Down syndrome (DS), or trisomy 21, is a common disorder associated with several complex clinical phenotypes. Although several hypotheses have been put forward, it is unclear as to whether particular gene loci on chromosome 21 (HSA21) are sufficient to cause DS and its associated features. Here we present a high-resolution genetic map of DS phenotypes based on an analysis of 30 subjects carrying rare segmental trisomies of various regions of HSA21. By using state-of-the-art genomics technologies we mapped segmental trisomies at exon-level resolution and identified discrete regions of 1.8-16.3 Mb likely to be involved in the development of 8 DS phenotypes, 4 of which are congenital malformations, including acute megakaryocytic leukemia, transient myeloproliferative disorder, Hirschsprung disease, duodenal stenosis, imperforate anus, severe mental retardation, DS-Alzheimer Disease, and DS-specific congenital heart disease (DSCHD). Our DS-phenotypic maps located DSCHD to a < 2-Mb interval. Furthermore, the map enabled us to present evidence against the necessary involvement of other loci as well as specific hypotheses that have been put forward in relation to the etiology of DS-i.e., the presence of a single DS consensus region and the sufficiency of DSCR1 and DYRK1A, or APP, in causing several severe DS phenotypes. Our study demonstrates the value of combining advanced genomics with cohorts of rare patients for studying DS, a prototype for the role of copy-number variation in complex disease. "
Wednesday, July 29, 2009
Wednesday, June 24, 2009
ERG Is a Megakaryocytic Oncogene
Ets-related gene (ERG) is a member of the ETS transcription factor gene family located on Hsa21. ERG is known to have a crucial role in establishing definitive hematopoiesis and is required for normal megakaryopoiesis. Truncated forms of ERG are associated with multiple cancers such as Ewing's sarcoma, prostate cancer, and leukemia as part of oncogenic fusion translocations. Increased expression of ERG is highly indicative of poor prognosis in acute myeloid leukemia and ERG is expressed in acute megakaryoblastic leukemia (AMKL); however, it is unclear if expression of ERG per se has a leukemogenic activity. We show that ectopic expression of ERG in fetal hematopoietic progenitors promotes megakaryopoiesis and that ERG alone acts as a potent oncogene in vivo leading to rapid onset of leukemia in mice. We observe that the endogenous ERG is required for the proliferation and maintenance of AMKL cell lines. ERG also strongly cooperates with the GATA1s mutated protein, found in Down syndrome AMKL, to immortalize megakaryocyte progenitors, suggesting that the additional copy of ERG in trisomy 21 may have a role in Down syndrome AMKL. These data suggest that ERG is a hematopoietic oncogene that may play a direct role in myeloid leukemia pathogenesis. [Cancer Res 2009;69(11):4665-73]
[Medical_Sciences] Functional genomic analysis of amniotic fluid cell-free mRNA suggests that oxidative stress is significant in Down syndrome fetuses
To characterize the differences between second trimester Down syndrome (DS) and euploid fetuses, we used Affymetrix microarrays to compare gene expression in uncultured amniotic fluid supernatant samples. Functional pathway analysis highlighted the importance of oxidative stress, ion transport, and G protein signaling in the DS fetuses. Further evidence supporting these results was derived by correlating the observed gene expression patterns to those of small molecule drugs via the Connectivity Map. Our results suggest that there are secondary adverse consequences of DS evident in the second trimester, leading to testable hypotheses about possible antenatal therapy for DS.
Inverted duplications on acentric markers: mechanism of formation
Acentric inverted duplication (inv dup) markers, the largest group of chromosomal abnormalities with neocentromere formation, are found in patients both with idiopathic mental retardation and with cancer. The mechanism of their formation has been investigated by analyzing the breakpoints and the genotypes of 12 inv dup marker cases (three trisomic, six tetrasomic, two polysomic and one X chromosome derived marker) using a combination of fluorescence in situ hybridization, quantitative SNP array and microsatellite analysis. Inv dup markers were found to form either symmetrically with one breakpoint or asymmetrically with two distinct breakpoints. Genotype analyses revealed that all inv dup markers formed from one single chromatid end. This observation is incompatible with the previously suggested model by which the acentric inv dup markers form through inter-chromosomal U-type exchange. On the basis of the identification of DNA sequence motifs with inverted homologies within all observed breakpoint regions, a new general mechanism is proposed for the acentric inv dup marker formation: following a double-strand break an acentric fragment forms, during either meiosis or mitosis. The open DNA end of the acentric fragment is stabilized by the formation of an intra-chromosomal loop promoted by the presence of sequences with inverted homologies. Likely coinciding with the neocentromere formation, this stabilized fragment is duplicated during an early mitotic event, insuring the marker’s survival during cell division and its presence in all cells.
"Statistical model for whole genome sequencing and its application to minimally invasive diagnosis of fetal genetic disease
There is currently great interest in the development of methods for the minimally invasive diagnosis of fetal genetic disease using cell-free DNA from maternal plasma samples obtained in the first trimester of pregnancy. With the rapid development of high-throughput sequencing technology, the possibility of detecting the presence of trisomy fetal genomes in the maternal plasma DNA sample has recently been explored. The major concern of this whole genome sequencing approach is that, while detecting the karyotype of the fetal genome from the maternal plasma requires extremely high accuracy of copy number estimation, the majority of the available high-throughput sequencing technologies require polymerase chain reaction (PCR) and are subject to the substantial bias that is inherent to the PCR process. We introduce a novel and sophisticated statistical model for the whole genome sequencing data, and based on this model, develop a highly sensitive method of Minimally Invasive Karyotyping (MINK) for the diagnosis of the fetal genetic disease. Specifically we demonstrate, by applying our statistical method to ultra high-throughput whole sequencing data, that trisomy 21 can be detected in a minor (‘fetal’) genome when it is mixed into a major (‘maternal’) background genome at frequencies as low as 5%. This observation provides additional proof of concept and justification for the further development of this method towards its eventual clinical application. Here, we describe the statistical and experimental methods that illustrate this approach and discuss future directions for technical development and potential clinical applications.
Contact:
Supplementary information:
DNA Methylation Analysis of Chromosome 21 Gene Promoters at Single Base Pair and Single Allele Resolution
Epigenetics is defined as the inheritance of changes in gene function without changing the DNA sequence. Epigenetic signals comprise methylation of cytosine bases of the DNA and chemical modifications of the histone proteins. DNA methylation plays important roles in development and disease processes. To investigate the biological role of DNA methylation, we analyzed DNA methylation patterns of 190 gene promoter regions on chromosome 21 in five human cell types. Our results show that average DNA methylation levels are distributed bimodally with enrichment of highly methylated and unmethylated sequences, indicating that DNA methylation acts in a switch-like manner. Consistent with the well-established role of DNA methylation in gene silencing, we found DNA methylation in promoter regions strongly correlated with absence of gene expression and low levels of additional activating epigenetic marks. Although methylation levels of individual cells in one tissue are very similar, we observed differences in DNA methylation when comparing different cell types in 43% of all regions analyzed. This finding is in agreement with a role of DNA methylation in cellular development. We identified three cases of genes that are differentially methylated in both alleles that illustrate the tight interplay of genetic and epigenetic processes.
Friday, March 20, 2009
Insights into the manifestations, outcomes, and mechanisms of leukemogenesis in Down syndrome
Children with Down syndrome (DS) show a spectrum of clinical anomalies, including cognitive impairment, cardiac malformations, and craniofacial dysmorphy. Moreover, hematologists have also noted that these children commonly show macrocytosis, abnormal platelet counts, and an increased incidence of transient myeloproliferative disease (TMD), acute megakaryocytic leukemia (AMKL), and acute lymphoid leukemia (ALL).
In this review, we summarize the clinical manifestations and characteristics of these leukemias, provide an update on therapeutic strategies and patient outcomes, and discuss the most recent advances in DS-leukemia research. With the increased knowledge of the way in which trisomy 21 affects hematopoiesis and the specific genetic mutations that are found in DS-associated leukemias, we are well on our way toward designing improved strategies for treating both myeloid and lymphoid malignancies in this high-risk population.
(Via Blood recent issues.)
Down syndrome--recent progress and future prospects
Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and is associated with a number of deleterious phenotypes, including learning disability, heart defects, early-onset Alzheimer's disease and childhood leukaemia. Individuals with DS are affected by these phenotypes to a variable extent; understanding the cause of this variation is a key challenge.
Here, we review recent research progress in DS, both in patients and relevant animal models. In particular, we highlight exciting advances in therapy to improve cognitive function in people with DS and the significant developments in understanding the gene content of Hsa21. Moreover, we discuss future research directions in light of new technologies. In particular, the use of chromosome engineering to generate new trisomic mouse models and large-scale studies of genotype-phenotype relationships in patients are likely to significantly contribute to the future understanding of DS.
Friday, March 13, 2009
Regulation of APC/C Activity in Oocytes by a Bub1-Dependent Spindle Assembly Checkpoint
Barry E. McGuinness, Martin Anger, Anna Kouznetsova, Ana M. Gil-Bernabé, Wolfgang Helmhart, Nobuaki R. Kudo, Annelie Wuensche, Stephen Taylor, Christer Hoog, Bela Novak, Kim Nasmyth.
Summary
Missegregation of chromosomes during meiosis in human females causes aneuploidy, including trisomy 21, and is thought also to be the major cause of age-related infertility [1]. Most errors are thought to occur at the first meiotic division. The high frequency of errors raises questions as to whether the surveillance mechanism known as the spindle assembly checkpoint (SAC) that controls the anaphase-promoting complex or cyclosome (APC/C) operates effectively in oocytes.
Experimental approaches hitherto used to inactivate the SAC in oocytes suffer from a number of drawbacks.Bub1 protein was depleted specifically in oocytes with a Zp3-Cre transgene to delete exons 7 and 8 from a floxed BUB1F allele. Loss of Bub1 greatly accelerates resolution ofchiasmata and extrusion of polar bodies. It also causes defective biorientation of bivalents, massive chromosome missegregation at meiosis I, and precocious loss of cohesion between sister centromeres.
By using a quantitative assay forAPC/C-mediated securin destruction, we show that the APC/C is activated in an exponential fashion, with activity peaking 1213 hr after GVBD, and that this process is advanced by 5hr in oocytes lacking Bub1. Importantly, premature chiasmata resolution does not occur in Bub1-deficient oocytes also lacking either the APC/C's Apc2 subunit or separase.
Finally, we show that Bub1's kinase domain is not required to delay APC/C activation.We conclude that far from being absent or ineffective, the SAC largely determines the timing of APC/C and hence separase activation in oocytes, delaying it for about 5 hr.
(Via Current Biology.)
Current use of medical eponyms - a need for global uniformity in scientific publications
Background:
Although eponyms are widely used in medicine, they arbitrarily alternate between the possessive and nonpossessive forms. As very little is known regarding extent and distribution of this variation, the present study was planned to assess current use of eponymous term taking 'Down syndrome' and 'Down's syndrome' as an example.
Methods:
This study was carried out in two phases - first phase in 1998 and second phase in 2008. In the first phase, we manually searched the terms 'Down syndrome' and 'Down's syndrome' in the indexes 70 medical books, and 46 medical journals. In second phase, we performed PubMed search with both the terms, followed by text-word search for the same.
Results:
In the first phase, there was an overall tilt towards possessive form - 62(53.4%) 'Down's syndrome' versus 54(46.6%) 'Down syndrome.' However, the American publications preferred the nonpossesive form when compared with their European counterpart (40/50 versus 14/66; P<0.001). In the second phase, PubMed search showed, compared to "Down syndrome," term "Down's syndrome" yielded approximately 5% more articles. The text-word search of both forms between January 1970 and June 2008 showed a gradual shift from "Down's syndrome" to "Down syndrome," and over the last 20 years, the frequency of the former was approximately halved (33.7% versus 16.5%; P<0.001). The abstracts having possessive form were mostly published from the European countries, while most American publications used nonpossesive form consistently.
Conclusions:
Inconsistency in the use of medical eponyms remains a major problem in literature search. Because of linguistic simplicity and technical advantages, the nonpossessive form should be used uniformly worldwide."
Wednesday, March 4, 2009
Heterochromatic Threads Connect Oscillating Chromosomes during Prometaphase I in Drosophila Oocytes
Proper chromosome segregation is essential during the production of eggs and sperm. Chromosome missegregation during meiosis results in the lethality of the offspring or in children carrying extra copies of a given chromosome (for example, Down syndrome). Recombination results in homologous chromosomes becoming physically interlocked in a manner that is normally sufficient to ensure proper segregation. Chromosomes that fail to undergo recombination require additional mechanisms to ensure their proper segregation.
In Drosophila melanogaster oocytes we show that chromosomes that fail to recombine undergo dynamic movements on the meiotic spindle prior to their proper segregation. Although previous studies had shown that non-recombinant chromosomes move to opposite sides of the developing meiotic spindle, we show that these chromosomes can cross the spindle and re-associate with their homologs to attempt reorientation.
Additionally, we observed threads connecting separated non-recombinant chromosomes that contained heterochromatic DNA and passenger complex proteins. These threads could assist the non-recombinant chromosomes in locating their homologs during their dynamic movements on the spindle. These chromosome movements and the heterochromatic threads are likely part of the mechanism ensuring proper segregation of nonexchange chromosomes.
[Reviews] Dscam and DSCAM: complex genes in simple animals, complex animals yet simple genes
Cadherins and the immunoglobulin (Ig) proteins give rise to a multitude of surface receptors, which function as diverse cell adhesion molecules (CAMs) or signal-transducing receptors. These functions are often interdependent, and rely on a high degree of specificity in homophilic binding as well as heterophilic interactions. The Drosophila receptor Dscam is an exceptional example of homophilic binding specificity involved in a number of important biological processes, such as neural wiring and innate immunity.
Combinatorial use of alternatively spliced Ig-domains enables the generation of an estimated 18,000 isoform-specific homophilic receptor pairs. Although isoform diversity of Dscam is unique to arthropods, recent genetic analysis of vertebrate DSCAM (Down Syndrome Cell Adhesion Molecule) genes has revealed an intriguing conservation of molecular functions underlying neural wiring.
This review covers the multiple functions of Dscam across different species highlighting its remarkable versatility as well as its conserved basic functions in neural development. We discuss how an unprecedented expansion of complex alternative splicing has been uniquely employed by arthropods to generate diverse surface receptors, important for cell-cell communication, molecular self-recognition in neurons, and innate immune defenses. We end with a speculative hypothesis reconciling the striking differences in Dscam and DSCAM gene structures with their conserved functions in molecular recognition underlying neural circuit formation.
Differential gene expression in ADAM10 and mutant ADAM10 transgenic mice
Background In a transgenic mouse model of Alzheimer disease (AD), cleavage of the amyloid precursor protein (APP) by the alpha-secretase ADAM10 prevented amyloid plaque formation, and alleviated cognitive deficits. Furthermore, ADAM10 overexpression increased the cortical synaptogenesis. These results suggest that upregulation of ADAM10 in the brain has beneficial effects on AD pathology.
Results: To assess the influence of ADAM10 on the gene expression profile in the brain, we performed a microarray analysis using RNA isolated from brains of five months old mice overexpressing either the alpha-secretase ADAM10, or a dominant-negative mutant (dn) of this enzyme. As compared to non-transgenic wild-type mice, in ADAM10 transgenic mice 355 genes, and in dnADAM10 mice 143 genes were found to be differentially expressed. A higher number of genes was differentially regulated in double-transgenic mouse strains additionally expressing the human APP[V717I] mutant.
Overexpression of proteolytically active ADAM10 affected several physiological pathways, such as cell communication, nervous system development, neuron projection as well as synaptic transmission. Although ADAM10 has been implicated in Notch and beta-catenin signaling, no significant changes in the respective target genes were observed in adult ADAM10 transgenic mice.
Real-time RT-PCR confirmed a downregulation of genes coding for the inflammation-associated proteins S100a8 and S100a9 induced by moderate ADAM10 overexpression. Overexpression of the dominant-negative form dnADAM10 led to a significant increase in the expression of the fatty acid-binding protein Fabp7, which also has been found in higher amounts in brains of Down Syndrome patients.
Conclusion: In general, there was only a moderate alteration of gene expression in ADAM10 overexpressing mice. Genes coding for pro-inflammatory or pro-apoptotic proteins were not over-represented among differentially regulated genes. Even a decrease of inflammation markers was observed. These results are further supportive for the strategy to treat AD by increasing the alpha-secretase activity.
[Neuroscience] DSCAM functions as a netrin receptor in commissural axon pathfinding
Down syndrome cell adhesion molecule (DSCAM) is required for axon guidance and dendrite arborization. How DSCAM functions in vertebrates is not well understood. Here we show that DSCAM is expressed on commissural axons and interacts with Netrin-1, a prototypical guidance cue for commissural axons. The knockdown of DSCAM by specific siRNA or blockage of DSCAM signaling by overexpression of a mutant lacking its intracellular domain inhibits netrin-induced axon outgrowth and commissural axon turning in vitro. SiRNA-mediated knockdown of DSCAM in ovo causes defects in commissural axon projection and pathfinding. In transfected cells, DSCAM by itself, in the absence of DCC, is capable of mediating netrin signaling in activating phosphorylation of Fyn and Pak1. These findings demonstrate an essential role of vertebrate DSCAM in axon guidance, indicating that DSCAM functions as a receptor of netrin-1. Our data suggest previously unexpected complexity in receptors that mediate vertebrate netrin signaling.
(Via Proceedings of the National Academy of Sciences recent issues.)
Sunday, February 22, 2009
CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets
Summary: MicroRNAs (miRNAs) are ~20- to 22-nt long endogenous RNA sequences that play a critical role in the regulation of gene expression in eukaryotic genomes. Confident identification of miRNA targets is vital to understand their functions.
Currently available computational algorithms for miRNA target prediction have diverse degrees of sensitivity and specificity and as a consequence each predicted target generally requires experimental confirmation. miRNAs and other small RNAs that direct endonucleolytic cleavage of target mRNAs produce diagnostic uncapped, polyadenylated mRNA fragments.
Degradome sequencing [also known as PARE (parallel analysis of RNA ends) and GMUCT (genome-wide mapping of uncapped transcripts)] samples the 5'-ends of uncapped mRNAs and can be used to discover in vivo miRNA targets independent of computational predictions.
Here, we describe a generalizable computational pipeline, CleaveLand, for the detection of cleaved miRNA targets from degradome data. CleaveLand takes as input degradome sequences, small RNAs and an mRNA database and outputs small RNA targets. CleaveLand can thus be applied to degradome data from any species provided a set of mRNA transcripts and a set of query miRNAs or other small RNAs are available.
Availability: The code and documentation for CleaveLand is freely available under a GNU license at
Contact:
Conservation and implications of eukaryote transcriptional regulatory regions across multiple species
Background: Increasing evidence shows that whole genomes of eukaryotes are almost entirely transcribed into both protein coding genes and an enormous number of non-protein-coding RNAs (ncRNAs). Therefore, revealing the underlying regulatory mechanisms of transcripts becomes imperative. However, for a complete understanding of transcriptional regulatory mechanisms, we need to identify the regions in which they are found. We will call these transcriptional regulation regions, or TRRs, which can be considered functional regions containing a cluster of regulatory elements that cooperatively recruit transcriptional factors for binding and then regulating the expression of transcripts.
Results: We constructed a hierarchical stochastic language (HSL) model for the identification of core TRRs in yeast based on regulatory cooperation among TRR elements. The HSL model trained based on yeast achieved comparable accuracy in predicting TRRs in other species, e.g., fruit fly, human, and rice, thus demonstrating the conservation of TRRs across species. The HSL model was also used to identify the TRRs of genes, such as p53 or OsALYL1, as well as microRNAs. In addition, the ENCODE regions were examined by HSL, and TRRs were found to pervasively locate in the genomes.
Conclusions: Our findings indicate that 1) the HSL model can be used to accurately predict core TRRs of transcripts across species and 2) identified core TRRs by HSL are proper candidates for the further scrutiny of specific regulatory elements and mechanisms. Meanwhile, the regulatory activity taking place in the abundant numbers of ncRNAs might account for the ubiquitous presence of TRRs across the genome. In addition, we also found that the TRRs of protein coding genes and ncRNAs are similar in structure, with the latter being more conserved than the former.
MicroRNA target prediction by expression analysis of host genes
MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression by inducing RNA cleavage or translational inhibition. Most human miRNAs are intragenic and are transcribed as part of their hosting transcription units.
We hypothesized that the expression profiles of miRNA host genes and of their targets are inversely correlated and devised a novel procedure, HOCTAR (Host gene Oppositely Correlated TARgets), which ranks predicted miRNA target genes based on their anti-correlated expression behaviour relative to their respective miRNA host genes. HOCTAR is the first tool for systematic miRNA target prediction that utilizes the same set of microarray experiments to monitor the expression of both miRNAs (through their host genes) and candidate targets.
We applied the procedure to 178 human intragenic miRNAs and found that it performs better than currently available prediction softwares in pinpointing previously validated miRNA targets. The high-scoring HOCTAR predicted targets were enriched in Gene Ontology categories, which were consistent with previously published data, as in the case of miR-106b and miR-93. By means of overexpression and loss-of-function assays, we also demonstrated that HOCTAR is efficient in predicting novel miRNA targets and we identified, by microarray and qRT-PCR procedures, 34 and 28 novel targets for miR-26b and miR-98, respectively.
Overall, we believe that the use of HOCTAR significantly reduces the number of candidate miRNA targets to be tested compared to the procedures based solely on target sequence recognition. Finally, our data further confirm that miRNAs have a significant impact on the mRNA levels of most of their targets.
(Via GR-in-Advance.)
Thermodynamic stability and Watson-Crick base pairing in the seed duplex are major determinants of the efficiency of the siRNA-based off-target effect
Short interfering RNA (siRNA) may down-regulate many unintended genes whose transcripts possess complementarity to the siRNA seed region, which contains 7 nt. The capability of siRNA to induce this off-target effect was highly correlated with the calculated melting temperature or standard free-energy change for formation of protein-free seed duplex, indicating that thermodynamic stability of seed duplex formed between the seed and target is one of the major factor in determining the degree of off-target effects.
Furthermore, unlike intended gene silencing (RNA interference), off-target effect was completely abolished by introduction of a G:U pair into the seed duplex, and this loss in activity was completely recovered by a second mutation regenerating Watson-Crick pairing, indicating that seed duplex Watson-Crick pairing is also essential for off-target gene silencing. The off-target effect was more sensitive to siRNA concentration compared to intended gene silencing, which requires a near perfect sequence match between the siRNA guide strand and target mRNA.
Protein kinase C epsilon is important for migration of neuroblastoma cells
Background: Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility.
Methods: PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot.
Results: Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Go6976 inhibited migration while an inhibitor of PKC isoforms did not have an effect. Downregulation of PKC epsilon, but not of PKC alpha or PKC delta, with siRNA led to a suppression of both basal and TPA-stimulated migration.
Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKC epsilon. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS.
Conclusions: PKC epsilon is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKC epsilon but they may be involved in TPA-mediated migration.
Genome-Wide siRNA-Based Functional Genomics of Pigmentation Identifies Novel Genes and Pathways That Impact Melanogenesis in Human Cells
by Anand K. Ganesan, Hsiang Ho, Brian Bodemann, Sean Petersen, Jayavani Aruri, Shiney Koshy, Zachary Richardson, Lu Q. Le, Tatiana Krasieva, Michael G. Roth, Pat Farmer, Michael A. White
Author SummaryAberrant pigment regulation correlates with skin disorders, opthalmologic disorders, and neurologic disorders. While extensive studies have identified regulators of mouse coat color, the regulation of human skin phenotypic variation is less well understood. To give a broader picture of the molecular regulators of melanogenesis in human cells, we used a genome-wide siRNA functional genomics approach to identify 92 novel regulators of melanin production in heavily pigmented MNT-1 melanoma cells.
Our screen identified several genes that converge to regulate tyrosinase, the rate-limiting step in pigment production, in both MNT-1 cells and primary melanocytes. Some of the identified genes were selectively active in different genetic backgrounds, suggesting that they may regulate human phenotypic variation. Small molecule inhibition of a family of novel pigment regulators was sufficient to impair pigment production in melanocytes.
Additionally, our screen identified molecular machinery known to support autophagosome biosynthesis as putative regulators of melanogenesis. In vitro co-localization studies and autophagy-deficient mice provided evidence that normal melanogenesis requires the same molecular machinery used by the autophagy pathway.
Taken together, these results illustrate the utility of genome wide siRNA screening approaches for identifying genes, novel pharmacologic agents, and pathways that regulate differentiated cellular phenotypes.
Modular control of endothelial sheet migration
Growth factor-induced migration of endothelial cell monolayers enables embryonic development, wound healing, and angiogenesis. Although collective migration is widespread and therapeutically relevant, the underlying mechanism by which cell monolayers respond to growth factor, sense directional signals, induce motility, and coordinate individual cell movements is only partially understood.
Here we used RNAi to identify 100 regulatory proteins that enhance or suppress endothelial sheet migration into cell-free space. We measured multiple live-cell migration parameters for all siRNA perturbations and found that each targeted protein primarily regulates one of four functional outputs: cell motility, directed migration, cell-cell coordination, or cell density. We demonstrate that cell motility regulators drive random, growth factor-independent motility in the presence or absence of open space.
In contrast, directed migration regulators selectively transduce growth factor signals to direct cells along the monolayer boundary toward open space. Lastly, we found that regulators of cell-cell coordination are growth factor-independent and reorient randomly migrating cells inside the sheet when boundary cells begin to migrate.
Thus, cells transition from random to collective migration through a modular control system, whereby growth factor signals convert boundary cells into pioneers, while cells inside the monolayer reorient and follow pioneers through growth factor-independent migration and cell-cell coordination.
Most mammalian mRNAs are conserved targets of microRNAs
MicroRNAs (miRNAs) are small endogenous RNAs that pair to sites in mRNAs to direct post-transcriptional repression. Many sites that match the miRNA seed (nucleotides 2-7), particularly those in 3' untranslated regions (3'UTRs), are preferentially conserved. Here we overhauled our tool for finding preferential conservation of sequence motifs and applied it to the analysis of human 3'UTRs, increasing by nearly threefold the detected number of preferentially conserved miRNA target sites.
The new tool more efficiently incorporates new genomes and more completely controls for background conservation by accounting for mutational biases, dinucleotide conservation rates, and the conservation rates of individual UTRs. The improved background model enabled preferential conservation of a new site type, the 'offset 6mer,' to be detected. In total, >45,000 miRNA target sites within human 3'UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.
Mammalian-specific miRNAs have far fewer conserved targets than do the more broadly conserved miRNAs, even when considering only more recently emerged targets. Although pairing to the 3' end of miRNAs can compensate for seed mismatches, this class of sites constitutes less than 2% of all preferentially conserved sites detected. The new tool enables statistically powerful analysis of individual miRNA target sites, with the probability of preferentially conserved targeting (PCT) correlating with experimental measurements of repression.
Our expanded set of target predictions (including conserved 3'-compensatory sites), are available at the TargetScan website, which displays the PCT for each site and each predicted target.
(Via GR-in-Advance.)
Regulation of neural macroRNAs by the transcriptional repressor REST
The essential transcriptional repressor REST (repressor element 1-silencing transcription factor) plays central roles in development and human disease by regulating a large cohort of neural genes. These have conventionally fallen into the class of known, protein-coding genes; recently, however, several noncoding microRNA genes were identified as REST targets.
Given the widespread transcription of messenger RNA-like, noncoding RNAs ('macroRNAs'), some of which are functional and implicated in disease in mammalian genomes, we sought to determine whether this class of noncoding RNAs can also be regulated by REST. By applying a new, unbiased target gene annotation pipeline to computationally discovered REST binding sites, we find that 23% of mammalian REST genomic binding sites are within 10 kb of a macroRNA gene. These putative target genes were overlooked by previous studies.
Focusing on a set of 18 candidate macroRNA targets from mouse, we experimentally demonstrate that two are regulated by REST in neural stem cells. Flanking protein-coding genes are, at most, weakly repressed, suggesting specific targeting of the macroRNAs by REST. Similar to the majority of known REST target genes, both of these macroRNAs are induced during nervous system development and have neurally restricted expression profiles in adult mouse.
We observe a similar phenomenon in human: the DiGeorge syndrome-associated noncoding RNA, DGCR5, is repressed by REST through a proximal upstream binding site. Therefore neural macroRNAs represent an additional component of the REST regulatory network. These macroRNAs are new candidates for understanding the role of REST in neuronal development, neurodegeneration, and cancer.
(Via In Advance.)
Position dependent mismatch discrimination on DNA microarrays - experiments and model
Background: The propensity of oligonucleotide strands to form stable duplexes with complementary sequences is fundamental to a variety of biological and biotechnological processes as various as microRNA signalling, microarray hybridization and PCR. Yet our understanding of oligonucleotide hybridization, in particular in presence of surfaces, is rather limited. Here we use oligonucleotide microarrays made in-house by optically controlled DNA synthesis to produce probe sets comprising all possible single base mismatches and base bulges for each of 20 sequence motifs under study.
Results: We observe that mismatch discrimination is mostly determined by the defect position (relative to the duplex ends) as well as by the sequence context. We investigate the thermodynamics of the oligonucleotide duplexes on the basis of double-ended molecular zipper. Theoretical predictions of defect positional influence as well as long range sequence influence agree well with the experimental results.
Conclusions: Molecular zipping at thermodynamic equilibrium explains the binding affinity of mismatched DNA duplexes on microarrays well. The position dependent nearest neighbor model (PDNN) can be inferred from it. Quantitative understanding of microarray experiments from first principles is in reach.
MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts
Authors: Thomas Thum, Carina Gross, Jan Fiedler, Thomas Fischer, Stephan Kissler, Markus Bussen, Paolo Galuppo, Steffen Just, Wolfgang Rottbauer, Stefan Frantz, Mirco Castoldi, Jürgen Soutschek, Victor Koteliansky, Andreas Rosenwald, M. Albert Basson, Jonathan D. Licht, John T. R. Pena, Sara H. Rouhanifard, Martina U. Muckenthaler, Thomas Tuschl, Gail R. Martin, Johann Bauersachs & Stefan Engelhardt
MicroRNAs comprise a broad class of small non-coding RNAs that control expression of complementary target messenger RNAs. Dysregulation of microRNAs by several mechanisms has been described in various disease states including cardiac disease. Whereas previous studies of cardiac disease have focused on microRNAs that are primarily expressed in cardiomyocytes, the role of microRNAs expressed in other cell types of the heart is unclear.
Here we show that microRNA-21 (miR-21, also known as Mirn21) regulates the ERK-MAP kinase signalling pathway in cardiac fibroblasts, which has impacts on global cardiac structure and function. miR-21 levels are increased selectively in fibroblasts of the failing heart, augmenting ERK-MAP kinase activity through inhibition of sprouty homologue 1 (Spry1). This mechanism regulates fibroblast survival and growth factor secretion, apparently controlling the extent of interstitial fibrosis and cardiac hypertrophy. In vivo silencing of miR-21 by a specific antagomir in a mouse pressure-overload-induced disease model reduces cardiac ERK-MAP kinase activity, inhibits interstitial fibrosis and attenuates cardiac dysfunction.
These findings reveal that microRNAs can contribute to myocardial disease by an effect in cardiac fibroblasts. Our results validate miR-21 as a disease target in heart failure and establish the therapeutic efficacy of microRNA therapeutic intervention in a cardiovascular disease setting.
(Via Nature.)
High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA
MicroRNAs (miRNAs) are an emerging class of small non-coding RNAs implicated in a wide variety of cellular processes. Research in this field is accelerating, and the growing number of miRNAs emphasizes the need for high-throughput and sensitive detection methods.
Here we present the successful evaluation of the Megaplex reverse transcription format of the stem-loop primer-based real-time quantitative polymerase chain reaction (RT-qPCR) approach to quantify miRNA expression. The Megaplex reaction provides simultaneous reverse transcription of 450 mature miRNAs, ensuring high-throughput detection. Further, the introduction of a complementary DNA pre-amplification step significantly reduces the amount of input RNA needed, even down to single-cell level. To evaluate possible pre-amplification bias, we compared the expression of 384 miRNAs in three different cancer cell lines with Megaplex RT, with or without an additional pre-amplification step.
The normalized Cq values of all three sample pairs showed a good correlation with maintenance of differential miRNA expression between the cell lines. Moreover, pre-amplification using 10 ng of input RNA enabled the detection of miRNAs that were undetectable when using Megaplex alone with 400 ng of input RNA. The high specificity of RT-qPCR together with a superior sensitivity makes this approach the method of choice for high-throughput miRNA expression profiling.
Annotation of Mammalian Primary microRNAs
Background: MicroRNAs (miRNAs) are important regulators of gene expression and have been implicated in development, differentiation and pathogenesis. Hundreds of miRNAs have been discovered in mammalian genomes. Approximately 50% of mammalian miRNAs are expressed from introns of protein-coding genes; the primary transcript (pri-miRNA) is therefore assumed to be the host transcript. However, very little is known about the structure of pri-miRNAs expressed from intergenic regions. Here we annotate transcript boundaries of miRNAs in human, mouse and rat genomes using various transcription features. The 5' end of the pri-miRNA is predicted from transcription start sites, CpG islands and 5' CAGE tags mapped in the upstream flanking region surrounding the precursor miRNA (pre-miRNA). The 3' end of the pri-miRNA is predicted based on the mapping of polyA signals, and supported by cDNA/EST and ditags data. The predicted pri-miRNAs are also analyzed for promoter and insulator-associated regulatory regions.
Results: We define sets of conserved and non-conserved human, mouse and rat pre-miRNAs using bidirectional BLAST and synteny analysis. Transcription features in their flanking regions are used to demarcate the 5' and 3' boundaries of the pri-miRNAs. The lengths and boundaries of primary transcripts are highly conserved between orthologous miRNAs. A significant fraction of pri-miRNAs have lengths between 1 and 10kb, with very few introns. We annotate a total of 59 pri-miRNA structures, which include 82 pre-miRNAs. 36 pri-miRNAs are conserved in all 3 species. In total, 18 of the confidently annotated transcripts express more than one pre-miRNA. The upstream regions of 54% of the predicted pri-miRNAs are found to be associated with promoter and insulator regulatory sequences.
Conclusions: Little is known about the primary transcripts of intergenic miRNAs. Using comparative data, we are able to identify the boundaries of a significant proportion of human, mouse and rat pri-miRNAs. We confidently predict the transcripts including a total of 77, 58 and 47 human, mouse and rat pre-miRNAs respectively. Our computational annotations provide a basis for subsequent experimental validation of predicted pri-miRNAs.
Human multipotent stromal cells from bone marrow and microRNA: Regulation of differentiation and leukemia inhibitory factor expression
"We observed that microRNAs (miRNAs) that regulate differentiation in a variety of simpler systems also regulate differentiation of human multipotent stromal cells (hMSCs) from bone marrow. Differentiation of hMSCs into osteoblasts and adipocytes was inhibited by using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA.
Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were up-regulated during osteogenic differentiation and 20 during adipogenic differentiation, 11 of which were commonly up-regulated in both osteogenic and adipogenic differentiation. In silico models predicted that five of the up-regulated miRNAs targeted leukemia inhibitory factor (LIF) expression.
The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF."
(Via Proceedings of the National Academy of Sciences recent issues.)
[ARTICLE] Deadenylation is a widespread effect of miRNA regulation
miRNAs silence gene expression by repressing translation and/or by promoting mRNA decay. In animal cells, degradation of partially complementary miRNA targets occurs via deadenylation by the CAF1-CCR4-NOT1 deadenylase complex, followed by decapping and subsequent exonucleolytic digestion.
To determine how generally miRNAs trigger deadenylation, we compared mRNA expression profiles in D. melanogaster cells depleted of AGO1, CAF1, or NOT1. We show that ~60% of AGO1 targets are regulated by CAF1 and/or NOT1, indicating that deadenylation is a widespread effect of miRNA regulation. However, neither a poly(A) tail nor mRNA circularization are required for silencing, because mRNAs whose 3' ends are generated by a self-cleaving ribozyme are also silenced in vivo.
We show further that miRNAs trigger mRNA degradation, even when binding by 40S ribosomal subunits is inhibited in cis. These results indicate that miRNAs promote mRNA decay by altering mRNP composition and/or conformation, rather than by directly interfering with the binding and function of ribosomal subunits.
(Via In Advance.)
[Research Papers] Chromatin structure analyses identify miRNA promoters
Although microRNAs (miRNAs) are key regulators of gene expression in normal human physiology and disease, transcriptional regulation of miRNAs is poorly understood, because most miRNA promoters have not yet been characterized. We identified the proximal promoters of 175 human miRNAs by combining nucleosome mapping with chromatin signatures for promoters.
We observe that one-third of intronic miRNAs have transcription initiation regions independent from their host promoters and present a list of RNA polymerase II- and III-occupied miRNAs. Nucleosome mapping and linker sequence analyses in miRNA promoters permitted accurate prediction of transcription factors regulating miRNA expression, thus identifying nine miRNAs regulated by the MITF transcription factor/oncoprotein in melanoma cells.
Furthermore, DNA sequences encoding mature miRNAs were found to be preferentially occupied by positioned-nucleosomes, and the 3' end sites of known genes exhibited nucleosome depletion. The high-throughput identification of miRNA promoter and enhancer regulatory elements sheds light on evolution of miRNA transcription and permits rapid identification of transcriptional networks of miRNAs.