Whole genome RNAi screens reveal a critical role of REV3 in coping with replication stress

REV3, the catalytic subunit of translesion polymerase zeta (polζ), is commonly associated with DNA damage bypass and repair. Despite sharing accessory subunits with replicative polymerase δ, very little is known about the role of polζ in DNA replication. We previously demonstrated that inhibition of REV3 expression induces persistent DNA damage and growth arrest in cancer cells. To reveal determinants of this sensitivity and obtain insights into the cellular function of REV3, we performed whole human genome RNAi library screens aimed at identification of synthetic lethal interactions with REV3 in A549 lung cancer cells. The top confirmed hit was RRM1, the large subunit of ribonucleotide reductase (RNR), a critical enzyme of de novo nucleotide synthesis. Treatment with the RNR-inhibitor hydroxyurea (HU) synergistically increased the fraction of REV3-deficient cells containing single stranded DNA (ssDNA) as indicated by an increase in replication protein A (RPA). However, this increase was not accompanied by accumulation of the DNA damage marker γH2AX suggesting a role of REV3 in counteracting HU-induced replication stress (RS). Consistent with a role of REV3 in DNA replication, increased RPA staining was confined to HU-treated S-phase cells. Additionally, we found genes related to RS to be significantly enriched among the top hits of the synthetic sickness/lethality (SSL) screen further corroborating the importance of REV3 for DNA replication under conditions of RS.

The effect of DNA methylation in carcinogenesis

CpG island methylation within single gene promoters can silence expression of associated genes. We first extended these studies to bidirectional gene pairs controlled by single promoters. We showed that hypermethylation of bidirectional promoter-associated CpG island silences gene pairs (WNT9A/CD558500, CTDSPL/BC040563, and KCNK15/BF 195580) simultaneously. Hypomethylation of these promoters by 5-aza-2'-deoxycytidine treatment reactivated or enhanced gene expression bidirectionally. These results were further confirmed by luciferase assays. Methylation of WNT9A/CD558500 and CTDSPL/BC040563 promoters occurs frequently in primary colon cancers and acute lymphoid leukemia, respectively. ^ Next we sought to understand the origins of hypermethylation in cancer. CpG islands associated with tumor suppressor genes are normally free from methylation, but can be hypermethylated in cancer. It remains poorly understood how these genes are protected from methylation in normal tissues. In our studies, we aimed to determine if cis-acting elements in these genes are responsible for this protection, using the tumor suppressor gene p16 as a model. We found that Alu repeats located both upstream and downstream of the p16 promoter become hypermethylated with age. In colon cancer samples, the methylation level is particularly high, and the promoter can also be affected. Therefore, the protection in the promoter against methylation spreading could fail during tumorigenesis. This methylation pattern in p16 was also observed in cell lines of different tissue origins, and their methylation levels were found to be inversely correlated with that of active histone modification markers (H3K4-3me and H3K9-Ac). To identify the mechanism of protection against methylation spreading, we constructed serial deletions of the p16 protected region and used silencing of a neomycin reporter gene to evaluate the protective effects of these fragments. A 126 bp element was identified within the region which exerts bidirectional protection against DNA methylation, independently of its transcriptional activity. The protective strength of this element is comparable to that of the HS4 insulator. During long-term culture, the presence of this element significantly slowed methylation spreading. In conclusion, we have found that an element located in the p16 promoter is responsible for protection against DNA methylation spreading in normal tissues. The failure of protective cis-elements may be a general feature of tumor-suppressor gene silencing during tumorigenesis. ^

Human activity recognition by inertial signals obtained from a smartphone

Human Activity Recognition (HAR) is an emerging research field with the aim to identify the actions carried out by a person given a set of observations and the surrounding environment. The wide growth in this research field inside the scientific community is mainly explained by the high number of applications that are arising in the last years. A great part of the most promising applications are related to the healthcare field, where it is possible to track the mobility of patients with motor dysfunction as also the physical activity in patients with cardiovascular risk. Until a few years ago, by using distinct kind of sensors, a patient follow-up was possible. However, far from being a long-term solution and with the smartphone irruption, that monitoring can be achieved in a non-invasive way by using the embedded smartphone’s sensors. For these reasons this Final Degree Project arises with the main target to evaluate new feature extraction techniques in order to carry out an activity and user recognition, and also an activity segmentation. The recognition is done thanks to the inertial signals integration obtained by two widespread sensors in the greater part of smartphones: accelerometer and gyroscope. In particular, six different activities are evaluated walking, walking-upstairs, walking-downstairs, sitting, standing and lying. Furthermore, a segmentation task is carried out taking into account the activities performed by thirty users. This can be done by using Hidden Markov Models and also a set of tools tested satisfactory in speech recognition: HTK (Hidden Markov Model Toolkit).

Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3′ end target sequence

Unique, small sequences (sequence tag sites) have been identified at the 3′ ends of most human genes that serve as landmarks in genome mapping. We investigated whether a single copy gene could be isolated directly from total human DNA by transformation-associated recombination (TAR) cloning in yeast using a short, 3′ unique target. A TAR cloning vector was constructed that, when linearized, contained a small amount (381 bp) of 3′ hypoxanthine phosphoribosyltransferase (HPRT) sequence at one end and an 189-bp Alu repeat at the other end. Transformation with this vector along with human DNA led to selective isolations of the entire HPRT gene as yeast artificial chromosomes (YACs) that extended from the 3′ end sequence to various Alu positions as much as 600 kb upstream. These YACs were retrofitted with a NeoR and a bacterial artificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection. Most of the HPRT isolates were functional, demonstrating that TAR cloning retains the functional integrity of the isolated material. Thus, this modified version of TAR cloning, which we refer to as radial TAR cloning, can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information.

Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction

Nuclear receptors regulate metabolic pathways in response to changes in the environment by appropriate alterations in gene expression of key metabolic enzymes. Here, a computational search approach based on iteratively built hidden Markov models of nuclear receptors was used to identify a human nuclear receptor, termed hPAR, that is expressed in liver and intestines. hPAR was found to be efficiently activated by pregnanes and by clinically used drugs including rifampicin, an antibiotic known to selectively induce human but not murine CYP3A expression. The CYP3A drug-metabolizing enzymes are expressed in gut and liver in response to environmental chemicals and clinically used drugs. Interestingly, hPAR is not activated by pregnenolone 16α-carbonitrile, which is a potent inducer of murine CYP3A genes and an activator of the mouse receptor PXR.1. Furthermore, hPAR was found to bind to and trans-activate through a conserved regulatory sequence present in human but not murine CYP3A genes. These results provide evidence that hPAR and PXR.1 may represent orthologous genes from different species that have evolved to regulate overlapping target genes in response to pharmacologically distinct CYP3A activators, and have potential implications for the in vitro identification of drug interactions important to humans.

Complete sequencing of the Fugu WAGR region from WT1 to PAX6: Dramatic compaction and conservation of synteny with human chromosome 11p13

The pufferfish Fugu rubripes has a genome ≈7.5 times smaller than that of mammals but with a similar number of genes. Although conserved synteny has been demonstrated between pufferfish and mammals across some regions of the genome, there is some controversy as to what extent Fugu will be a useful model for the human genome, e.g., [Gilley, J., Armes, N. & Fried, M. (1997) Nature (London) 385, 305–306]. We report extensive conservation of synteny between a 1.5-Mb region of human chromosome 11 and <100 kb of the Fugu genome in three overlapping cosmids. Our findings support the idea that the majority of DNA in the region of human chromosome 11p13 is intergenic. Comparative analysis of three unrelated genes with quite different roles, WT1, RCN1, and PAX6, has revealed differences in their structural evolution. Whereas the human WT1 gene can generate 16 protein isoforms via a combination of alternative splicing, RNA editing, and alternative start site usage, our data predict that Fugu WT1 is capable of generating only two isoforms. This raises the question of the extent to which the evolution of WT1 isoforms is related to the evolution of the mammalian genitourinary system. In addition, this region of the Fugu genome shows a much greater overall compaction than usual but with significant noncoding homology observed at the PAX6 locus, implying that comparative genomics has identified regulatory elements associated with this gene.

Cluster analysis and display of genome-wide expression patterns

A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is described that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression. The output is displayed graphically, conveying the clustering and the underlying expression data simultaneously in a form intuitive for biologists. We have found in the budding yeast Saccharomyces cerevisiae that clustering gene expression data groups together efficiently genes of known similar function, and we find a similar tendency in human data. Thus patterns seen in genome-wide expression experiments can be interpreted as indications of the status of cellular processes. Also, coexpression of genes of known function with poorly characterized or novel genes may provide a simple means of gaining leads to the functions of many genes for which information is not available currently.

Multiple-complete-digest restriction fragment mapping: Generating sequence-ready maps for large-scale DNA sequencing

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of ×15–30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a “shotgun” method.

Yeast and human genes that affect the Escherichia coli SOS response

The sequencing of the human genome has led to the identification of many genes whose functions remain to be determined. Because of conservation of genetic function, microbial systems have often been used for identification and characterization of human genes. We have investigated the use of the Escherichia coli SOS induction assay as a screen for yeast and human genes that might play a role in DNA metabolism and/or in genome stability. The SOS system has previously been used to analyze bacterial and viral genes that directly modify DNA. An initial screen of meiotically expressed yeast genes revealed several genes associated with chromosome metabolism (e.g., RAD51 and HHT1 as well as others). The SOS induction assay was then extended to the isolation of human genes. Several known human genes involved in DNA metabolism, such as the Ku70 end-binding protein and DNA ligase IV, were identified, as well as a large number of previously unknown genes. Thus, the SOS assay can be used to identify and characterize human genes, many of which may participate in chromosome metabolism.

Target DNA chromatinization modulates nicking by L1 endonuclease

L1 elements are human transposons which replicate via an RNA intermediate. At least 15% of the human genome is composed of L1 sequence. An important initial step in the transposition reaction is nicking of the genomic DNA by L1 endonuclease (L1 EN). In vivo much of the genome exists in the form of chromatin or is undergoing biochemical transactions such as transcription, replication or repair, which may alter the accessibility of the L1 transposition machinery to DNA. To investigate this possibility we have examined the effect of substrate chromatinization on the ability of L1 EN to nick DNA. We find that DNA incorporated into nucleosomes is generally refractory to nicking by L1 EN. Interestingly, nicking of a minority of DNA sequences is enhanced when included in chromatin. Thus, dynamic epigenetic factors such as chromatinization are likely to influence the relatively permanent placement of L1 and other retroelements in the human genome.

The Mouse Genome Database (MGD): integration nexus for the laboratory mouse

The Mouse Genome Database (MGD) is the community database resource for the laboratory mouse, a key model organism for interpreting the human genome and for understanding human biology and disease (http://www.informatics.jax.org). MGD provides standard nomenclature and consensus map positions for mouse genes and genetic markers; it provides a curated set of mammalian homology records, user-defined chromosomal maps, experimental data sets and the definitive mouse ‘gene to sequence’ reference set for the research community. The integration and standardization of these data sets facilitates the transition between mouse DNA sequence, gene and phenotype annotations. A recent focus on allele and phenotype representations enhances the ability of MGD to organize and present data for exploring the relationship between genotype and phenotype. This link between the genome and the biology of the mouse is especially important as phenotype information grows from large mutagenesis projects and genotype information grows from large-scale sequencing projects.

Genetic evidence for the involvement of DNA ligase IV in the DNA-PK-dependent pathway of non-homologous end joining in mammalian cells

Cells of vertebrates remove DNA double-strand breaks (DSBs) from their genome predominantly utilizing a fast, DNA-PKcs-dependent form of non-homologous end joining (D-NHEJ). Mutants with inactive DNA-PKcs remove the majority of DNA DSBs utilizing a slow, DNA-PKcs-independent pathway that does not utilize genes of the RAD52 epistasis group, is error-prone and can therefore be classified as a form of NHEJ (termed basic or B-NHEJ). We studied the role of DNA ligase IV in these pathways of NHEJ. Although biochemical studies show physical and functional interactions between the DNA-PKcs/Ku and the DNA ligase IV/Xrcc4 complexes suggesting operation within the same pathway, genetic evidence to support this notion is lacking in mammalian cells. Primary human fibroblasts (180BR) with an inactivating mutation in DNA ligase IV, rejoined DNA DSBs predominantly with slow kinetics similar to those observed in cells deficient in DNA-PKcs, or in wild-type cells treated with wortmannin to inactivate DNA-PK. Treatment of 180BR cells with wortmannin had only a small effect on DNA DSB rejoining and no effect on cell radiosensitivity to killing although it sensitized control cells to 180BR levels. This is consistent with DNA ligase IV functioning as a component of the D-NHEJ, and demonstrates the unperturbed operation of the DNA-PKcs-independent pathway (B-NHEJ) at significantly reduced levels of DNA ligase IV. In vitro, extracts of 180BR cells supported end joining of restriction endonuclease-digested plasmid to the same degree as extracts of control cells when tested at 10 mM Mg2+. At 0.5 mM Mg2+, where only DNA ligase IV is expected to retain activity, low levels of end joining (∼10% of 10 mM) were seen in the control but there was no detectable activity in 180BR cells. Antibodies raised against DNA ligase IV did not measurably inhibit end joining at 10 mM Mg2+ in either cell line. Thus, in contrast to the situation in vivo, end joining in vitro is dominated by pathways with properties similar to B-NHEJ that do not display a strong dependence on DNA ligase IV, with D-NHEJ retaining only a limited contribution. The implications of these observations to studies of NHEJ in vivo and in vitro are discussed.

Complete structure of the human alpha-albumin gene, a new member of the serum albumin multigene family.

The nucleotide sequence of the human alpha-albumin gene, including 887 bp of the 5'-flanking region and 1311 bp of the 3-flanking region (24,454 in total), was determined from three overlapping lambda phage clones. The sequence spans 22,256 bp from the cap site to the polyadenylylation site, revealing a gene structure of 15 exons separated by 14 introns. The methionine initiation codon ATG is within exon 1; the termination codon TGA is within exon 14. Exon 15 is entirely untranslated and contains the polyadenylylation signal AATAAA. The deduced polypeptide chain is composed of a 21-amino-acid leader peptide, followed by 578 amino acids of the mature protein. There are seven repetitive DNA elements (Alu and Kpn) in the introns and 3-flanking region. The sizes of the 15 alpha-albumin exons match closely those of the albumin, alpha-fetoprotein, and vitamin D-binding protein genes. The exons are symmetrically placed within the three domains of the individual proteins, and they share a characteristic codon splitting pattern that is conserved among members of the gene family. The results provide strong evidence that alpha-albumin belongs to, and most likely completes with, the serum albumin gene family. Based on structural similarity, alpha-albumin appears to be most closely related to alpha-fetoprotein. The complete structure of this family of four tandemly linked genes provides a well-characterized approximately 200 kb locus in the 4q subcentromeric region of the human genome.

A high-resolution physical map of human chromosome 11.

The development of a highly reliable physical map with landmark sites spaced an average of 100 kbp apart has been a central goal of the Human Genome Project. We have approached the physical mapping of human chromosome 11 with this goal as a primary target. We have focused on strategies that would utilize yeast artificial chromosome (YAC) technology, thus permitting long-range coverage of hundreds of kilobases of genomic DNA, yet we sought to minimize the ambiguities inherent in the use of this technology, particularly the occurrence of chimeric genomic DNA clones. This was achieved through the development of a chromosome 11-specific YAC library from a human somatic cell hybrid line that has retained chromosome 11 as its sole human component.To maximize the efficiency of YAC contig assembly and extension, we have employed an Alu-PCR-based hybridization screening system. This system eliminates many of the more costly and time-consuming steps associated with sequence tagged site content mapping such as sequencing, primer production, and hierarchical screening, resulting in greater efficiency with increased throughput and reduced cost. Using these approaches, we have achieved YAC coverage for >90% of human chromosome 11, with an average intermarker distance of <100 kbp. Cytogenetic localization has been determined for each contig by fluorescent in situ hybridization and/or sequence tagged site content. The YAC contigs that we have generated should provide a robust framework to move forward to sequence-ready templates for the sequencing efforts of the Human Genome Project as well as more focused positional cloning on chromosome 11.

Conservation of synteny between the genome of the pufferfish (Fugu rubripes) and the region on human chromosome 14 (14q24.3) associated with familial Alzheimer disease (AD3 locus)

The genome of the pufferfish (Fugu rubripes) (400 Mb) is approximately 7.5 times smaller than the human genome, but it has a similar gene repertoire to that of man. If regions of the two genomes exhibited conservation of gene order (i.e., were syntenic), it should be possible to reduce dramatically the effort required for identification of candidate genes in human disease loci by sequencing syntenic regions of the compact Fugu genome. We have demonstrated that three genes (dihydrolipoamide succinyltransferase, S31iii125, and S20i15), which are linked to FOS in the familial Alzheimer disease focus (AD3) on human chromosome 14, have homologues in the Fugu genome adjacent to Fugu cFOS. The relative gene order of cFOS, S31iii125, and S20i15 was the same in both genomes, but in Fugu these three genes lay within a 12.4-kb region, compared to >600 kb in the human AD3 locus. These results demonstrate the conservation of synteny between the genomes of Fugu and man and highlight the utility of this approach for sequence-based identification of genes in human disease loci.