Target Genomic Regions Research Articles

Scale and direction of adaptive introgression between black cottonwood (Populus trichocarpa) and balsam poplar (P.balsamifera).

Introgression can introduce novel genetic variation at a faster rate than mutation alone and result in adaptive introgression when adaptive alleles are maintained in the recipient genome over time by natural selection. A previous study from our group demonstrated adaptive introgression from Populus balsamifera into P.trichocarpa in a target genomic region. Here we expand our local ancestry analysis to the whole genome of both parents to provide a comprehensive view of introgression patterns and to identify additional candidate regions for adaptive introgression genomewide. Populus trichocarpa is a large, fast-growing tree of mild coastal regions of the Pacific Northwest, whereas P.balsamifera is a smaller stature tree of continental and boreal regions with intense winter cold. The species hybridize where they are parapatric. We detected asymmetric patterns of introgression across the whole genome of these two poplar species adapted to contrasting environments, with stronger introgression from P.balsamifera to P.trichocarpa than vice versa. Admixed P.trichocarpa individuals contained more genomic regions with unusually high levels of introgression (19 regions) and also the largest introgressed genome fragment (1.02Mb) compared with admixed P.balsamifera (nine regions). Our analysis also revealed numerous candidate regions for adaptive introgression with strong signals of selection, notably related to disease resistance, and enriched for genes that may play crucial roles in survival and adaptation. Furthermore, we detected a potential overrepresentation of subtelomeric regions in P.balsamifera introgressed into P.trichocarpa and possible protection of sex-determining regions from interspecific gene flow.

RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis.

Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide (nt) small RNAs (siRNAs) that bind ARGONAUTE4 (AGO4) and target genomic regions for silencing. It also requires non-coding RNAs transcribed by RNA POLYMERASE V (Pol V) that likely serve as scaffolds for binding of AGO4/siRNA complexes. Here we utilized a modified global nuclear run-on (GRO) protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5′ adenine found in many AGO4-bound 24-nt siRNAs and was eliminated in a siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expressing wild-type AGO4 in ago4/6/9 was able to restore slicing of Pol V transcripts but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required the little understood elongation factor SPT5L. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.

Revealing Alzheimer\u2019s disease genes spectrum in the whole-genome by machine learning

BackgroundAlzheimer’s disease (AD) is an important, progressive neurodegenerative disease, with a complex genetic architecture. A key goal of biomedical research is to seek out disease risk genes, and to elucidate the function of these risk genes in the development of disease. For this purpose, expanding the AD-associated gene set is necessary. In past research, the prediction methods for AD related genes has been limited in their exploration of the target genome regions. We here present a genome-wide method for AD candidate genes predictions.MethodsWe present a machine learning approach (SVM), based upon integrating gene expression data with human brain-specific gene network data, to discover the full spectrum of AD genes across the whole genome.ResultsWe classified AD candidate genes with an accuracy and the area under the receiver operating characteristic (ROC) curve of 84.56% and 94%. Our approach provides a supplement for the spectrum of AD-associated genes extracted from more than 20,000 genes in a genome wide scale.ConclusionsIn this study, we have elucidated the whole-genome spectrum of AD, using a machine learning approach. Through this method, we expect for the candidate gene catalogue to provide a more comprehensive annotation of AD for researchers.

Genetic Diversity Assessment of Four Rice Varieties using SNP Markers

Markers assisted selection is a tool for improving the speed and efficiency of crop improvement. The identification of SNPs within the target genomic region of a QTL is necessary for efficient breeding of quantitative traits such as salt tolerance. The study was conducted to characterize parental lines using SNP markers and to identify the polymorphic SNPs for salt tolerance in QTL study of offspring. Four rice lines were evaluated under five levels of salt conditions including the control. The experimental design was a split-plot with two replications. Leaf samples were collected from four rice lines at heading stage and sent to LGC Genomics laboratory for genotyping using rice SNP platform. DNA extraction and SNP genotyping were performed using an internal protocol. The phenotypic data recorded were the visual scoring, tiller number, green leaf number, panicle number, panicle weight and dry weight. Phenotypic data were analyzed using SAS 9.2 software. The genotypic data were analyzed using MEGA6 and GGT2 software. Significant diversity was revealed among lines concerning all the phenotypic traits. Significant diversity, divergence and substitution pattern were observed among lines. 200 markers out of 1896 were polymorphic and selected for the next step.

Genetic Diversity Assessment of Four Rice Varieties using SNP Markers

Markers assisted selection is a tool for improving the speed and efficiency of crop improvement. The identification of SNPs within the target genomic region of a QTL is necessary for efficient breeding of quantitative traits such as salt tolerance. The study was conducted to characterize parental lines using SNP markers and to identify the polymorphic SNPs for salt tolerance in QTL study of offspring. Four rice lines were evaluated under five levels of salt conditions including the control. The experimental design was a split-plot with two replications. Leaf samples were collected from four rice lines at heading stage and sent to LGC Genomics laboratory for genotyping using rice SNP platform. DNA extraction and SNP genotyping were performed using an internal protocol. The phenotypic data recorded were the visual scoring, tiller number, green leaf number, panicle number, panicle weight and dry weight. Phenotypic data were analyzed using SAS 9.2 software. The genotypic data were analyzed using MEGA6 and GGT2 software. Significant diversity was revealed among lines concerning all the phenotypic traits. Significant diversity, divergence and substitution pattern were observed among lines. 200 markers out of 1896 were polymorphic and selected for the next step.

Redundancy analysis allows improved detection of methylation changes in large genomic regions

BackgroundDNA methylation is an epigenetic process that regulates gene expression. Methylation can be modified by environmental exposures and changes in the methylation patterns have been associated with diseases. Methylation microarrays measure methylation levels at more than 450,000 CpGs in a single experiment, and the most common analysis strategy is to perform a single probe analysis to find methylation probes associated with the outcome of interest. However, methylation changes usually occur at the regional level: for example, genomic structural variants can affect methylation patterns in regions up to several megabases in length. Existing DMR methods provide lists of Differentially Methylated Regions (DMRs) of up to only few kilobases in length, and cannot check if a target region is differentially methylated. Therefore, these methods are not suitable to evaluate methylation changes in large regions. To address these limitations, we developed a new DMR approach based on redundancy analysis (RDA) that assesses whether a target region is differentially methylated.ResultsUsing simulated and real datasets, we compared our approach to three common DMR detection methods (Bumphunter, blockFinder, and DMRcate). We found that Bumphunter underestimated methylation changes and blockFinder showed poor performance. DMRcate showed poor power in the simulated datasets and low specificity in the real data analysis. Our method showed very high performance in all simulation settings, even with small sample sizes and subtle methylation changes, while controlling type I error. Other advantages of our method are: 1) it estimates the degree of association between the DMR and the outcome; 2) it can analyze a targeted or region of interest; and 3) it can evaluate the simultaneous effects of different variables. The proposed methodology is implemented in MEAL, a Bioconductor package designed to facilitate the analysis of methylation data.ConclusionsWe propose a multivariate approach to decipher whether an outcome of interest alters the methylation pattern of a region of interest. The method is designed to analyze large target genomic regions and outperforms the three most popular methods for detecting DMRs. Our method can evaluate factors with more than two levels or the simultaneous effect of more than one continuous variable, which is not possible with the state-of-the-art methods.

Exploring the genetics of fertility restoration controlled by Rf1 in common wheat (Triticum aestivum L.) using high-density linkage maps.

Hybrid wheat breeding has the potential to significantly increase wheat productivity compared to line breeding. The induction of male sterility by the cytoplasm of Triticum timopheevii Zhuk. is a widely discussed approach to ensure cross-pollination between parental inbred lines in hybrid wheat seed production. As fertility restoration in hybrids with this cytoplasm is often incomplete, understanding the underlying genetics is a prerequisite to apply this technology. A promising component for fertility restoration is the restorer locus Rf1, which was first detected on chromosome 1A of the restorer accession R3. In the present study, we performed quantitative trait locus (QTL) analyses to locate Rf1 and estimate its effect in populations involving the restorer lines R3, R113 and L19. Molecular markers linked to Rf1 in these populations were used to analyse the genomic target region in T. timopheevii accessions and common wheat breeding lines. The QTL analyses revealed that Rf1 interacted with a modifier locus on chromosome 1BS and the restorer locus Rf4 on chromosome 6B. The modifier locus significantly influenced both the penetrance and expressivity of Rf1. Whereas Rf1 exhibited expressivity higher than that of Rf4, the effects of these loci were not additive. Evaluating the marker haplotype for the Rf1 region, we propose that the restoring Rf1 allele may be derived exclusively from T. timopheevii. The present study demonstrates that interactions between restorer and modifier loci play a critical role in fertility restoration of common wheat with the cytoplasm of T. timopheevii.

Simultaneous Detection of Multiple Salmonella Serovars from Milk and Chicken Meat by Real-Time PCR Using Unique Genomic Target Regions

A novel genomic and plasmid target-based PCR platform was developed for the detection of Salmonella serovars Heidelberg, Dublin, Hadar, Kentucky, and Enteritidis. Unique genome loci were obtained through extensive genome mining of protein databases and comparative genomic analysis of these serovars. Assays targeting Salmonella serovars Hadar, Heidelberg, Kentucky, and Dublin had 100% specificity and sensitivity, whereas those for Salmonella Enteritidis had 97% specificity and 88% sensitivity. The limits of detection for Salmonella serovars Heidelberg, Kentucky, Hadar, Enteritidis, and Dublin were 12, 9, 40, 13, and 5,280 CFU, respectively. A sensitivity assay was also performed by using milk artificially inoculated with pooled Salmonella serovars, yielding a detection limit of 1 to10 CFU/25 mL of milk samples after enrichment. The minimum DNA detected using the multiplexed TaqMan assay was 75.8 fg (1.53 × 101 genomic equivalents [GE]) for Salmonella Heidelberg, 140.8 fg (2.8 × 101 GE) for Salmonella Enteritidis, and 3.48 pg (6.96 × 102 GE) for Salmonella Dublin. PCR efficiencies were 89.8% for Salmonella Heidelberg, 94.5% for Salmonella Enteritidis, and 75.5% for Salmonella Dublin. Four types of 30 pasteurized milk samples were tested negative by culture techniques and with a genus-specific Salmonella invA gene PCR assay. Among 30 chicken samples similarly tested, 12 (40%) were positive by both culture and the invA PCR. Testing of these 12 samples with the serovar-specific PCR assay detected single and mixed contamination with Salmonella Kentucky, Salmonella Enteritidis, and Salmonella Heidelberg. Five unique primers were designed and tested by multiplex conventional PCR in conjunction with the use of the multiplex TaqMan assay with three of the primers. The diagnostic assays developed in this study could be used as tools for routine detection of these five Salmonella serovars and for epidemiological investigations of foodborne disease outbreaks.

Whole genome population genetics analysis of Sudanese goats identifies regions harboring genes associated with major traits

BackgroundSudan is endowed with a variety of indigenous goat breeds which are used for meat and milk production and which are well adapted to the local environment. The aim of the present study was to determine the genetic diversity and relationship within and between the four main Sudanese breeds of Nubian, Desert, Taggar and Nilotic goats. Using the 50 K SNP chip, 24 animals of each breed were genotyped.ResultsMore than 96% of high quality SNPs were polymorphic with an average minor allele frequency of 0.3. In all breeds, no significant difference between observed (0.4) and expected (0.4) heterozygosity was found and the inbreeding coefficients (FIS) did not differ from zero. Fst coefficients for the genetic distance between breeds also did not significantly deviate from zero. In addition, the analysis of molecular variance revealed that 93% of the total variance in the examined population can be explained by differences among individuals, while only 7% result from differences between the breeds. These findings provide evidence for high genetic diversity and little inbreeding within breeds on one hand, and low diversity between breeds on the other hand. Further examinations using Nei’s genetic distance and STRUCTURE analysis clustered Taggar goats distinct from the other breeds. In a principal component (PC) analysis, PC1 could separate Taggar, Nilotic and a mix of Nubian and Desert goats into three groups. The SNPs that contributed strongly to PC1 showed high Fst values in Taggar goat versus the other goat breeds. PCA allowed us to identify target genomic regions which contain genes known to influence growth, development, bone formation and the immune system.ConclusionsThe information on the genetic variability and diversity in this study confirmed that Taggar goat is genetically different from the other goat breeds in Sudan. The SNPs identified by the first principal components show high Fst values in Taggar goat and allowed to identify candidate genes which can be used in the development of breed selection programs to improve local breeds and find genetic factors contributing to the adaptation to harsh environments.

Promoter architecture and transcriptional regulation of musculoskeletal embryonic nuclear protein 1b (mustn1b) gene in zebrafish.

Mustn1 is a specific musculoskeletal protein that plays a critical role in myogenesis and chondrogenesis in vertebrates. Whole-mount in situ hybridization revealed that mustn1b mRNAs are specifically expressed in skeletal and cardiac muscles in Zebrafish embryos. However, the precise function and the regulatory elements required for its muscle-specific expression are largely unknown. The purpose of this study was to explore and uncover the target genomic regions that regulate mustn1b gene expression by in vivo functional characterization of the mustn1b promoter. We report here stable expression analyses of eGFP from fluorescent transgenic reporter Zebrafish line containing a 0.8kb_mustn1b-Tol2-eGFP construct. eGFP expression was specifically found in the skeletal and cardiac muscle tissues. We show that reporter Zebrafish lines generated replicate the endogenous mustn1b expression pattern in early Zebrafish embryos. Specific site directed-mutagenesis analysis revealed that promoter activity resides in two annotated genomic regulatory regions, each one corresponding to a specific functional transcription factor binding site. Our data indicate that mustn1b is specifically expressed in skeletal and cardiac muscle tissues and its muscle specificity is controlled by the 0.2-kb promoter and flanking sequences and in vivo regulated by the action of two sequence-specific families of transcription factors. Developmental Dynamics 246:992-1000, 2017. © 2017 Wiley Periodicals, Inc.

Tissue-specific genome editing of laminA/C in the posterior silk glands of Bombyx mori

The RNA-guided CRISPR/Cas9 system has been shown to be a powerful tool for genome editing in various organisms. A comprehensive toolbox for multiplex genome editing has been developed for the silkworm, Bombyx mori, a lepidopteran model insect of economic importance. However, as previous methods mainly relied on delivery of transient Cas9/guide RNA (gRNA), they could not be used in loss-of-function studies of essential genes. Here, we report a simple and versatile tissue-specific genome editing strategy. We perform a proof-of-principle demonstration by establishing and crossing two transgenic B. mori lines, one expressing Cas9 protein in the posterior silk glands (PSGs) and the other constitutively expressing BmlaminA/C (BmLMN) gRNA. All BmLMN alleles in the PSG cells were edited precisely at the target genome region, resulting in diverse mutations. mRNA expression of BmLMN was reduced by up to 75%, and only very low levels of BmLaminA/C protein were detected. Knockout of BmLMN produced obvious defects in gland cell development and cocoon production. In this study, we developed an efficient strategy for spatially controlled genome editing, providing unprecedented opportunities for investigating the function of essential/lethal genes in B. mori, with potential application for other insects.

Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA.

Cystic Fibrosis is an autosomal recessive disorder caused by mutations in the CFTR gene. CRISPR mediated, template-dependent homology-directed gene editing has been used to correct the most common mutation, c.1521_1523delCTT / p.Phe508del (F508del) which affects ~70% of individuals, but the efficiency was relatively low. Here, we describe a high efficiency strategy for editing of three different rare CFTR mutations which together account for about 3% of individuals with Cystic Fibrosis. The mutations cause aberrant splicing of CFTR mRNA due to the creation of cryptic splice signals that result in the formation of pseudoexons containing premature stop codons c.1679+1634A>G (1811+1.6kbA>G) and c.3718-2477C>T (3849+10kbC>T), or an out-of-frame 5’ extension to an existing exon c.3140-26A>G (3272-26A>G). We designed pairs of Cas9 guide RNAs to create targeted double-stranded breaks in CFTR either side of each mutation which resulted in high efficiency excision of the target genomic regions via non-homologous end-joining repair. When evaluated in a mini-gene splicing assay, we showed that targeted excision restored normal splicing for all three mutations. This approach could be used to correct aberrant splicing signals or remove disruptive transcription regulatory motifs caused by deep-intronic mutations in a range of other genetic disorders.

Locus-specific ChIP combined with NGS analysis reveals genomic regulatory regions that physically interact with the Pax5 promoter in a chicken B cell line.

Chromosomal interactions regulate genome functions, such as transcription, via dynamic chromosomal organization in the nucleus. In this study, we attempted to identify genomic regions that physically bind to the promoter region of the Pax5 gene, which encodes a master regulator for B cell lineage commitment, in a chicken B cell line, DT40, with the goal of obtaining mechanistic insight into transcriptional regulation through chromosomal interaction. We found that the Pax5 promoter bound to multiple genomic regions using locus-specific chromatin immunoprecipitation (locus-specific ChIP), a method for locus-specific isolation of target genomic regions, in combination with next-generation sequencing (NGS). Comparing chromosomal interactions in wild-type DT40 with those in a macrophage-like counterpart, we found that some of the identified chromosomal interactions were organized in a B cell-specific manner. In addition, deletion of a B cell-specific interacting genomic region in chromosome 11, which was marked by active enhancer histone modifications, resulted in moderate but significant down-regulation of Pax5 transcription. Together, these results suggested that Pax5 transcription in DT40 is regulated by B cell-specific inter-chromosomal interactions. Moreover, these analyses showed that locus-specific ChIP combined with NGS analysis is useful for non-biased identification of functional genomic regions that physically interact with a locus of interest.

Identification of physical interactions between genomic regions by enChIP-Seq.

Physical interactions between genomic regions play critical roles in the regulation of genome functions, including gene expression. Here, we show the feasibility of using engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) in combination with next-generation sequencing (NGS) (enChIP-Seq) to detect such interactions. In enChIP-Seq, the target genomic region is captured by an engineered DNA-binding complex, such as a clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a catalytically inactive form of Cas9 and a single guide RNA. Subsequently, the genomic regions that physically interact with the target genomic region in the captured complex are sequenced by NGS. Using enChIP-Seq, we found that the 5'HS5 locus, which is involved in the regulation of globin genes expression at the β-globin locus, interacts with multiple genomic regions upon erythroid differentiation in the human erythroleukemia cell line K562. Genes near the genomic regions inducibly associated with the 5'HS5 locus were transcriptionally up-regulated in the differentiated state, suggesting the existence of a coordinated transcription mechanism mediated by physical interactions between these loci. Thus, enChIP-Seq might be a potentially useful tool for detecting physical interactions between genomic regions in a nonbiased manner, which would facilitate elucidation of the molecular mechanisms underlying regulation of genome functions.

Systematic Mapping of RNA-Chromatin Interactions In Vivo

RNA molecules can attach to chromatin. It remains difficult to know what RNAs are associated with chromatin and where the genomic target loci of these RNAs are. Here, we present MARGI (mapping RNA-genome interactions), a technology to massively reveal native RNA-chromatin interactions from unperturbed cells. The gist of this technology is to ligate chromatin-associated RNAs (caRNAs) with their target genomic sequences by proximity ligation, forming RNA-DNA chimeric sequences, which are converted to a sequencing library for paired-end sequencing. Using MARGI, we produced RNA-genome interaction maps for human embryonic stem cells (ESCs) and human embryonic kidney (HEK) cells. MARGI revealed hundreds of caRNAs, including previously known XIST, SNHG1, NEAT1, and MALAT1, as well as each caRNA's genomic interaction loci. Using a cross-species experiment, we estimated that approximately 2.2% of MARGI-identified interactions were false positives. In ESCs and HEK cells, the RNA ends of more than 5% of MARGI read pairs were mapped to distal or inter-chromosomal locations as compared to the locations of theircorresponding DNA ends. The majority of transcription start sites are associated with distal or inter-chromosomal caRNAs. Chromatin-immunoprecipitation-sequencing (ChIP-seq)-reported H3K27ac and H3K4me3 levels are positively correlated, while H3K9me3 is negatively correlated, with MARGI-reported RNA attachment levels. The MARGI technology should facilitate revealing novel RNA functions and their genomic target regions.

High-Throughput Resequencing of Maize Landraces at Genomic Regions Associated with Flowering Time.

Despite the reduction in the price of sequencing, it remains expensive to sequence and assemble whole, complex genomes of multiple samples for population studies, particularly for large genomes like those of many crop species. Enrichment of target genome regions coupled with next generation sequencing is a cost-effective strategy to obtain sequence information for loci of interest across many individuals, providing a less expensive approach to evaluating sequence variation at the population scale. Here we evaluate amplicon-based enrichment coupled with semiconductor sequencing on a validation set consisting of three maize inbred lines, two hybrids and 19 landrace accessions. We report the use of a multiplexed panel of 319 PCR assays that target 20 candidate loci associated with photoperiod sensitivity in maize while requiring 25 ng or less of starting DNA per sample. Enriched regions had an average on-target sequence read depth of 105 with 98% of the sequence data mapping to the maize ‘B73’ reference and 80% of the reads mapping to the target interval. Sequence reads were aligned to B73 and 1,486 and 1,244 variants were called using SAMtools and GATK, respectively. Of the variants called by both SAMtools and GATK, 30% were not previously reported in maize. Due to the high sequence read depth, heterozygote genotypes could be called with at least 92.5% accuracy in hybrid materials using GATK. The genetic data are congruent with previous reports of high total genetic diversity and substantial population differentiation among maize landraces. In conclusion, semiconductor sequencing of highly multiplexed PCR reactions is a cost-effective strategy for resequencing targeted genomic loci in diverse maize materials.

Manipulations with early mouse embryos for generation of genetically modified animals

Recently, genome-editing technologies have become more efficient and accessible. The discovery of nucleases for directional genome editing (CRISPR/Cas9, TALEN, ZFNs) significantly accelerated and simplified the production of mice with targeted gene editing in the genome. Until last time, the CRISPR/Cas9 system noticeably simplified the preparation of knockout or transgenic mice. CRISPR/Cas9 technology was successfully applied for gene knockout and knock-in, generation of large deletions or directed insertions in targeted genome regions in embryonic stem cells (ESCs).When injected into blastocysts, such modified ESCs are able to generate chimeras producing gametes with an identical genotype with ESC. Thus, it can identify animals with modified genomes. More recently, CRISPR/Cas9 technology was successfully applied to mouse zygotes and the birth of genetic modified mice was observed, i. e., the time required for generating genome-modified animals decreased significantly. The CRISPR/Cas9 system allows making gene knockout, large deletions or directed insertions into the target region of the genome by cytoplasm or pronuclear microinjection into zygotes. In addition, this is faster and simpler than similar work with mouse ESCs. Meanwhile, methods of manipulation with early embryos and their transplantation to surrogate mothers may be somewhat tricky. Therefore, it is important to use modern technologies for directional genome editing and perfect mastery in the embryological technics. In this article, we describe the protocols of microinjection into the pronucleus or cytoplasm of zygotes and injection of embryonic stem cells into the blastocyst cavity. We also describe embryological methods, such as superovulation, preparation of early stage embryos, surgical operation, production of foster mice. In addition, we describe the assembly and necessary components for the isoflurane anesthetic apparatus and isoflurane anesthesia.

HDAC Inhibitors As Novel Targeted Therapies for NUP98-HOXA9 AML Patients

Background: The chromosomal translocation t(7;11)(p15,p15) encodes the oncogenic transcription factor NUP98-HOXA9 which results in a fusion of the nucleoporin 98kDa (NUP98) and homeobox A9 (HOXA9) genes. The oncogenic mechanisms underlying this translocation remain poorly understood and patients are currently inadequately served by traditional cytotoxic chemotherapy regimens.Aims:To decipher the underlying biology of the NUP98-HOXA9 fusion protein and develop rational therapeutic strategies targeting its oncogenic mechanism.Methods: Human cellular models expressing NUP98-HOXA9, HOXA9 wt or NUP98 wt were established by retroviral transduction of HEK293FT human cell line and human hematopoietic progenitors (CD34+, hHP) isolated from donor cord blood. Chromatin immunoprecepitation experiments followed by sequencing (ChIP-seq) and quantitative ChIP (qChIP) were used to define fusion specific binding locations. Cloning regulatory regions of selected target genes in a luciferase vectorconfirmed the direct involvement of NUP98-HOXA9 in their regulation. RTQ-PCR and gene expression microarrays were used to evaluate expression levels. Co-Immunoprecipitation experiments validated protein-protein interactions and drug treatments were performed at IC50. Cell viability was analysed by apoptosis, proliferation and Colony Forming Unit assays.Results:Comparison of ChIP-seq data from HEK293FTmodels of NUP98-HOXA9, HOXA9 wt or NUP98 wt respectively, identified 4,471 target genomic regions of the fusion protein (FDR < 0.05), located within +4/-4 kb from the annotated Transcription Start Site (TSS) of 1,363 genes, with 399 genes common to HOXA9 wt and 5 to NUP98 wt. The NUP98-HOXA9 binding sites included enhancers of MEIS1, HOXA9 and PBX3 (PBX3 and HOXA9 were common to NUP98 wt and MEIS1 to HOXA9 wt). Together these transcription factors form a key activator complex that regulates the expression of genes involved in leukemogenesis and its overexpression is significant related to adverse prognosis in AML. Luciferase assays showed that the upregulation of this leukemic axis was directly induced by the interaction of NUP98-HOXA9 with the corresponding enhancer regions of MEIS1, HOXA9 and PBX3. Treatment of cells with HXR9, a specific peptide inhibitor of HOXA9 and PBX3 interaction, led to a selective decrease in the proliferation of hHP expressing NUP98-HOXA9, confirming the relevance of these target genes to its oncogenic mechanism. Combining ChIP-seq and gene expression data of three independent clones of hHP expressing NUP98-HOXA9 and patient samples (n = 5) harbouring t(7;11)(p15,p15) revealed a dual regulatory role of the fusion protein, in both repressing and activating target gene transcription where, for example, MEIS1, HOXA9, PBX3 and AFF3 were found overexpressed and BIRC3, SMAD1, FILIP1L and PTEN downregulated. Interactions of NUP98-HOXA9 with p300 and HDAC1 were shown to drive this transcriptional activation and repression, respectively. We found using qChIP experiments that p300 bound to the regulatory regions of the overexpressed genes only when NUP98-HOXA9 was present, whereas we observed significant enrichment of HDAC1 binding to the promoter regions of the downregulated genes when the fusion protein was expressed. Taking advantage of this latter observation, we demonstrated a dramatic inhibitory effect on the viability of hHP expressing NUP98-HOXA9after the treatment with subtherapeutic doses (IC50 = 4nM) of the HDAC inhibitor LBH-589 (Panobinostat) with no effect in control hHP transduced with an empty vector.Conclusion: An improved understanding of the pathobiology underlying recurrent translocation events in AML is a critical first step for the development of rational, targeted therapies. Here, we identify upregulation of the targetable MEIS1-HOXA9-PBX3 complex underpinning the leukemogenic activity of NUP98-HOXA9. Its activity in repressing transcription mediated through interaction with HDAC1, has been shown to be also a key pathogenic mechanism that can be exploited through use of HDAC inhibitors and potentially lead to a promising new therapy for this high-risk group of patients. DisclosuresNo relevant conflicts of interest to declare.

Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing.

Mutation breeding is based on the induction of genetic variations; hence knowledge of the frequency and type of induced mutations is of paramount importance for the design and implementation of a mutation breeding program. Although γ ray irradiation has been widely used since the 1960s in the breeding of about 200 economically important plant species, molecular elucidation of its genetic effects has so far been achieved largely by analysis of target genes or genomic regions. In the present study, the whole genomes of six γ-irradiated M2 rice plants were sequenced; a total of 144-188 million high-quality (Q>20) reads were generated for each M2 plant, resulting in genome coverage of >45 times for each plant. Single base substitution (SBS) and short insertion/deletion (Indel) mutations were detected at the average frequency of 7.5×10-6-9.8×10-6 in the six M2 rice plants (SBS being about 4 times more frequent than Indels). Structural and copy number variations, though less frequent than SBS and Indel, were also identified and validated. The mutations were scattered in all genomic regions across 12 rice chromosomes without apparent hotspots. The present study is the first genome-wide single-nucleotide resolution study on the feature and frequency of γ irradiation-induced mutations in a seed propagated crop; the findings are of practical importance for mutation breeding of rice and other crop species.

Dysregulation of TET2 in hematologic malignancies.

The TET dioxygenases, TET1, TET2, and TET3, catalyze transfer of an oxygen atom to the methyl group of 5-methylcytocine (5-mC), converting it to 5-hydroxymethylcytocine (5-hmC). Among the genes encoding these enzymes, ten-eleven translocation 2 (TET2) is frequently mutated somatically in both myeloid and lymphoid malignancies. Because these TET2 mutations result in the impairment of the dioxygenase activity of TET2, it is thought that these mutations interfere with 5-mC to 5-hmC conversion. There is ample evidence indicating that TET2 mutations are a driver of tumorigenesis in blood cells and that TET2 mutations are often acquired at the hematopoietic stem/early progenitor cell stage. In addition, TET2 is the second-most frequently mutated gene in clonal hematopoiesis in individuals with no apparent blood cancers, suggesting that while TET2 mutations alone are insufficient to cause hematologic malignancy, they represent an early event during tumorigenesis. A number of questions, including the precise target genome regions of TET2, and the importance of the balance of 5-mC and 5-hmC in the regulatory regions in transcriptional control, remain.