xcore: an R package for inference of gene expression regulators.

BACKGROUND: Elucidating the Transcription Factors (TFs) that drive the gene expression changes in a given experiment is a common question asked by researchers. The existing methods rely on the predicted Transcription Factor Binding Site (TFBS) to model the changes in the motif activity. Such methods only work for TFs that have a motif and assume the TF binding profile is the same in all cell types. RESULTS: Given the wealth of the ChIP-seq data available for a wide range of the TFs in various cell types, we propose that gene expression modeling can be done using ChIP-seq "signatures" directly, effectively skipping the motif finding and TFBS prediction steps. We present xcore, an R package that allows TF activity modeling based on ChIP-seq signatures and the user's gene expression data. We also provide xcoredata a companion data package that provides a collection of preprocessed ChIP-seq signatures. We demonstrate that xcore leads to biologically relevant predictions using transforming growth factor beta induced epithelial-mesenchymal transition time-courses, rinderpest infection time-courses, and embryonic stem cells differentiated to cardiomyocytes time-course profiled with Cap Analysis Gene Expression. CONCLUSIONS: xcore provides a simple analytical framework for gene expression modeling using linear models that can be easily incorporated into differential expression analysis pipelines. Taking advantage of public ChIP-seq databases, xcore can identify meaningful molecular signatures and relevant ChIP-seq experiments.

Development of p53 knockout U87MG cell line for unbiased drug delivery testing system using CRISPR-Cas9 and transcriptomic analysis.

Antibody-drug conjugates offers many advantages as a drug delivery platform that allows for highly specific targeting of cell types and genes. Ideally, testing the efficacy of these systems requires two cell types to be different only in the gene targeted by the drug, with the rest of the cellular machinery unchanged, in order to minimize other potential differences from obscuring the effects of the drug. In this study, we created multiple variants of U87MG cells with targeted mutation in the TP53 gene using the CRISPR-Cas9 system, and determined that their major transcriptional differences stem from the loss of p53 function. Using the transcriptome data, we predicted which mutant clones would have less divergent phenotypes from the wild type and thereby serve as the best candidates to be used as drug delivery testing platforms. Further in vitro and in vivo assays of cell morphology, proliferation rate and target antigen-mediated uptake supported our predictions. Based on the combined analysis results, we successfully selected the best qualifying mutant clone. This study serves as proof-of-principle of the approach and paves the way for extending to additional cell types and target genes.

Spliceostatin A interaction with SF3B limits U1 snRNP availability and causes premature cleavage and polyadenylation.

RNA splicing, a highly conserved process in eukaryotic gene expression, is seen as a promising target for anticancer agents. Splicing is associated with other RNA processing steps, such as transcription and nuclear export; however, our understanding of the interaction between splicing and other RNA regulatory mechanisms remains incomplete. Moreover, the impact of chemical splicing inhibition on long non-coding RNAs (lncRNAs) has been poorly understood. Here, we demonstrate that spliceostatin A (SSA), a chemical splicing modulator that binds to the SF3B subcomplex of the U2 small nuclear ribonucleoprotein particle (snRNP), limits U1 snRNP availability in splicing, resulting in premature cleavage and polyadenylation of MALAT1, a nuclear lncRNA, as well as protein-coding mRNAs. Therefore, truncated transcripts are exported into the cytoplasm and translated, resulting in aberrant protein products. Our work demonstrates that active recycling of the splicing machinery maintains homeostasis of RNA processing beyond intron excision.

A framework for identification of on- and off-target transcriptional responses to drug treatment.

Owing to safety concerns or insufficient efficacy, few drug candidates are approved for marketing. Drugs already on the market may be withdrawn due to adverse effects (AEs) discovered after market introduction. Comprehensively investigating the on-/off-target effects of drugs can help expose AEs during the drug development process. We have developed an integrative framework for systematic identification of on-/off-target pathways and elucidation of the underlying regulatory mechanisms, by combining promoter expression profiling after drug treatment with gene perturbation of the primary drug target. Expression profiles from statin-treated cells and HMG-CoA reductase knockdowns were analyzed using the framework, allowing for identification of not only reported adverse effects but also novel candidates of off-target effects from statin treatment, including key regulatory elements of on- and off-targets. Our findings may provide new insights for finding new usages or potential side effects of drug treatment.

C1 CAGE detects transcription start sites and enhancer activity at single-cell resolution.

Single-cell transcriptomic profiling is a powerful tool to explore cellular heterogeneity. However, most of these methods focus on the 3'-end of polyadenylated transcripts and provide only a partial view of the transcriptome. We introduce C1 CAGE, a method for the detection of transcript 5'-ends with an original sample multiplexing strategy in the C1TM microfluidic system. We first quantifiy the performance of C1 CAGE and find it as accurate and sensitive as other methods in the C1 system. We then use it to profile promoter and enhancer activities in the cellular response to TGF-ß of lung cancer cells and discover subpopulations of cells differing in their response. We also describe enhancer RNA dynamics revealing transcriptional bursts in subsets of cells with transcripts arising from either strand in a mutually exclusive manner, validated using single molecule fluorescence in situ hybridization.

CD157 Marks Tissue-Resident Endothelial Stem Cells with Homeostatic and Regenerative Properties.

The generation of new blood vessels via angiogenesis is critical for meeting tissue oxygen demands. A role for adult stem cells in this process remains unclear. Here, we identified CD157 (bst1, bone marrow stromal antigen 1) as a marker of tissue-resident vascular endothelial stem cells (VESCs) in large arteries and veins of numerous mouse organs. Single CD157+ VESCs form colonies in vitro and generate donor-derived portal vein, sinusoids, and central vein endothelial cells upon transplantation in the liver. In response to injury, VESCs expand and regenerate entire vasculature structures, supporting the existence of an endothelial hierarchy within blood vessels. Genetic lineage tracing revealed that VESCs maintain large vessels and sinusoids in the normal liver for more than a year, and transplantation of VESCs rescued bleeding phenotypes in a mouse model of hemophilia. Our findings show that tissue-resident VESCs display self-renewal capacity and that vascular regeneration potential exists in peripheral blood vessels.

FANTOM5 CAGE profiles of human and mouse samples.

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

Global analysis of pre-mRNA subcellular localization following splicing inhibition by spliceostatin A.

Spliceostatin A (SSA) is a methyl ketal derivative of FR901464, a potent antitumor compound isolated from a culture broth of Pseudomonas sp no. 2663. These compounds selectively bind to the essential spliceosome component SF3b, a subcomplex of the U2 snRNP, to inhibit pre-mRNA splicing. However, the mechanism of SSA's antitumor activity is unknown. It is noteworthy that SSA causes accumulation of a truncated form of the CDK inhibitor protein p27 translated from CDKN1B pre-mRNA, which is involved in SSA-induced cell-cycle arrest. However, it is still unclear whether pre-mRNAs are uniformly exported from the nucleus following SSA treatment. We performed RNA-seq analysis on nuclear and cytoplasmic fractions of SSA-treated cells. Our statistical analyses showed that intron retention is the major consequence of SSA treatment, and a small number of intron-containing pre-mRNAs leak into the cytoplasm. Using a series of reporter plasmids to investigate the roles of intronic sequences in the pre-mRNA leakage, we showed that the strength of the 5' splice site affects pre-mRNA leakage. Additionally, we found that the level of pre-mRNA leakage is related to transcript length. These results suggest that the strength of the 5' splice site and the length of the transcripts are determinants of the pre-mRNA leakage induced by SF3b inhibitors.

CCT2 Mutations Evoke Leber Congenital Amaurosis due to Chaperone Complex Instability.

Leber congenital amaurosis (LCA) is a hereditary early-onset retinal dystrophy that is accompanied by severe macular degeneration. In this study, novel compound heterozygous mutations were identified as LCA-causative in chaperonin-containing TCP-1, subunit 2 (CCT2), a gene that encodes the molecular chaperone protein, CCTß. The zebrafish mutants of CCTß are known to exhibit the eye phenotype while its mutation and association with human disease have been unknown. The CCT proteins (CCT α-θ) forms ring complex for its chaperon function. The LCA mutants of CCTß, T400P and R516H, are biochemically instable and the affinity for the adjacent subunit, CCTγ, was affected distinctly in both mutants. The patient-derived induced pluripotent stem cells (iPSCs), carrying these CCTß mutants, were less proliferative than the control iPSCs. Decreased proliferation under Cct2 knockdown in 661W cells was significantly rescued by wild-type CCTß expression. However, the expression of T400P and R516H didn't exhibit the significant effect. In mouse retina, both CCTß and CCTγ are expressed in the retinal ganglion cells and connecting cilium of photoreceptor cells. The Cct2 knockdown decreased its major client protein, transducing ß1 (Gß1). Here we report the novel LCA mutations in CCTß and the impact of chaperon disability by these mutations in cellular biology.

Reduced rod electroretinograms in carrier parents of two Japanese siblings with autosomal recessive retinitis pigmentosa associated with PDE6B gene mutations.

PURPOSE: To present the clinical and genetic findings in two siblings with autosomal recessive retinitis pigmentosa (RP) and their non-symptomatic parents. METHODS: We studied two siblings, a 48-year-old woman and her 44-year-old brother, and their parents. They had general ophthalmic examinations including ophthalmoscopy, perimetry, and electroretinography (ERG). Their whole exomes were analyzed by the next-generation sequence technique. RESULTS: The two siblings had night blindness for a long time, and clinical examinations revealed diffuse retinal degeneration with bone spicule pigmentation, constriction of the visual field, and non-recordable ERGs. Their parents were non-symptomatic and had normal fundi; however, their rod ERGs were reduced. Genetic examination revealed compound heterozygous mutations of I535N and H557Y in the PDE6B gene in the siblings, and the parents were heterozygous carriers of the mutations. CONCLUSIONS: Heterozygous mutation in the PDE6B gene can cause a reduction in the rod function to different degrees. The retinal function of non-symptomatic carriers of autosomal recessive RP should be evaluated with care.

Congenital Achromatopsia and Macular Atrophy Caused by a Novel Recessive PDE6C Mutation (p.E591K).

PURPOSE: We have previously reported clinical features of two siblings, a sister with complete achromatopsia (ACHM) and a brother with incomplete ACHM, in a consanguineous Japanese family. With the current study, we intended to identify a disease-causing mutation in the siblings and to investigate why the phenotypes of the siblings differed. METHODS: We performed a comprehensive ophthalmic examination for each sibling and parent. Whole-exome and Sanger sequencing were performed on genomic DNA. Molecular modeling was analyzed in an in silico study. RESULTS: The ophthalmic examination revealed severe macular atrophy in the older female sibling at 30 years of age and mild macular atrophy in the brother at 26 years of age. The genetic analysis identified a novel homozygous PDE6C mutation (p.E591K) as the disease-causing allele in the siblings. Each parent was heterozygous for the mutation. Molecular modeling showed that the mutation could cause a conformational change in the PDE6C protein and result in reduced phosphodiesterase activity. We also identified an OPN1SW mutation (p.G79R), which is associated with congenital tritan deficiencies, in the sister and the father but not in the brother. CONCLUSIONS: A novel homozygous PDE6C mutation was identified as the cause of ACHM. In addition, we identified an OPN1SW mutation in the sibling with complete ACHM, which might explain the difference in phenotype (complete versus incomplete ACHM) between the siblings.

Novel nonsense and splice site mutations in CRB1 gene in two Japanese patients with early-onset retinal dystrophy.

PURPOSE: To report novel mutations in the CRB1 gene in two patients with early-onset retinal dystrophy (EORD) and the longitudinal clinical course of EORD. PATIENTS AND METHODS: The patients were two unrelated Japanese children. Standard ophthalmic examinations including perimetry, electroretinography, and optical coherence tomography were performed on both patients. Whole exomes of the patients and their nonsymptomatic parents were analyzed using a next-generation sequence (NGS) technique. RESULTS: Patient 1 was noted to have esotropia and hyperopia at age 3. His decimal best-corrected visual acuity (BCVA) was 0.6 OD and 0.3 OS at age 6 with de-pigmentation of the retinal pigment epithelium (RPE). At age 19, his central vision was still preserved; however, numerous pigment granules were present in the retina. NGS analysis revealed a p.R632X nonsense and c.652 + 1_652 + 4delGTAA splice site mutations in the CRB1 gene. Patient 2 was noted to have hyperopia at age 3. His decimal BCVA at age 6 was 0.3 OD and 0.4 OS with de-pigmented RPE. The degree of retinal pigmentation was increased but his BCVA was good until the age of 14 years. NGS analysis revealed c.652 + 1_652 + 4delGTAA and c.652 + 1_652 + 2insT splice site mutations in the CRB1 gene. CONCLUSIONS: The phenotypes of these novel mutations for EORD are typical of CRB1-associated EORD (LCA8). They were slowly progressive until the second decade of life.

RHO Mutations (p.W126L and p.A346P) in Two Japanese Families with Autosomal Dominant Retinitis Pigmentosa.

Purpose. To investigate genetic and clinical features of patients with rhodopsin (RHO) mutations in two Japanese families with autosomal dominant retinitis pigmentosa (adRP). Methods. Whole-exome sequence analysis was performed in ten adRP families. Identified RHO mutations for the cosegregation analysis were confirmed by Sanger sequencing. Ophthalmic examinations were performed to evaluate the RP phenotypes. The impact of the RHO mutation on the rhodopsin conformation was examined by molecular modeling analysis. Results. In two adRP families, we identified two RHO mutations (c.377G>T (p.W126L) and c.1036G>C (p.A346P)), one of which was novel. Complete cosegregation was confirmed for each mutation exhibiting the RP phenotype in both families. Molecular modeling predicted that the novel mutation (p.W126L) might impair rhodopsin function by affecting its conformational transition in the light-adapted form. Clinical phenotypes showed that patients with p.W126L exhibited sector RP, whereas patients with p.A346P exhibited classic RP. Conclusions. Our findings demonstrated that the novel mutation (p.W126L) may be associated with the phenotype of sector RP. Identification of RHO mutations is a very useful tool for predicting disease severity and providing precise genetic counseling.

Whole exome analysis identifies frequent CNGA1 mutations in Japanese population with autosomal recessive retinitis pigmentosa.

OBJECTIVE: The purpose of this study was to investigate frequent disease-causing gene mutations in autosomal recessive retinitis pigmentosa (arRP) in the Japanese population. METHODS: In total, 99 Japanese patients with non-syndromic and unrelated arRP or sporadic RP (spRP) were recruited in this study and ophthalmic examinations were conducted for the diagnosis of RP. Among these patients, whole exome sequencing analysis of 30 RP patients and direct sequencing screening of all CNGA1 exons of the other 69 RP patients were performed. RESULTS: Whole exome sequencing of 30 arRP/spRP patients identified disease-causing gene mutations of CNGA1 (four patients), EYS (three patients) and SAG (one patient) in eight patients and potential disease-causing gene variants of USH2A (two patients), EYS (one patient), TULP1 (one patient) and C2orf71 (one patient) in five patients. Screening of an additional 69 arRP/spRP patients for the CNGA1 gene mutation revealed one patient with a homozygous mutation. CONCLUSIONS: This is the first identification of CNGA1 mutations in arRP Japanese patients. The frequency of CNGA1 gene mutation was 5.1% (5/99 patients). CNGA1 mutations are one of the most frequent arRP-causing mutations in Japanese patients.

Longitudinal clinical course of three Japanese patients with Leber congenital amaurosis/early-onset retinal dystrophy with RDH12 mutation.

PURPOSE: To report the longitudinal clinical course of three Japanese patients from two families with Leber congenital amaurosis/early-onset retinal dystrophy (LCA/EORD), and the results of next-generation DNA sequences on them. PATIENTS AND METHODS: The patients were three Japanese children: a 4-year-old girl, a 6-year-old boy, and a 3-year-old girl. Patients 1 and 2 were siblings, and patient 3 was from an unrelated family. Standard ophthalmic examinations including perimetry, electroretinography, optical coherence tomography, and ultrasonography were performed on each patient. The patients were observed for 28, 16, and 10 years. Whole exomes of the patients and their non-symptomatic parents were analyzed using a next-generation sequence technique. RESULTS: The decimal visual acuity varied between 0.07 and 0.6 at the initial visit and decreased to counting finger to hand motion in their teens. Funduscopy showed diffuse retinal and macular degeneration. During the follow-up period, a posterior staphyloma developed and the macular area became atrophic. Patient 1 developed cataracts in her early twenties. Genetic analysis revealed a homozygous A126V substitution in the RDH12 gene in all patients. CONCLUSIONS: The three patients with LCA/EORD had a progressive decrease of their vision with the formation of a posterior staphyloma. This is the first report of Japanese patients with LCA/EORD with a RDH12 mutation.

Whole-exome sequencing identifies a novel ALMS1 mutation (p.Q2051X) in two Japanese brothers with Alström syndrome.

PURPOSE: No mutations associated with Alström syndrome (AS), a rare autosomal recessive disease, have been reported in the Japanese population. The purpose of this study was to investigate the genetic and clinical features of two brothers with AS in a consanguineous Japanese family. METHODS: Whole-exome sequencing analysis was performed on two brothers with AS and their unaffected parents. We performed a complete ophthalmic examination, including decimal best-corrected visual acuity, slit-lamp and funduscopic examination, visual-field and color-vision testing, full-field electroretinography, and optical coherence tomography. Fasting blood tests and systemic examinations were also performed. RESULTS: A novel mutation (c.6151C>T in exon 8) in the Alström syndrome 1 (ALMS1) gene that causes a premature termination codon at amino acid 2051 (p.Q2051X), was identified in the homozygous state in the affected brothers and in the heterozygous state in the parents. The ophthalmologic findings for both brothers revealed infantile-onset severe retinal degeneration and visual impairment, marked macular thinning, and severe cataracts. Systemic findings showed hepatic dysfunction, hyperlipidemia, hypogonadism, short stature, and wide feet in both brothers, whereas hearing loss, renal failure, abnormal digits, history of developmental delay, scoliosis, hypertension, and alopecia were not observed in either brother. The older brother exhibited type 2 diabetic mellitus and obesity, whereas the younger brother had hyperinsulinemia and subclinical hypothyroidism. CONCLUSIONS: A novel ALMS1 mutation was identified by using whole-exome sequencing analysis, which is useful not only to identify a disease causing mutation but also to exclude other gene mutations. Although characteristic ophthalmologic findings and most systemic findings were similar between the brothers, the brothers differed slightly in terms of glucose tolerance and thyroid function.

Competition between a noncoding exon and introns: Gomafu contains tandem UACUAAC repeats and associates with splicing factor-1.

Gomafu (also referred to as RNCR2/MIAT) was originally identified as a noncoding RNA expressed in a particular set of neurons. Unlike protein-coding mRNAs, the Gomafu RNA escapes nuclear export and stably accumulates in the nucleus, making a unique nuclear compartment. Although recent studies have revealed the functional relevance of Gomafu in a series of physiological processes, the underlying molecular mechanism remains largely uncharacterized. In this report, we identified a chicken homologue of Gomafu using a comparative genomic approach to search for functionally important and conserved sequence motifs among evolutionarily distant species. Unexpectedly, we found that all Gomafu RNA examined shared a distinctive feature: tandem repeats of UACUAAC, a sequence that has been identified as a conserved intron branch point in the yeast Saccharomyces cerevisiae. The tandem UACUAAC Gomafu RNA repeats bind to the SF1 splicing factor with a higher affinity than the divergent branch point sequence in mammals, which affects the kinetics of the splicing reaction in vitro. We propose that the Gomafu RNA regulates splicing efficiency by changing the local concentration of splicing factors within the nucleus.

The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line.

Using deep sequencing (deepCAGE), the FANTOM4 study measured the genome-wide dynamics of transcription-start-site usage in the human monocytic cell line THP-1 throughout a time course of growth arrest and differentiation. Modeling the expression dynamics in terms of predicted cis-regulatory sites, we identified the key transcription regulators, their time-dependent activities and target genes. Systematic siRNA knockdown of 52 transcription factors confirmed the roles of individual factors in the regulatory network. Our results indicate that cellular states are constrained by complex networks involving both positive and negative regulatory interactions among substantial numbers of transcription factors and that no single transcription factor is both necessary and sufficient to drive the differentiation process.

Discrimination of non-protein-coding transcripts from protein-coding mRNA.

Several recent studies indicate that mammals and other organisms produce large numbers of RNA transcripts that do not correspond to known genes. It has been suggested that these transcripts do not encode proteins, but may instead function as RNAs. However, discrimination of coding and non-coding transcripts is not straightforward, and different laboratories have used different methods, whose ability to perform this discrimination is unclear. In this study, we examine ten bioinformatic methods that assess protein-coding potential and compare their ability and congruency in the discrimination of non-coding from coding sequences, based on four underlying principles: open reading frame size, sequence similarity to known proteins or protein domains, statistical models of protein-coding sequence, and synonymous versus non-synonymous substitution rates. Despite these different approaches, the methods show broad concordance, suggesting that coding and non-coding transcripts can, in general, be reliably discriminated, and that many of the recently discovered extra-genic transcripts are indeed non-coding. Comparison of the methods indicates reasons for unreliable predictions, and approaches to increase confidence further. Conversely and surprisingly, our analyses also provide evidence that as much as approximately 10% of entries in the manually curated protein database Swiss-Prot are erroneous translations of actually non-coding transcripts.

Clusters of internally primed transcripts reveal novel long noncoding RNAs.

Non-protein-coding RNAs (ncRNAs) are increasingly being recognized as having important regulatory roles. Although much recent attention has focused on tiny 22- to 25-nucleotide microRNAs, several functional ncRNAs are orders of magnitude larger in size. Examples of such macro ncRNAs include Xist and Air, which in mouse are 18 and 108 kilobases (Kb), respectively. We surveyed the 102,801 FANTOM3 mouse cDNA clones and found that Air and Xist were present not as single, full-length transcripts but as a cluster of multiple, shorter cDNAs, which were unspliced, had little coding potential, and were most likely primed from internal adenine-rich regions within longer parental transcripts. We therefore conducted a genome-wide search for regional clusters of such cDNAs to find novel macro ncRNA candidates. Sixty-six regions were identified, each of which mapped outside known protein-coding loci and which had a mean length of 92 Kb. We detected several known long ncRNAs within these regions, supporting the basic rationale of our approach. In silico analysis showed that many regions had evidence of imprinting and/or antisense transcription. These regions were significantly associated with microRNAs and transcripts from the central nervous system. We selected eight novel regions for experimental validation by northern blot and RT-PCR and found that the majority represent previously unrecognized noncoding transcripts that are at least 10 Kb in size and predominantly localized in the nucleus. Taken together, the data not only identify multiple new ncRNAs but also suggest the existence of many more macro ncRNAs like Xist and Air.