find that the addition of a strict consensus GABP site into a set of promoters that normally function in only one direction significantly increases activity in the opposite direction in 67% of cases. Our findings demonstrate that GABP regulates the majority of divergent promoters and suggest that bidirectional transcriptional activity is mediated through GABP binding and transactivation at both divergent and nondivergent promoters.
Author Summary
Surveys of the locations of genes in the human genome have revealed that a surprising number of genes, greater than 10%, have transcription start sites within 1 kb of one another on opposite strands. These divergent gene pairs, sometimes referred to as bidirectional genes, are common in organisms such as bacteria and yeast, but it is unknown why such an arrangement exists in large, mammalian genomes. Recently, it has become apparent that the promoters of these divergent genes are regulated by a subset of transcription factors, and we have focused on one of these, GA-binding protein (GABP). We find that it regulates a large number of human genes, including the majority of divergent genes, and that its binding is associated with, correlated with, and sufficient for bidirectional transcriptional activity. Although clearly GABP is a major regulator of divergent genes, which carry out a variety of roles critical for the function and survival of the cell, these data also propose novel roles for GABP as a transcription factor. For example, the ability of GABP to promote bidirectional transcription may prove to be biologically relevant in generating many of the transcripts that have been observed outside of protein coding genes.
doi:10.1371/journal.pgen.0030208
PMCID: PMC2077898  PMID: 18020712
19.  ZIPK: A Unique Case of Murine-Specific Divergence of a Conserved Vertebrate Gene 
PLoS Genetics  2007;3(10):e180.
Zipper interacting protein kinase (ZIPK, also known as death-associated protein kinase 3 [DAPK3]) is a Ser/Thr kinase that functions in programmed cell death. Since its identification eight years ago, contradictory findings regarding its intracellular localization and molecular mode of action have been reported, which may be attributed to unpredicted differences among the human and rodent orthologs. By aligning the sequences of all available ZIPK orthologs, from fish to human, we discovered that rat and mouse sequences are more diverged from the human ortholog relative to other, more distant, vertebrates. To test experimentally the outcome of this sequence divergence, we compared rat ZIPK to human ZIPK in the same cellular settings. We found that while ectopically expressed human ZIPK localized to the cytoplasm and induced membrane blebbing, rat ZIPK localized exclusively within nuclei, mainly to promyelocytic leukemia oncogenic bodies, and induced significantly lower levels of membrane blebbing. Among the unique murine (rat and mouse) sequence features, we found that a highly conserved phosphorylation site, previously shown to have an effect on the cellular localization of human ZIPK, is absent in murines but not in earlier diverging organisms. Recreating this phosphorylation site in rat ZIPK led to a significant reduction in its promyelocytic leukemia oncogenic body localization, yet did not confer full cytoplasmic localization. Additionally, we found that while rat ZIPK interacts with PAR-4 (also known as PAWR) very efficiently, human ZIPK fails to do so. This interaction has clear functional implications, as coexpression of PAR-4 with rat ZIPK caused nuclear to cytoplasm translocation and induced strong membrane blebbing, thus providing the murine protein a possible adaptive mechanism to compensate for its sequence divergence. We have also cloned zebrafish ZIPK and found that, like the human and unlike the murine orthologs, it localizes to the cytoplasm, and fails to bind the highly conserved PAR-4 protein. This further supports the hypothesis that murine ZIPK underwent specific divergence from a conserved consensus. In conclusion, we present a case of species-specific divergence occurring in a specific branch of the evolutionary tree, accompanied by the acquisition of a unique protein–protein interaction that enables conservation of cellular function.
Author Summary
Mammals are a fairly young class of animals, first appearing about 70 million years ago. Such recent common descent does not allow the evolutionary process to create much diversity within the class, and indeed, the physiology among different mammals is remarkably similar. This similarity enables the use of various small mammals, especially rats and mice, as model systems for the study of biological phenomenon and disease. Experiments unfeasible or unethical to perform on humans are conducted on these model animals, with the postulation that insights gained from them are applicable to the human system. In this article, we present an exception to this rule. We bring evidence that ZIPK, a gene with important roles in programmed cell death, has undergone accelerated evolution in the rat and mouse, thus diverging considerably from a well-conserved consensus in all vertebrates, from fish to man. We also show that this sequence divergence caused changes in the protein's properties, including its localization within the cell, and the proteins with which it interacts. Still, the basic biologic function of ZIPK is conserved in both systems, and we propose an adaptive mechanism that compensates for the sequence divergence in rodents.
doi:10.1371/journal.pgen.0030180
PMCID: PMC2041995  PMID: 17953487
20.  ZIPK: A Unique Case of Murine-Specific Divergence of a Conserved Vertebrate Gene 
PLoS Genetics  2007;3(10):e180.
Zipper interacting protein kinase (ZIPK, also known as death-associated protein kinase 3 [DAPK3]) is a Ser/Thr kinase that functions in programmed cell death. Since its identification eight years ago, contradictory findings regarding its intracellular localization and molecular mode of action have been reported, which may be attributed to unpredicted differences among the human and rodent orthologs. By aligning the sequences of all available ZIPK orthologs, from fish to human, we discovered that rat and mouse sequences are more diverged from the human ortholog relative to other, more distant, vertebrates. To test experimentally the outcome of this sequence divergence, we compared rat ZIPK to human ZIPK in the same cellular settings. We found that while ectopically expressed human ZIPK localized to the cytoplasm and induced membrane blebbing, rat ZIPK localized exclusively within nuclei, mainly to promyelocytic leukemia oncogenic bodies, and induced significantly lower levels of membrane blebbing. Among the unique murine (rat and mouse) sequence features, we found that a highly conserved phosphorylation site, previously shown to have an effect on the cellular localization of human ZIPK, is absent in murines but not in earlier diverging organisms. Recreating this phosphorylation site in rat ZIPK led to a significant reduction in its promyelocytic leukemia oncogenic body localization, yet did not confer full cytoplasmic localization. Additionally, we found that while rat ZIPK interacts with PAR-4 (also known as PAWR) very efficiently, human ZIPK fails to do so. This interaction has clear functional implications, as coexpression of PAR-4 with rat ZIPK caused nuclear to cytoplasm translocation and induced strong membrane blebbing, thus providing the murine protein a possible adaptive mechanism to compensate for its sequence divergence. We have also cloned zebrafish ZIPK and found that, like the human and unlike the murine orthologs, it localizes to the cytoplasm, and fails to bind the highly conserved PAR-4 protein. This further supports the hypothesis that murine ZIPK underwent specific divergence from a conserved consensus. In conclusion, we present a case of species-specific divergence occurring in a specific branch of the evolutionary tree, accompanied by the acquisition of a unique protein–protein interaction that enables conservation of cellular function.
Author Summary
Mammals are a fairly young class of animals, first appearing about 70 million years ago. Such recent common descent does not allow the evolutionary process to create much diversity within the class, and indeed, the physiology among different mammals is remarkably similar. This similarity enables the use of various small mammals, especially rats and mice, as model systems for the study of biological phenomenon and disease. Experiments unfeasible or unethical to perform on humans are conducted on these model animals, with the postulation that insights gained from them are applicable to the human system. In this article, we present an exception to this rule. We bring evidence that ZIPK, a gene with important roles in programmed cell death, has undergone accelerated evolution in the rat and mouse, thus diverging considerably from a well-conserved consensus in all vertebrates, from fish to man. We also show that this sequence divergence caused changes in the protein's properties, including its localization within the cell, and the proteins with which it interacts. Still, the basic biologic function of ZIPK is conserved in both systems, and we propose an adaptive mechanism that compensates for the sequence divergence in rodents.
doi:10.1371/journal.pgen.0030180
PMCID: PMC2041995  PMID: 17953487
21.  Determinants of Cell- and Gene-Specific Transcriptional Regulation by the Glucocorticoid Receptor 
PLoS Genetics  2007;3(6):e94.
The glucocorticoid receptor (GR) associates with glucocorticoid response elements (GREs) and regulates selective gene transcription in a cell-specific manner. Native GREs are typically thought to be composite elements that recruit GR as well as other regulatory factors into functional complexes. We assessed whether GR occupancy is commonly a limiting determinant of GRE function as well as the extent to which core GR binding sequences and GRE architecture are conserved at functional loci. We surveyed 100-kb regions surrounding each of 548 known or potentially glucocorticoid-responsive genes in A549 human lung cells for GR-occupied GREs. We found that GR was bound in A549 cells predominately near genes responsive to glucocorticoids in those cells and not at genes regulated by GR in other cells. The GREs were positionally conserved at each responsive gene but across the set of responsive genes were distributed equally upstream and downstream of the transcription start sites, with 63% of them >10 kb from those sites. Strikingly, although the core GR binding sequences across the set of GREs varied extensively around a consensus, the precise sequence at an individual GRE was conserved across four mammalian species. Similarly, sequences flanking the core GR binding sites also varied among GREs but were conserved at individual GREs. We conclude that GR occupancy is a primary determinant of glucocorticoid responsiveness in A549 cells and that core GR binding sequences as well as GRE architecture likely harbor gene-specific regulatory information.
Author Summary
The glucocorticoid receptor (GR) regulates a myriad of physiological functions, such as cell differentiation and metabolism, achieved through modulating transcription in a cell- and gene-specific manner. However, the determinants that specify cell- and gene-specific GR transcriptional regulation are not well established. We describe three properties that contribute to this specificity: (1) GR occupancy at genomic glucocorticoid response elements (GREs) appears to be a primary determinant of glucocorticoid responsiveness; (2) the DNA sequences bound by GR vary widely around a consensus, but the precise sequences of individual GREs are highly conserved, suggesting a role for these sequences in gene-specific GR transcriptional regulation; and (3) native chromosomal GREs were generally found to be composite elements, comprised of multiple factor binding sites that were highly variable in composition, but as with the GR binding sequences, highly conserved at individual GREs. In addition, we discovered that most GREs were positioned far from their GR target genes and that they were equally distributed upstream and downstream of the target genes. These findings, which may be applicable to other regulatory factors, provide fundamental insights for understanding cell- and gene-specific transcriptional regulation.
doi:10.1371/journal.pgen.0030094
PMCID: PMC1904358  PMID: 17559307
22.  Determinants of Cell- and Gene-Specific Transcriptional Regulation by the Glucocorticoid Receptor 
PLoS Genetics  2007;3(6):e94.
The glucocorticoid receptor (GR) associates with glucocorticoid response elements (GREs) and regulates selective gene transcription in a cell-specific manner. Native GREs are typically thought to be composite elements that recruit GR as well as other regulatory factors into functional complexes. We assessed whether GR occupancy is commonly a limiting determinant of GRE function as well as the extent to which core GR binding sequences and GRE architecture are conserved at functional loci. We surveyed 100-kb regions surrounding each of 548 known or potentially glucocorticoid-responsive genes in A549 human lung cells for GR-occupied GREs. We found that GR was bound in A549 cells predominately near genes responsive to glucocorticoids in those cells and not at genes regulated by GR in other cells. The GREs were positionally conserved at each responsive gene but across the set of responsive genes were distributed equally upstream and downstream of the transcription start sites, with 63% of them >10 kb from those sites. Strikingly, although the core GR binding sequences across the set of GREs varied extensively around a consensus, the precise sequence at an individual GRE was conserved across four mammalian species. Similarly, sequences flanking the core GR binding sites also varied among GREs but were conserved at individual GREs. We conclude that GR occupancy is a primary determinant of glucocorticoid responsiveness in A549 cells and that core GR binding sequences as well as GRE architecture likely harbor gene-specific regulatory information.
Author Summary
The glucocorticoid receptor (GR) regulates a myriad of physiological functions, such as cell differentiation and metabolism, achieved through modulating transcription in a cell- and gene-specific manner. However, the determinants that specify cell- and gene-specific GR transcriptional regulation are not well established. We describe three properties that contribute to this specificity: (1) GR occupancy at genomic glucocorticoid response elements (GREs) appears to be a primary determinant of glucocorticoid responsiveness; (2) the DNA sequences bound by GR vary widely around a consensus, but the precise sequences of individual GREs are highly conserved, suggesting a role for these sequences in gene-specific GR transcriptional regulation; and (3) native chromosomal GREs were generally found to be composite elements, comprised of multiple factor binding sites that were highly variable in composition, but as with the GR binding sequences, highly conserved at individual GREs. In addition, we discovered that most GREs were positioned far from their GR target genes and that they were equally distributed upstream and downstream of the target genes. These findings, which may be applicable to other regulatory factors, provide fundamental insights for understanding cell- and gene-specific transcriptional regulation.
doi:10.1371/journal.pgen.0030094
PMCID: PMC1904358  PMID: 17559307
23.  New Small Nuclear RNA Gene-Like Transcriptional Units as Sources of Regulatory Transcripts 
PLoS Genetics  2007;3(2):e1.
By means of a computer search for upstream promoter elements (distal sequence element and proximal sequence element) typical of small nuclear RNA genes, we have identified in the human genome a number of previously unrecognized, putative transcription units whose predicted products are novel noncoding RNAs with homology to protein-coding genes. By elucidating the function of one of them, we provide evidence for the existence of a sense/antisense-based gene-regulation network where part of the polymerase III transcriptome could control its polymerase II counterpart.
Author Summary
After the sequence of the human genome was determined, it was immediately recognized that a large part of the regulation of the gene expression occurring in the cells under physiological, as well as under pathological conditions, is carried out by RNA molecules that do not code for proteins (the “noncoding portion” of the genome). Here, we focus on small RNA molecules transcribed by the RNA polymerase III and identify a novel set of approximately 30 noncoding (nc) RNA genes. We propose that these RNA transcripts play a key role in regulating the expression of specific protein-coding genes transcribed by the RNA polymerase II, thus constituting an unprecedented example of cogene/gene pairs. Furthermore, we provide evidence that the RNA polymerase III, in addition to the well-known task in the constitutive synthesis of small RNAs (such as 5S rRNA and tRNAs), also plays a key role in the area of gene-expression control. A detailed investigation of the function of one of the novel ncRNA genes, called 21A, revealed that its transcript plays a role in the control of the proliferation of some tumor cells. The above findings significantly expand our understanding of the ncRNA universe and open the way to further studies aimed at the elucidation of the molecular pathways involving this novel class of regulatory RNAs.
doi:10.1371/journal.pgen.0030001
PMCID: PMC1790723  PMID: 17274687
24.  New Small Nuclear RNA Gene-Like Transcriptional Units as Sources of Regulatory Transcripts 
PLoS Genetics  2007;3(2):e1.
By means of a computer search for upstream promoter elements (distal sequence element and proximal sequence element) typical of small nuclear RNA genes, we have identified in the human genome a number of previously unrecognized, putative transcription units whose predicted products are novel noncoding RNAs with homology to protein-coding genes. By elucidating the function of one of them, we provide evidence for the existence of a sense/antisense-based gene-regulation network where part of the polymerase III transcriptome could control its polymerase II counterpart.
Author Summary
After the sequence of the human genome was determined, it was immediately recognized that a large part of the regulation of the gene expression occurring in the cells under physiological, as well as under pathological conditions, is carried out by RNA molecules that do not code for proteins (the “noncoding portion” of the genome). Here, we focus on small RNA molecules transcribed by the RNA polymerase III and identify a novel set of approximately 30 noncoding (nc) RNA genes. We propose that these RNA transcripts play a key role in regulating the expression of specific protein-coding genes transcribed by the RNA polymerase II, thus constituting an unprecedented example of cogene/gene pairs. Furthermore, we provide evidence that the RNA polymerase III, in addition to the well-known task in the constitutive synthesis of small RNAs (such as 5S rRNA and tRNAs), also plays a key role in the area of gene-expression control. A detailed investigation of the function of one of the novel ncRNA genes, called 21A, revealed that its transcript plays a role in the control of the proliferation of some tumor cells. The above findings significantly expand our understanding of the ncRNA universe and open the way to further studies aimed at the elucidation of the molecular pathways involving this novel class of regulatory RNAs.
doi:10.1371/journal.pgen.0030001
PMCID: PMC1790723  PMID: 17274687
25.  Constitutive Nucleosome Depletion and Ordered Factor Assembly at the GRP78 Promoter Revealed by Single Molecule Footprinting 
PLoS Genetics  2006;2(9):e160.
Chromatin organization and transcriptional regulation are interrelated processes. A shortcoming of current experimental approaches to these complex events is the lack of methods that can capture the activation process on single promoters. We have recently described a method that combines methyltransferase M.SssI treatment of intact nuclei and bisulfite sequencing allowing the representation of replicas of single promoters in terms of protected and unprotected footprint modules. Here we combine this method with computational analysis to study single molecule dynamics of transcriptional activation in the stress inducible GRP78 promoter. We show that a 350–base pair region upstream of the transcription initiation site is constitutively depleted of nucleosomes, regardless of the induction state of the promoter, providing one of the first examples for such a promoter in mammals. The 350–base pair nucleosome-free region can be dissected into modules, identifying transcription factor binding sites and their combinatorial organization during endoplasmic reticulum stress. The interaction of the transcriptional machinery with the GRP78 core promoter is highly organized, represented by six major combinatorial states. We show that the TATA box is frequently occupied in the noninduced state, that stress induction results in sequential loading of the endoplasmic reticulum stress response elements, and that a substantial portion of these elements is no longer occupied following recruitment of factors to the transcription initiation site. Studying the positioning of nucleosomes and transcription factors at the single promoter level provides a powerful tool to gain novel insights into the transcriptional process in eukaryotes.
Synopsis
Control of gene expression and transcription are complex and well-coordinated processes. Most current experimental approaches to understanding the underlying mechanisms, which include binding of transcription factors to regulatory regions of genes, and changes in the structure and composition of chromatin, rely on studies of populations of cells and cannot capture the transcription activation process on single promoters. The authors describe the use of a footprinting method which enables analysis of chromatin structure and binding of factors on single DNA molecules. This is applied to study the activation process of GRP78, a protein which is important for the induction of a response to endoplasmic reticulum stress. By combining the footprinting method and computational analyses, the authors define functional modules on the GRP78 promoter and show that it exists in few major combinatorial states, reflecting its high level of organization. These results provide novel insights into the activation of GRP78 which could not be gleaned using conventional methods. They also demonstrate the use of the method as a unique and powerful tool to study the transcriptional process in eukaryotes, which remains a major source of interest and challenge for the scientific community.
doi:10.1371/journal.pgen.0020160
PMCID: PMC1574359  PMID: 17002502

Results 1-25 (38)