DNA bending by an adenine–thymine tract and its role in gene regulation

To gain insight into the structural basis of DNA bending by adenine–thymine tracts (A-tracts) and their role in DNA recognition by gene-regulatory proteins, we have determined the crystal structure of the high-affinity DNA target of the cancer-associated human papillomavirus E2 protein. The three independent B-DNA molecules of the crystal structure determined at 2.2-Å resolution are examples of A-tract-containing helices where the global direction and magnitude of curvature are in accord with solution data, thereby providing insights, at the base pair level, into the mechanism of DNA bending by such sequence motifs. A comparative analysis of E2–DNA conformations with respect to other structural and biochemical studies demonstrates that (i) the A-tract structure of the core region, which is not contacted by the protein, is critical for the formation of the high-affinity sequence-specific protein–DNA complex, and (ii) differential binding affinity is regulated by the intrinsic structure and deformability encoded in the base sequence of the DNA target.

CDP/cut is the DNA-binding subunit of histone gene transcription factor HiNF-D: a mechanism for gene regulation at the G1/S phase cell cycle transition point independent of transcription factor E2F.

Transcription of the genes for the human histone proteins H4, H3, H2A, H2B, and H1 is activated at the G1/S phase transition of the cell cycle. We have previously shown that the promoter complex HiNF-D, which interacts with cell cycle control elements in multiple histone genes, contains the key cell cycle factors cyclin A, CDC2, and a retinoblastoma (pRB) protein-related protein. However, an intrinsic DNA-binding subunit for HiNF-D was not identified. Many genes that are up-regulated at the G1/S phase boundary are controlled by E2F, a transcription factor that associates with cyclin-, cyclin-dependent kinase-, and pRB-related proteins. Using gel-shift immunoassays, DNase I protection, and oligonucleotide competition analyses, we show that the homeodomain protein CDP/cut, not E2F, is the DNA-binding subunit of the HiNF-D complex. The HiNF-D (CDP/cut) complex with the H4 promoter is immunoreactive with antibodies against CDP/cut and pRB but not p107, whereas the CDP/cut complex with a nonhistone promoter (gp91-phox) reacts only with CDP and p107 antibodies. Thus, CDP/cut complexes at different gene promoters can associate with distinct pRB-related proteins. Transient coexpression assays show that CDP/cut modulates H4 promoter activity via the HiNF-D-binding site. Hence, DNA replication-dependent histone H4 genes are regulated by an E2F-independent mechanism involving a complex of CDP/cut with cyclin A/CDC2/ RB-related proteins.

DNA sequence insertion and evolutionary variation in gene regulation.

Current evidence on the long-term evolutionary effect of insertion of sequence elements into gene regions is reviewed, restricted to cases where a sequence derived from a past insertion participates in the regulation of expression of a useful gene. Ten such examples in eukaryotes demonstrate that segments of repetitive DNA or mobile elements have been inserted in the past in gene regions, have been preserved, sometimes modified by selection, and now affect control of transcription of the adjacent gene. Included are only examples in which transcription control was modified by the insert. Several cases in which merely transcription initiation occurred in the insert were set aside. Two of the examples involved the long terminal repeats of mammalian endogenous retroviruses. Another two examples were control of transcription by repeated sequence inserts in sea urchin genomes. There are now six published examples in which Alu sequences were inserted long ago into human gene regions, were modified, and now are central in control/enhancement of transcription. The number of published examples of Alu sequences affecting gene control has grown threefold in the last year and is likely to continue growing. Taken together, all of these examples show that the insertion of sequence elements in the genome has been a significant source of regulatory variation in evolution.

Arabidopsis mutant analysis and gene regulation define a nonredundant role for glutamate dehydrogenase in nitrogen assimilation.

Glutamate dehydrogenase (GDH) is ubiquitous to all organisms, yet its role in higher plants remains enigmatic. To better understand the role of GDH in plant nitrogen metabolism, we have characterized an Arabidopsis mutant (gdh1-1) defective in one of two GDH gene products and have studied GDH1 gene expression. GDH1 mRNA accumulates to highest levels in dark-adapted or sucrose-starved plants, and light or sucrose treatment each repress GDH1 mRNA accumulation. These results suggest that the GDH1 gene product functions in the direction of glutamate catabolism under carbon-limiting conditions. Low levels of GDH1 mRNA present in leaves of light-grown plants can be induced by exogenously supplied ammonia. Under such conditions of carbon and ammonia excess, GDH1 may function in the direction of glutamate biosynthesis. The Arabidopsis gdh-deficient mutant allele gdh1-1 cosegregates with the GDH1 gene and behaves as a recessive mutation. The gdh1-1 mutant displays a conditional phenotype in that seedling growth is specifically retarded on media containing exogenously supplied inorganic nitrogen. These results suggest that GDH1 plays a nonredundant role in ammonia assimilation under conditions of inorganic nitrogen excess. This notion is further supported by the fact that the levels of mRNA for GDH1 and chloroplastic glutamine synthetase (GS2) are reciprocally regulated by light.

Transcriptional activation by Sp1 and post-transcriptional repression by muscle-specific microRNA miR-133 of expression of human ERG1 and KCNQ1 genes and potential implication in arrhythmogenesis

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Posttranscriptional Regulation of Embryonic Neurogenesis by the Exon Junction Complex

The six-layered neuron structure in the cerebral cortex is the foundation for human mental abilities. In the developing cerebral cortex, neural stem cells undergo proliferation and differentiate into intermediate progenitors and neurons, a process known as embryonic neurogenesis. Disrupted embryonic neurogenesis is the root cause of a wide range of neurodevelopmental disorders, including microcephaly and intellectual disabilities. Multiple layers of regulatory networks have been identified and extensively studied over the past decades to understand this complex but extremely crucial process of brain development. In recent years, post-transcriptional RNA regulation through RNA binding proteins has emerged as a critical regulatory nexus in embryonic neurogenesis. The exon junction complex (EJC) is a highly conserved RNA binding complex composed of four core proteins, Magoh, Rbm8a, Eif4a3, and Casc3. The EJC plays a major role in regulating RNA splicing, nuclear export, subcellular localization, translation, and nonsense mediated RNA decay. Human genetic studies have associated individual EJC components with various developmental disorders. We showed previously that haploinsufficiency of Magoh causes microcephaly and disrupted neural stem cell differentiation in mouse. However, it is unclear if other EJC core components are also required for embryonic neurogenesis. More importantly, the molecular mechanism through which the EJC regulates embryonic neurogenesis remains largely unknown. Here, we demonstrated with genetically modified mouse models that both Rbm8a and Eif4a3 are required for proper embryonic neurogenesis and the formation of a normal brain. Using transcriptome and proteomic analysis, we showed that the EJC posttranscriptionally regulates genes involved in the p53 pathway, splicing and translation regulation, as well as ribosomal biogenesis. This is the first in vivo evidence suggesting that the etiology of EJC associated neurodevelopmental diseases can be ribosomopathies. We also showed that, different from other EJC core components, depletion of Casc3 only led to mild neurogenesis defects in the mouse model. However, our data suggested that Casc3 is required for embryo viability, development progression, and is potentially a regulator of cardiac development. Together, data presented in this thesis suggests that the EJC is crucial for embryonic neurogenesis and that the EJC and its peripheral factors may regulate development in a tissue-specific manner.

Quantifying Eukaryotic Gene Regulation in Hormone Response and Disease.

Quantifying the function of mammalian enhancers at the genome or population scale has been longstanding challenge in the field of gene regulation. Studies of individual enhancers have provided anecdotal evidence on which many foundational assumptions in the field are based. Genome-scale studies have revealed that the number of sites bound by a given transcription factor far outnumber the genes that the factor regulates. In this dissertation we describe a new method, chromatin immune-enriched reporter assays (ChIP-reporters), and use that approach to comprehensively test the enhancer activity of genomic loci bound by the glucocorticoid receptor (GR). Integrative genomics analyses of our ChIP-reporter data revealed an unexpected mechanism of glucocorticoid (GC)-induced gene regulation. In that mechanism, only the minority of GR bound sites acts as GC-inducible enhancers. Many non-GC-inducible GR binding sites interact with GC-induced sites via chromatin looping. These interactions can increase the activity of GC-induced enhancers. Finally, we describe a method that enables the detection and characterization of the functional effects of non-coding genetic variation on enhancer activity at the population scale. Taken together, these studies yield both mechanistic and genetic evidence that provides context that informs the understanding of the effects of multiple enhancer variants on gene expression.

Transcriptional activation by Sp1 and post-transcriptional repression by muscle-specific microRNA miR-133 of expression of human ERG1 and KCNQ1 genes and potential implication in arrhythmogenesis

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

A rep-based hairpin inhibits replication of diverse maize streak virus isolates in a transient assay

Centre for High-Performance Computing, Rosebank, Cape Town, South Africa Maize streak disease, caused by the A strain of the African endemic geminivirus, maize streak mastrevirus (MSV-A), threatens the food security and livelihoods of subsistence farmers throughout sub-Saharan Africa. Using a well-established transient expression assay, this study investigated the potential of a spliceable-intron hairpin RNA (hpRNA) approach to interfere with MSV replication. Two strategies were explored: (i) an inverted repeat of a 662 bp region of the MSV replication-associated protein gene (rep), which is essential for virus replication and is therefore a good target for post-transcriptional gene silencing; and (ii) an inverted repeat of the viral long intergenic region (LIR), considered for its potential to trigger transcriptional silencing of the viral promoter region. After co-bombardment of cultured maize cells with each construct and an infectious partial dimer of the cognate virus genome (MSV-Kom), followed by viral replicativeform-specific PCR, it was clear that, whilst the hairpin rep construct (pHPrepDI662) completely inhibited MSV replication, the LIR hairpin construct was ineffective in this regard. In addition, pHPrepDI662 inhibited or reduced replication of six MSV-A genotypes representing the entire breadth of known MSV-A diversity. Further investigation by real-time PCR revealed that the pHPrepDI662 inverted repeat was 22-fold more effective at reducing virus replication than a construct containing the sense copy, whilst the antisense copy had no effect on replication when compared with the wild type. This is the first indication that an hpRNA strategy targeting MSV rep has the potential to protect transgenic. © 2011 SGM.

The brain-specific microRNA miR-128b regulates the formation of fear-extinction memory

MicroRNAs are small non-coding RNAs that mediate post-transcriptional gene silencing. Fear-extinction learning in C57/Bl6J mice led to increased expression of the brain-specific microRNA miR-128b, which disrupted stability of several plasticity-related target genes and regulated formation of fear-extinction memory. Increased miR-128b activity may therefore facilitate the transition from retrieval of the original fear memory toward the formation of a new fear-extinction memory.

A Comparative Kinetic and Thermodynamic Perspective of the sigma-Competition Model in Escherichia coli

Transcription is the most fundamental step in gene expression in any living organism. Various environmental cues help in the maturation of core RNA polymerase (RNAP; alpha(2)beta beta'omega) with different sigma-factors, leading to the directed recruitment of RNAP to different promoter DNA sequences. Thus it is essential to determine the sigma-factors that affect the preferential partitioning of core RNAP among various a-actors, and the role of sigma-switching in transcriptional gene regulation. Further, the macromolecular assembly of holo RNAP takes place in an extremely crowded environment within a cell, and thus far the kinetics and thermodynamics of this molecular recognition process have not been well addressed. In this study we used a site-directed bioaffinity immobilization method to evaluate the relative binding affinities of three different Escherichia coli sigma-factors to the same core RNAP with variations in temperature and ionic strength while emulating the crowded cellular milieu. Our data indicate that the interaction of core RNAP-sigma is susceptible to changes in external stimuli such as osmolytic and thermal stress, and the degree of susceptibility varies among different sigma-factors. This allows for a reversible sigma-switching from housekeeping factors to alternate sigma-factors when the organism senses a change in its physiological conditions.

Towards conditional RNAi : shape and sequence transduction with small conditional RNAs

RNA interference (RNAi) is a powerful biological pathway allowing for sequence-specific knockdown of any gene of interest. While RNAi is a proven tool for probing gene function in biological circuits, it is limited by being constitutively ON and executes the logical operation: silence gene Y. To provide greater control over post-transcriptional gene silencing, we propose engineering a biological logic gate to implement “conditional RNAi.” Such a logic gate would silence gene Y only upon the expression of gene X, a completely unrelated gene, executing the logic: if gene X is transcribed, silence independent gene Y. Silencing of gene Y could be confined to a specific time and/or tissue by appropriately selecting gene X.

To implement the logic of conditional RNAi, we present the design and experimental validation of three nucleic acid self-assembly mechanisms which detect a sub-sequence of mRNA X and produce a Dicer substrate specific to gene Y. We introduce small conditional RNAs (scRNAs) to execute the signal transduction under isothermal conditions. scRNAs are small RNAs which change conformation, leading to both shape and sequence signal transduction, in response to hybridization to an input nucleic acid target. While all three conditional RNAi mechanisms execute the same logical operation, they explore various design alternatives for nucleic acid self-assembly pathways, including the use of duplex and monomer scRNAs, stable versus metastable reactants, multiple methods of nucleation, and 3-way and 4-way branch migration.

We demonstrate the isothermal execution of the conditional RNAi mechanisms in a test tube with recombinant Dicer. These mechanisms execute the logic: if mRNA X is detected, produce a Dicer substrate targeting independent mRNA Y. Only the final Dicer substrate, not the scRNA reactants or intermediates, is efficiently processed by Dicer. Additional work in human whole-cell extracts and a model tissue-culture system delves into both the promise and challenge of implementing conditional RNAi in vivo.

Investigação de alterações em genes de microRNAs expressos no cérebro como causa de deficiência intelectual ligada ao cromossomo X

A Deficiência Intelectual (DI) é uma condição definida como um funcionamento intelectual significativamente prejudicado, expresso juntamente com limitações em pelo menos duas áreas do comportamento adaptativo que se manifestam antes dos 18 anos de idade. A prevalência estimada da DI na população em geral é de 2-3% e um número expressivo de casos permanece sem um diagnóstico definitivo. Há um consenso geral de que a DI é mais comum em indivíduos do sexo masculino em relação aos do sexo feminino. Entre as explicações para este excesso está a concentração de genes específicos para a habilidade cognitiva no cromossomo X. MicroRNAs (miRNAs) são pequenas moléculas de RNA não codificador que modulam a expressão gênica pós-transcricional de RNAs mensageiros alvo. Recentemente, estudos têm demonstrado a importância essencial dos miRNAs para o desenvolvimento e funcionamento cerebrais e sabe-se que o cromossomo X tem uma alta densidade de genes de miRNAs. Neste contexto, os miRNAs são candidatos potenciais como fatores genéticos envolvidos na Deficiência Intelectual Ligada ao X (DILX). Neste estudo, foram analisadas as regiões genômicas de 17 genes de miRNAs expressos no cérebro localizados no cromossomo X, com o objetivo de investigar o possível envolvimento de variantes na sequência destes miRNAs na DILX. Para este fim, selecionamos amostras de DNA genômico (sangue periférico) de 135 indivíduos do sexo masculino portadores de DI sugestiva de DILX de um grupo de mais de 1.100 pacientes com DI encaminhados ao Serviço de Genética Humana da UERJ. O critério de inclusão para este estudo era de que os probandos apresentassem um ou mais parentes do sexo masculino afetados pela DI que fossem interligados por via materna. As amostras de DNA dos pacientes foram amplificadas utilizando a técnica de reação em cadeia da polimerase, seguida por purificação e sequenciamento direto pelo método de Sanger dos fragmentos amplificados. Para avaliar a conservação dos 17 miRNAs foi realizada uma análise filogenética in silico incluindo sequências dos miRNAs selecionados de humanos e de outras 8 espécies de primatas estreitamente relacionadas. Não foram encontradas alterações nas sequências nos genes de 17 miRNAs analisados, mesmo diante do padrão genético altamente heterogêneo da população brasileira. Adicionalmente, a análise filogenética destes miRNAs revelou uma alta conservação entre as espécies comparadas. Considerando o papel dos miRNAs como reguladores da expressão gênica, a ausência de alterações e a alta conservação entre primatas sugerem uma forte pressão seletiva sobre estas moléculas, reforçando a sua importância funcional para o organismo em geral. Apesar de não termos encontrado variantes de sequência nos miRNAs estudados, o envolvimento de miRNAs na DI não pode ser completamente descartado. Alterações fora da molécula de miRNA precursor, nos fatores de processamento, nos sítios alvo e variações no número de cópias de genes de miRNAs podem implicar em alteração na expressão dos miRNAs e, consequentemente, na funcionalidade do miRNA maduro. Sendo assim, uma análise sistemática da expressão de miRNAs em pacientes com DILX é urgentemente necessária, a fim de desvendar novos genes/mecanismos moleculares relacionados a esta condição.

3'-UTR SIRF: A database for identifying clusters of short interspersed repeats in 3' untranslated regions

BACKGROUND:Short (~5 nucleotides) interspersed repeats regulate several aspects of post-transcriptional gene expression. Previously we developed an algorithm (REPFIND) that assigns P-values to all repeated motifs in a given nucleic acid sequence and reliably identifies clusters of short CAC-containing motifs required for mRNA localization in Xenopus oocytes.DESCRIPTION:In order to facilitate the identification of genes possessing clusters of repeats that regulate post-transcriptional aspects of gene expression in mammalian genes, we used REPFIND to create a database of all repeated motifs in the 3' untranslated regions (UTR) of genes from the Mammalian Gene Collection (MGC). The MGC database includes seven vertebrate species: human, cow, rat, mouse and three non-mammalian vertebrate species. A web-based application was developed to search this database of repeated motifs to generate species-specific lists of genes containing specific classes of repeats in their 3'-UTRs. This computational tool is called 3'-UTR SIRF (Short Interspersed Repeat Finder), and it reveals that hundreds of human genes contain an abundance of short CAC-rich and CAG-rich repeats in their 3'-UTRs that are similar to those found in mRNAs localized to the neurites of neurons. We tested four candidate mRNAs for localization in rat hippocampal neurons by in situ hybridization. Our results show that two candidate CAC-rich (Syntaxin 1B and Tubulin beta4) and two candidate CAG-rich (Sec61alpha and Syntaxin 1A) mRNAs are localized to distal neurites, whereas two control mRNAs lacking repeated motifs in their 3'-UTR remain primarily in the cell body.CONCLUSION:Computational data generated with 3'-UTR SIRF indicate that hundreds of mammalian genes have an abundance of short CA-containing motifs that may direct mRNA localization in neurons. In situ hybridization shows that four candidate mRNAs are localized to distal neurites of cultured hippocampal neurons. These data suggest that short CA-containing motifs may be part of a widely utilized genetic code that regulates mRNA localization in vertebrate cells. The use of 3'-UTR SIRF to search for new classes of motifs that regulate other aspects of gene expression should yield important information in future studies addressing cis-regulatory information located in 3'-UTRs.