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Multidrug resistance (MDR) is a major problem in the treatment of breast cancer. In the present study, next-generation sequencing technology was employed to identify differentially expressed genes in MCF‑7/MDR cells and MCF‑7 cells, and aimed to investigate the underlying molecular mechanisms of MDR in breast cancer. Differentially expressed genes between MCF‑7/MDR and MCF‑7 cells were selected using software; a total of 2085 genes were screened as differentially expressed in MCF‑7/MDR cells. Furthermore, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the DAVID database. Finally, a protein‑protein interaction network was constructed and the hub genes in the network were analyzed using the STRING database. GO annotation demonstrated that the differentially expressed genes were enriched in various biological processes, including ‘regulation of cell differentiation’, ‘cell development’, ‘neuron development’, ‘movement of cell or subcellular component’ and ‘cell morphogenesis involved in neuron differentiation’. Cellular component analysis by GO revealed that differentially expressed genes were enriched in ‘plasma membrane region’ and ‘extracellular matrix’ terms. Furthermore, KEGG analysis demonstrated that the target genes were enriched in various pathways, including ‘cell adhesion molecules (CAMs)’, ‘calcium signaling pathway’, ‘tight junction’, ‘Wnt signaling pathway’ and ‘pathways in cancer’ terms. A protein‑protein interaction network demonstrated that certain hub genes, including cyclin D1, nitric oxide synthase 3 (NOS3), NOTCH3, brain‑derived neurotrophic factor (BDNF), paired box 6, neuropeptide Y, phospholipase C β (PLCB) 4, PLCB2 and actin α cardiac muscle 1, may be associated with MDR in breast cancer. Subsequently, RT‑qPCR confirmed that the expression of these 9 hub genes was higher in MCF‑7/MDR cells compared with MCF‑7 cells, consistent with the RNA‑sequencing analysis. Additionally, a Cell Counting Kit‑8 assay demonstrated that specific inhibitors of NOS3 and BDNF/neurotrophic receptor tyrosine kinase, type 2 signaling reduced the IC50 of MCF‑7/MDR cells in response to various anticancer drugs, including adriamycin, cisplatin and 5‑fluorouracil. The results of the present study provide novel insights into the mechanism underlying MDR in MCF‑7 cells and may identify novel targets for the treatment of breast cancer. [ABSTRACT FROM AUTHOR]

High-throughput sequencing has rapidly gained popularity for transcriptome analysis in mammalian cells because of its ability to generate digital and quantitative information on annotated genes and to detect transcripts and mRNA isoforms. Here, we described a double- random priming method for deep sequencing to profile double poly(A)-selected RNA from LNCaP cells before and after androgen stimulation. From ≈20 million sequence tags, we uncovered 71% of annotated genes and identified hormone-regulated gene expression events that are highly correlated with quantitative real time PCR measurement. A fraction of the sequence tags were mapped to constitutive and alternative splicing events to detect known and new mRNA isoforms expressed in the cell. Finally, curve fitting was used to estimate the number of tags necessary to reach a "saturating" discovery rate among individual applications. This study provides a general guide for analysis of gene expression and alternative splicing by deep sequencing. [ABSTRACT FROM AUTHOR]

Higher eukaryotic genomes are bound by a large number of coding and non-coding RNAs, but approaches to comprehensively map the identity and binding sites of these RNAs are lacking. Here we report a method to capture in situ global RNA interactions with DNA by deep sequencing (GRID-seq), which enables the comprehensive identification of the entire repertoire of chromatin-interacting RNAs and their respective binding sites. In human, mouse, and Drosophila cells, we detected a large set of tissue-specific coding and non-coding RNAs that are bound to active promoters and enhancers, especially super-enhancers. Assuming that most mRNA-chromatin interactions indicate the physical proximity of a promoter and an enhancer, we constructed a three-dimensional global connectivity map of promoters and enhancers, revealing transcription-activity-linked genomic interactions in the nucleus. [ABSTRACT FROM AUTHOR]

FUS is an RNA-binding protein involved in amyotrophic lateral sclerosis ( ALS) and frontotemporal dementia ( FTD). Cytoplasmic FUS-containing aggregates are often associated with concomitant loss of nuclear FUS. Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell-specific CRE-mediated expression of wild-type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons. [ABSTRACT FROM AUTHOR]

DNA methylation at selective cytosine residues (5-methylcytosine (5mC)) and their removal by TET-mediated DNA demethylation are critical for setting up pluripotent states in early embryonic development. TET enzymes successively convert 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), with 5fC and 5caC subject to removal by thymine DNA glycosylase (TDG) in conjunction with base excision repair. Early reports indicate that 5fC and 5caC could be stably detected on enhancers, promoters and gene bodies, with distinct effects on gene expression, but the mechanisms have remained elusive. Here we determined the X-ray crystal structure of yeast elongating RNA polymerase II (Pol II) in complex with a DNA template containing oxidized 5mCs, revealing specific hydrogen bonds between the 5-carboxyl group of 5caC and the conserved epi-DNA recognition loop in the polymerase. This causes a positional shift for incoming nucleoside 5′-triphosphate (NTP), thus compromising nucleotide addition. To test the implication of this structural insight in vivo, we determined the global effect of increased 5fC/5caC levels on transcription, finding that such DNA modifications indeed retarded Pol II elongation on gene bodies. These results demonstrate the functional impact of oxidized 5mCs on gene expression and suggest a novel role for Pol II as a specific and direct epigenetic sensor during transcription elongation. [ABSTRACT FROM AUTHOR]

The U2AF heterodimer has been well studied for its role in defining functional 3′ splice sites in pre-mRNA splicing, but many fundamental questions still remain unaddressed regarding the function of U2AF in mammalian genomes. Through genome-wide analysis of U2AF-RNA interactions, we report that U2AF has the capacity to directly define ~88% of functional 3′ splice sites in the human genome, but numerous U2AF binding events also occur in intronic locations. Mechanistic dissection reveals that upstream intronic binding events interfere with the immediate downstream 3′ splice site associated either with the alternative exon, to cause exon skipping, or with the competing constitutive exon, to induce exon inclusion. We further demonstrate partial functional impairment with leukemia-associated mutations in U2AF35, but not U2AF65, in regulated splicing. These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states. [ABSTRACT FROM AUTHOR]

The surface of the cornea consists of a unique type of non-keratinized epithelial cells arranged in an orderly fashion, and this is essential for vision by maintaining transparency for light transmission. Cornea epithelial cells (CECs) undergo continuous renewal from limbal stem or progenitor cells (LSCs), and deficiency in LSCs or corneal epithelium-which turns cornea into a non-transparent, keratinized skin-like epithelium-causes corneal surface disease that leads to blindness in millions of people worldwide. How LSCs are maintained and differentiated into corneal epithelium in healthy individuals and which key molecular events are defective in patients have been largely unknown. Here we report establishment of an in vitro feeder-cell-free LSC expansion and three-dimensional corneal differentiation protocol in which we found that the transcription factors p63 (tumour protein 63) and PAX6 (paired box protein PAX6) act together to specify LSCs, and WNT7A controls corneal epithelium differentiation through PAX6. Loss of WNT7A or PAX6 induces LSCs into skin-like epithelium, a critical defect tightly linked to common human corneal diseases. Notably, transduction of PAX6 in skin epithelial stem cells is sufficient to convert them to LSC-like cells, and upon transplantation onto eyes in a rabbit corneal injury model, these reprogrammed cells are able to replenish CECs and repair damaged corneal surface. These findings suggest a central role of the WNT7A-PAX6 axis in corneal epithelial cell fate determination, and point to a new strategy for treating corneal surface diseases. [ABSTRACT FROM AUTHOR]

Summary: RNAP II is frequently paused near gene promoters in mammals, and its transition to productive elongation requires active recruitment of P-TEFb, a cyclin-dependent kinase for RNAP II and other key transcription elongation factors. A fraction of P-TEFb is sequestered in an inhibitory complex containing the 7SK noncoding RNA, but it has been unclear how P-TEFb is switched from the 7SK complex to RNAP II during transcription activation. We report that SRSF2 (also known as SC35, an SR-splicing factor) is part of the 7SK complex assembled at gene promoters and plays a direct role in transcription pause release. We demonstrate RNA-dependent, coordinated release of SRSF2 and P-TEFb from the 7SK complex and transcription activation via SRSF2 binding to promoter-associated nascent RNA. These findings reveal an unanticipated SR protein function, a role for promoter-proximal nascent RNA in gene activation, and an analogous mechanism to HIV Tat/TAR for activating cellular genes. [ABSTRACT FROM AUTHOR]

Summary: SR proteins are well-characterized RNA binding proteins that promote exon inclusion by binding to exonic splicing enhancers (ESEs). However, it has been unclear whether regulatory rules deduced on model genes apply generally to activities of SR proteins in the cell. Here, we report global analyses of two prototypical SR proteins, SRSF1 (SF2/ASF) and SRSF2 (SC35), using splicing-sensitive arrays and CLIP-seq on mouse embryo fibroblasts (MEFs). Unexpectedly, we find that these SR proteins promote both inclusion and skipping of exons in vivo, but their binding patterns do not explain such opposite responses. Further analyses reveal that loss of one SR protein is accompanied by coordinated loss or compensatory gain in the interaction of other SR proteins at the affected exons. Therefore, specific effects on regulated splicing by one SR protein actually depend on a complex set of relationships with multiple other SR proteins in mammalian genomes. [ABSTRACT FROM AUTHOR]

Summary: The induction of pluripotency or trans-differentiation of one cell type to another can be accomplished with cell-lineage-specific transcription factors. Here, we report that repression of a single RNA binding polypyrimidine-tract-binding (PTB) protein, which occurs during normal brain development via the action of miR-124, is sufficient to induce trans-differentiation of fibroblasts into functional neurons. Besides its traditional role in regulated splicing, we show that PTB has a previously undocumented function in the regulation of microRNA functions, suppressing or enhancing microRNA targeting by competitive binding on target mRNA or altering local RNA secondary structure. A key event during neuronal induction is the relief of PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby derepressing a large array of neuronal genes, including miR-124 and multiple neuronal-specific transcription factors, in nonneuronal cells. This converts a negative feedback loop to a positive one to elicit cellular reprogramming to the neuronal lineage. [ABSTRACT FROM AUTHOR]

Summary: The Hippo pathway is crucial in organ size control, and its dysregulation contributes to tumorigenesis. However, upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here, we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2, thereby activating YAP and TAZ transcription coactivators, which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression, cell migration, and proliferation. In contrast, stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity, thereby inhibiting YAP function. Thus, GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR. [ABSTRACT FROM AUTHOR]

Summary: Pre-mRNA splicing is regulated by developmental and environmental cues, but little is known about how specific signals are transduced in mammalian cells to regulate this critical gene expression step. Here, we report massive reprogramming of alternative splicing in response to EGF signaling. By blocking individual branches in EGF signaling, we found that Akt activation plays a major role, while other branches, such as the JAK/STAT and ERK pathways, make minor contributions to EGF-induced splicing. Activated Akt next branches to SR protein-specific kinases, rather than mTOR, by inducing SRPK autophosphorylation that switches the splicing kinases from Hsp70- to Hsp90-containing complexes. This leads to enhanced SRPK nuclear translocation and SR protein phosphorylation. These findings reveal a major signal transduction pathway for regulated splicing and place SRPKs in a central position in the pathway, consistent with their reputed roles in a large number of human cancers. [Copyright &y& Elsevier]

Summary: The nuclear matrix-associated hnRNP U/SAF-A protein has been implicated in diverse pathways from transcriptional regulation to telomere length control to X inactivation, but the precise mechanism underlying each of these processes has remained elusive. Here, we report hnRNP U as a regulator of SMN2 splicing from a custom RNAi screen. Genome-wide analysis by CLIP-seq reveals that hnRNP U binds virtually to all classes of regulatory noncoding RNAs, including all snRNAs required for splicing of both major and minor classes of introns, leading to the discovery that hnRNP U regulates U2 snRNP maturation and Cajal body morphology in the nucleus. Global analysis of hnRNP U-dependent splicing by RNA-seq coupled with bioinformatic analysis of associated splicing signals suggests a general rule for splice site selection through modulating the core splicing machinery. These findings exemplify hnRNP U/SAF-A as a potent regulator of nuclear ribonucleoprotein particles in diverse gene expression pathways. [ABSTRACT FROM AUTHOR]

Abstract: RNA-seq technologies are now replacing microarrays for profiling gene expression. Here we describe a robust RNA-seq strategy for multiplex analysis of RNA samples based on deep sequencing. First, an oligo-dT linked to an adaptor sequence is used to prime cDNA synthesis. Upon solid phase selection, second strand synthesis is initiated using a random primer linked to another adaptor sequence. Finally, the library is released from the beads and amplified using a bar-coded primer together with a common primer. This method, referred to as Multiplex Analysis of PolyA-linked Sequences (MAPS), preserves strand information, permits rapid identification of potentially new polyadenylation sites, and profiles gene expression in a highly cost effective manner. We have applied this technology to determine the transcriptome response to knockdown of the RNA binding protein TLS, and compared the result to current microarray technology, demonstrating the ability of MAPS to robustly detect regulated gene expression. [Copyright &y& Elsevier]

Simple Summary: Aberrant alternative splicing (AS) regulation plays a pivotal role in breast cancer development, progression, and resistance to therapeutical interventions. Indeed, cancer cells can adapt their own transcriptome by changing different AS programs, thus generating cancer-specific AS isoforms involved in every hallmark of cancer. Here, we investigated global AS errors occurring in human breast cancer cells by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing. Our results identified several dysregulated AS events potentially relevant for breast cancer-related biological processes and that provide a better comprehension of the molecular mechanisms that orchestrate the malignant transformation. Breast cancer is the most frequently occurred cancer type and the second cause of death in women worldwide. Alternative splicing (AS) is the process that generates more than one mRNA isoform from a single gene, and it plays a major role in expanding the human protein diversity. Aberrant AS contributes to breast cancer metastasis and resistance to chemotherapeutic interventions. Therefore, identifying cancer-specific isoforms is the prerequisite for therapeutic interventions intended to correct aberrantly expressed AS events. Here, we performed RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq) in breast cancer cells, to identify global breast cancer-specific AS defects. By RT-PCR validation, we demonstrate the high accuracy of RASL-seq results. In addition, we analyzed identified AS events using the Cancer Genome Atlas (TCGA) database in a large number of non-pathological and breast tumor specimens and validated them in normal and breast cancer samples. Interestingly, aberrantly regulated AS cassette exons in cancer tissues do not encode for known functional domains but instead encode for amino acids constituting regions of intrinsically disordered protein portions characterized by high flexibility and prone to be subjected to post-translational modifications. Collectively, our results reveal novel AS errors occurring in human breast cancer, potentially affecting breast cancer-related biological processes. [ABSTRACT FROM AUTHOR]

Aberrant alternative splicing (AS) is a hallmark of cancer and a potential target for novel anti-cancer therapeutics. Breast cancer-associated AS events are known to be linked to disease progression, metastasis, and survival of breast cancer patients. To identify altered AS programs occurring in metastatic breast cancer, we perform a global analysis of AS events by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq). We demonstrate that, relative to low-metastatic, high-metastatic breast cancer cells show different AS choices in genes related to cancer progression. Supporting a global reshape of cancer-related splicing profiles in metastatic breast cancer we found an enrichment of RNA-binding motifs recognized by several splicing regulators, which have aberrant expression levels or activity during breast cancer progression, including SRSF1. Among SRSF1-regulated targets we found DCUN1D5, a gene for which skipping of exon 4 in its pre-mRNA introduces a premature termination codon (PTC), thus generating an unstable transcript degraded by nonsense-mediated mRNA decay (NMD). Significantly, distinct breast cancer subtypes show different DCUN1D5 isoform ratios with metastatic breast cancer expressing the highest level of the NMD-insensitive DCUN1D5 mRNA, thus showing high DCUN1D5 expression levels, which are ultimately associated with poor overall and relapse-free survival in breast cancer patients. Collectively, our results reveal global AS features of metastatic breast tumors, which open new possibilities for the treatment of these aggressive tumor types. [ABSTRACT FROM AUTHOR]

The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes. • SRPK1 phosphorylates protamine to initiate paternal genome reprogramming • Phosphorylation site mutations in protamine inhibit protamine-to-histone exchange • Phosphorylated protamine permits efficient recruitment of NPM2 and HIRA • Paternal and maternal genomes undergo synchronized reprogramming upon fertilization Upon fertilization, protamines that tightly pack DNA into sperm are phosphorylated by SRPK1, triggering DNA decondensation, protamine removal, and histone deposition. [ABSTRACT FROM AUTHOR]

Background: Self-renewal is dependent on an intrinsic gene regulatory network centered on OCT4 and on an atypical cell cycle G1/S transition, which is also regulated by OCT4. p21, a gene negatively associated with self-renewal and a senescence marker, is a member of the universal cyclin-dependent kinase inhibitors (CDKIs) and plays critical roles in the regulation of the G1/S transition. The expression of p21 can be regulated by OCT4-targeted DNA methyltransferases (DNMTs), which play distinct roles in gene regulation and maintaining pluripotency properties. The aim of this study was to determine the role of OCT4 in the regulation of self-renewal and senescence in human hair follicle mesenchymal stem cells (hHFMSCs) and to characterize the molecular mechanisms involved. Methods: A lentiviral vector was used to ectopically express OCT4. The influences of OCT4 on the self-renewal and senescence of hHFMSCs were investigated. Next-generation sequencing (NGS) was performed to identify the downstream genes of OCT4 in this process. Methylation-specific PCR (MSP) analysis was performed to measure the methylation level of the p21 promoter region. p21 was overexpressed in hHFMSCsOCT4 to test its downstream effect on OCT4. The regulatory effect of OCT4 on DNMTs was examined by ChIP assay. 5-aza-dC/zebularine was used to inhibit the expression of DNMTs, and then self-renewal properties and senescence in hHFMSCs were detected. Results: The overexpression of OCT4 promoted proliferation, cell cycle progression, and osteogenic differentiation capacity of hHFMSCs. The cell senescence of hHFMSCs was markedly suppressed due to the ectopic expression of OCT4. Through NGS, we identified 2466 differentially expressed genes (DEGs) between hHFMSCsOCT4 and hHFMSCsEGFP, including p21, which was downregulated. The overexpression of p21 abrogated the proliferation and osteogenic differentiation capacity of hHFMSCsOCT4 and promoted cell senescence. OCT4 enhanced the transcription of DNMT genes, leading to an elevation in the methylation of the p21 promoter. The inhibition of DNMTs reversed the OCT4-induced p21 reduction, depleted the self-renewal of hHFMSCsOCT4, and triggered cell senescence. Conclusions: OCT4 maintains the self-renewal ability of hHFMSCs and reverses senescence by suppressing the expression of p21 through the upregulation of DNMTs. [ABSTRACT FROM AUTHOR]

Summary Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1 , including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase—the positive transcription elongation factor complex (P-TEFb)—from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype. [ABSTRACT FROM AUTHOR]

Summary R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed. [ABSTRACT FROM AUTHOR]