Chimeric Transcripts Research Articles

A Novel Mutation of the NARROW LEAF 1 Gene Adversely Affects Plant Architecture in Rice (Oryza sativa L.).

Plant architecture is critical for enhancing the adaptability and productivity of crop plants. Mutants with an altered plant architecture allow researchers to elucidate the genetic network and the underlying mechanisms. In this study, we characterized a novel nal1 rice mutant with short height, small panicle, and narrow and thick deep green leaves that was identified from a cross between a rice cultivar and a weedy rice accession. Bulked segregant analysis coupled with genome re-sequencing and cosegregation analysis revealed that the overall mutant phenotype was caused by a 1395-bp deletion spanning over the last two exons including the transcriptional end site of the nal1 gene. This deletion resulted in chimeric transcripts involving nal1 and the adjacent gene, which were validated by a reference-guided assembly of transcripts followed by PCR amplification. A comparative transcriptome analysis of the mutant and the wild-type rice revealed 263 differentially expressed genes involved in cell division, cell expansion, photosynthesis, reproduction, and gibberellin (GA) and brassinosteroids (BR) signaling pathways, suggesting the important regulatory role of nal1. Our study indicated that nal1 controls plant architecture through the regulation of genes involved in the photosynthetic apparatus, cell cycle, and GA and BR signaling pathways.

Widespread Transcriptional Readthrough Caused by Nab2 Depletion Leads to Chimeric Transcripts with Retained Introns

Nascent RNA sequencing has revealed that pre-mRNA splicing can occur shortly after introns emerge from RNA polymerase II (RNA Pol II). Differences in co-transcriptional splicing profiles suggest regulation by cis- and/or trans-acting factors. Here, we use single-molecule intron tracking (SMIT) to identify a cohort of regulators by machine learning in budding yeast. Of these, Nab2 displays reduced co-transcriptional splicing when depleted. Unexpectedly, these splicing defects are attributable to aberrant "intrusive" transcriptional readthrough from upstream genes, as revealed by long-read sequencing. Transcripts that originate from the intron-containing gene's own transcription start site (TSS) are efficiently spliced, indicating no direct role of Nab2 in splicing per se. This work highlights the coupling between transcription, splicing, and 3' end formation in the context of gene organization along chromosomes. We conclude that Nab2 is required for proper 3' end processing, which ensures gene-specific control of co-transcriptional RNA processing.

G9a Plays Distinct Roles in Maintaining DNA Methylation, Retrotransposon Silencing, and Chromatin Looping

G9a is a lysine methyltransferase that regulates epigenetic modifications, transcription, and genome organization. However, whether these properties are dependent on one another or represent distinct functions of G9a remains unclear. In this study, we observe widespread DNA methylation loss in G9a depleted and catalytic mutant embryonic stem cells. Furthermore, we define how G9a regulates chromatin accessibility, epigenetic modifications, and transcriptional silencing in both catalytic-dependent and -independent manners. Reactivated retrotransposons provide alternative promoters and splice sites leading to the upregulation of neighboring genes and the production of chimeric transcripts. Moreover, while topologically associated domains and compartment A/B definitions are largely unaffected, the loss of G9a leads to altered chromatin states, aberrant CTCF and cohesin binding, and differential chromatin looping, especially at retrotransposons. Taken together, our findings reveal how G9a regulates the epigenome, transcriptome, and higher-order chromatin structures in distinct mechanisms.

Modelling t(8;21) acute myeloid leukaemia - What have we learned?

Acute myeloid leukaemia (AML) is a heterogeneous haematopoietic malignancy caused by recurrent mutations in haematopoietic stem and progenitor cells that affect both the epigenetic regulatory machinery and signalling molecules. The t(8;21) or RUNX1‐RUNX1T1 translocation generates the RUNX1‐ETO chimeric transcription factor which primes haematopoietic stem cells for further oncogenic mutational events that in their sum cause overt disease. Significant progress has been made in generating both in vitro and in vivo model systems to recapitulate t(8;21) AML which are crucial for the understanding of the biology of the disease and the development of effective treatment. This review provides a comprehensive overview of the in vivo and in vitro model systems that were developed to gain insights into the molecular mechanisms of RUNX1‐ETO oncogenic activity and their contribution to the advancement of knowledge in the t(8;21) AML field. Such models include transgenic mice, patient‐derived xenografts, RUNX1‐ETO transduced human progenitor cells, cell lines and human embryonic stem cell model systems, making the t(8;21) as one of the well‐characterized sub‐type of AML at the molecular level.

A G-quadruplex-forming RNA aptamer binds to the MTG8 TAFH domain and dissociates the leukemic AML1-MTG8 fusion protein from DNA.

MTG8 (RUNX1T1) is a fusion partner of AML1 (RUNX1) in the leukemic chromosome translocation t(8;21). The AML1-MTG8 fusion gene encodes a chimeric transcription factor. One of the highly conserved domains of MTG8 is TAFH which possesses homology with human TAF4 [TATA-box binding protein-associated factor]. To obtain specific inhibitors of the AML1-MTG8 fusion protein, we isolated RNA aptamers against the MTG8 TAFH domain using systematic evolution of ligands by exponential enrichment. All TAF aptamers contained guanine-rich sequences. Analyses of a TAF aptamer by NMR, CD, and mutagenesis revealed that it forms a parallel G-quadruplex structure in the presence of K+ . Furthermore, the aptamer could bind to the AML1-MTG8 fusion protein and dissociate the AML1-MTG8/DNA complex, suggesting that it can inhibit the dominant negative effects of AML1-MTG8 against normal AML1 function and serve as a potential therapeutic agent for leukemia.

Abstract 3905: Human liver organoids for disease modeling of fibrolamellar hepatocellular carcinoma

Abstract Fibrolamellar Hepatocellular Carcinoma (FLC) is a usually lethal liver cancer affecting adolescents and young adults without previous liver disease. FLC is characterized by a ~400kb nucleotide deletion resulting in a fusion of a heatshock protein cofactor, DNAJB1, with the catalytic subunit of protein kinase A (PRKACA). One limitation to the study of this disease is the lack of in vitro models. Organoids have shown utility as a model system for studying many diseases, including cancer. This study describes the development and characterization of FLC tumor organoids. With IRB approval, normal liver and FLC tumor tissue was obtained from patients undergoing surgical resection. Liver cells were isolated from the tissue samples and placed into a 3D basement membrane matrix and the media supplemented with growth factors specific to promote liver organoid expansion. Normal and tumor organoids were analyzed by microscopy for morphology, by PCR for presence of the DNAJB1-PRKACA chimeric transcript, by Western blot for chimeric protein expression, and by RNA sequencing for transcriptomic changes. Eight organoid lines have been developed from normal liver and eight from FLC tumor tissue, including multiple independent organoids developed from different metastases from the same patient. A distinct morphological difference between normal liver organoids and FLC tumor organoids can be seen with either brightfield microscopy or with H&E. The DNAJB1-PRKACA chimeric transcript was detected in all of the tumor tissue and tumor organoids by RT-PCR and not detected in any of the normal tissue or organoids. The presence of the DNAJB1-PRKACA fusion protein was verified by Western blot in tumor organoids and tissue but not detectable in the normal tissue. The RNA sequencing analysis shows clustering of tumor organoids with tumor tissue. Tumor organoids injected subcutaneously in mice grew as tumors. Now that the normal and tumor organoids have been developed and validated, they are being used for screening for potential therapeutics. Citation Format: Nicole J. Croteau, David Requena, Gadi Lalazar, Denise Ng, Solomon Levin, Bassem Shebl, Ruisi Wang, William J. Hammond, James A. Saltsman, Helmuth Gehart, Hans Clevers, Michael P. LaQuaglia, Sanford Simon. Human liver organoids for disease modeling of fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3905.

Activation of PLAG1 and HMGA2 by gene fusions involving the transcriptional regulator gene NFIB.

The pleomorphic adenoma (PA), which is the most common salivary gland neoplasm, is a benign tumor characterized by recurrent chromosome rearrangements involving 8q12 and 12q14-15. We have previously shown that the PLAG1 and HMGA2 oncogenes are the targets of these rearrangements. Here, we have identified previously unrecognized subsets of PAs with ins(9;8)/t(8;9) (n = 5) and ins(9;12)/t(9;12) (n = 8) and breakpoints located in the vicinity of the PLAG1 and HMGA2 loci. RNA-sequencing and reverse transcriptase (RT)-PCR analyses of a case with an ins(9;8) revealed a novel NFIB-PLAG1 fusion in which NFIB exon 4 is linked to PLAG1 exon 3. In contrast to the developmentally regulated PLAG1 gene, NFIB was highly expressed in normal salivary gland, indicating that PLAG1 in this case, as in other variant fusions, is activated by promoter swapping. RT-PCR analysis of three PAs with t(9;12) revealed two tumors with chimeric transcripts consisting of HMGA2 exon 4 linked to NFIB exons 9 or 3 and one case with a fusion linking HMGA2 exon 3 to NFIB exon 9. The NFIB fusion events resulted in potent activation of PLAG1 and HMGA2. Analysis of the chromatin landscape surrounding NFIB revealed several super-enhancers in the 5'- and 3'-parts of the NFIB locus and its flanking sequences. These findings indicate that PLAG1 and HMGA2, similar to MYB in adenoid cystic carcinoma, may be activated by enhancer-hijacking events, in which super-enhancers in NFIB are translocated upstream of PLAG1 or downstream of HMGA2. Our results further emphasize the role of NFIB as a fusion partner to multiple oncogenes in histopathologically different types of salivary gland tumors.

Fusion transcript detection using spatial transcriptomics

BackgroundFusion transcripts are involved in tumourigenesis and play a crucial role in tumour heterogeneity, tumour evolution and cancer treatment resistance. However, fusion transcripts have not been studied at high spatial resolution in tissue sections due to the lack of full-length transcripts with spatial information. New high-throughput technologies like spatial transcriptomics measure the transcriptome of tissue sections on almost single-cell level. While this technique does not allow for direct detection of fusion transcripts, we show that they can be inferred using the relative poly(A) tail abundance of the involved parental genes.MethodWe present a new method STfusion, which uses spatial transcriptomics to infer the presence and absence of poly(A) tails. A fusion transcript lacks a poly(A) tail for the 5′ gene and has an elevated number of poly(A) tails for the 3′ gene. Its expression level is defined by the upstream promoter of the 5′ gene. STfusion measures the difference between the observed and expected number of poly(A) tails with a novel C-score.ResultsWe verified the STfusion ability to predict fusion transcripts on HeLa cells with known fusions. STfusion and C-score applied to clinical prostate cancer data revealed the spatial distribution of the cis-SAGe SLC45A3-ELK4 in 12 tissue sections with almost single-cell resolution. The cis-SAGe occurred in disease areas, e.g. inflamed, prostatic intraepithelial neoplastic, or cancerous areas, and occasionally in normal glands.ConclusionsSTfusion detects fusion transcripts in cancer cell line and clinical tissue data, and distinguishes chimeric transcripts from chimeras caused by trans-splicing events. With STfusion and the use of C-scores, fusion transcripts can be spatially localised in clinical tissue sections on almost single cell level.

Abstract PR09: STAG2 mutations alter topologic organization of the genome and cis-mediated interactions

Abstract STAG2 is one of the most recurrently mutated genes in human cancer, including in Ewing sarcoma, an aggressive bone tumor driven by the chimeric EWSR1-FLI1 transcription factor. We and others have shown that STAG2 mutations occurs in approximately 20% of Ewing sarcoma, leading most frequently to complete loss of function (LOF) of its protein. We also showed that STAG2 mutations were associated with poor outcome for these patients. However, functional mechanisms associated with these mutations are still poorly understood. STAG2 encodes an integral member of the cohesin complex, a ring-shaped multiprotein structure, which is important for proper sister chromatid cohesion and release during mitosis. Cohesin, together with CTCF, is also essential to shape the architecture of the genome through its ring structure that allows for chromatin loops formation. Here, we investigated STAG2 and STAG1 functions using isogenic proficient and deficient Ewing sarcoma models. Using extensive characterization of these models by transcriptomics and epigenetics approaches, we demonstrate that STAG2 LOF has a major impact on the expression of genes associated with enhancer features, highlighting STAG2 and EWSR1-FLI1 convergent effects. Using chromatin conformation capture methods, we further show that this transcriptomic modulation results from a STAG2 LOF specific topologic alteration of the genome. This work therefore unravels a new fundamental role of STAG2 in topologic organization of the genome and in gene regulation. This abstract is also being presented as Poster B53. Citation Format: Didier Surdez, Sakina Zaidi, Sandrine Grossetête, Virginie Raynal, Sylvain Baulande, Véronique Hill, Olivier Delattre. STAG2 mutations alter topologic organization of the genome and cis-mediated interactions [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR09.

Molecular profiling and analysis of genetic aberrations aimed at identifying potential therapeutic targets in fibrolamellar carcinoma of the liver.

Fibrolamellar carcinoma (FLC) is a rare primary liver cancer of young adults. A functional chimeric transcript resulting from the in-frame fusion of the DNAJ homolog, subfamily B, member 1 (DNAJB1), and the catalytic subunit of protein kinase A (PRKACA) genes on chromosome 19 is believed to be unique in FLC, with a possible role in pathogenesis, yet with no established therapeutic value. The objective of the current study was to understand the molecular landscape of FLC and to identify potential novel therapeutic targets. Archival fresh, formalin-fixed, paraffin-embedded samples from patients with FLC who prospectively consented to an institutional review board-approved protocol were analyzed using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a next-generation sequencing assay encompassing up to 468 key cancer genes. Custom targeted RNA-Seq was performed in selected patients. Demographics, treatment, and outcome data were collected prospectively. Survival outcomes were estimated and correlated with mutation and/or copy number alterations. A total of 33 tumor samples from 31 patients with FLC were analyzed. The median age of the patients at the time of diagnosis was 18years and approximately 53% were women. The DNAJB1-PRKACA fusion transcript was detected in 100% of patients. In 10 of 31 patients in which MSK-IMPACT did not detect the fusion, its presence was confirmed by targeted RNA-Seq. TERT promoter mutation was the second most common, and was detected in 7 patients. The median follow up was 30months (range, 6-153months). The 3-year overall survival rate was 84% (95% CI, 61%-93%). The DNAJB1-PRKACA fusion transcript is nonspecific and nonsensitive to FLC. Its potential therapeutic value currently is under evaluation. Opportunities currently are under development for therapy that may be driven or related to the DNAJB1-PRKACA fusion transcript or any therapeutic target identified from next-generation sequencing in patients with FLC.

Dysregulation of cancer genes by recurrent intergenic fusions

BackgroundGene fusions have been studied extensively, as frequent drivers of tumorigenesis as well as potential therapeutic targets. In many well-known cases, breakpoints occur at two intragenic positions, leading to in-frame gene-gene fusions that generate chimeric mRNAs. However, fusions often occur with intergenic breakpoints, and the role of such fusions has not been carefully examined.ResultsWe analyze whole-genome sequencing data from 268 patients to catalog gene-intergenic and intergenic-intergenic fusions and characterize their impact. First, we discover that, in contrast to the common assumption, chimeric oncogenic transcripts—such as those involving ETV4, ERG, RSPO3, and PIK3CA—can be generated by gene-intergenic fusions through splicing of the intervening region. Second, we find that over-expression of an upstream or downstream gene by a fusion-mediated repositioning of a regulatory sequence is much more common than previously suspected, with enhancers sometimes located megabases away. We detect a number of recurrent fusions, such as those involving ANO3, RGS9, FUT5, CHI3L1, OR1D4, and LIPG in breast; IGF2 in colon; ETV1 in prostate; and IGF2BP3 and SIX2 in thyroid cancers.ConclusionOur findings elucidate the potential oncogenic function of intergenic fusions and highlight the wide-ranging consequences of structural rearrangements in cancer genomes.

Immunohistochemical Analysis of CD99 and PAX8 in a Series of 15 Molecularly Confirmed Cases of Ewing Sarcoma.

Ewing sarcomas are an uncommon group of malignant neoplasms. A multidisciplinary approach is highly recommended to reach a correct diagnosis, considering the clinical, radiological, and histopathological aspects. Since in up to 90% of cases, the translocation t (11; 22) (q24; q12) occurs resulting in a chimeric fusion transcript EWSR1-FLI-1. The pathologist has several tools in addition to conventional techniques (hematoxylin and eosin), such as immunohistochemistry, which plays a very important role in the differential diagnosis. We present a series of 15 cases of molecularly confirmed ES, in which we found a sensitivity of 100% for CD99 and 80% for PAX8 by immunohistochemistry. This indicates a high sensitivity; however, it is known that both CD99 and PAX8 are also expressed in other tumours. Therefore, molecular confirmation should be performed in all cases.

Fibrolamellar Carcinoma

This liver tumor in a young adult was diagnosed by pathology as fibrolamellar carcinoma. What is different about fibrolamellar carcinoma compared with conventional hepatocellular carcinoma?a.Fibrolamellar carcinoma usually affects patients in their sixth and seventh decadesb.Fibrolamellar carcinoma almost always occurs in a background of liver cirrhosisc.Fibrolamellar carcinoma is strongly associated with DNAJB1-PRKACA oncogenic fusiond.Fibrolamellar carcinoma almost exclusively occurs in females Answer: c. Fibrolamellar carcinoma is strongly associated with DNAJB1-PRKACA oncogenic fusion Fibrolamellar carcinoma is an uncommon malignancy, comprising 1% to 5% of liver cancers. This tumor occurs in younger patients, usually less than 40 years of age, and affects males and females. Conventional hepatocellular carcinoma most often affects older adults. Most conventional hepatocellular carcinomas occur in livers affected by cirrhosis, while fibrolamellar carcinoma occurs in noncirrhotic livers. Fibrolamellar carcinoma is strongly associated with DNAJB1-PRKACA oncogenic fusions, which were also recently reported in intraductal oncocytic neoplasms of the pancreas and bile duct.1Honeyman J.N. Simon E.P. Robine N. et al.Detection of a recurrent DNAJB1-PRKACA chimeric transcript in fibrolamellar hepatocellular carcinoma.Science. 2014; 343: 1010-1014Crossref PubMed Scopus (292) Google Scholar,2Singhi A.D. Wood L.D. Parks E. et al.Recurrent rearrangements in PRKACA and PRKACB in intraductal oncocytic papillary neoplasms of the pancreas and bile duct.Gastroenterology. 2020; 158: 573-582Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar

CICERO: a versatile method for detecting complex and diverse driver fusions using cancer RNA sequencing data

To discover driver fusions beyond canonical exon-to-exon chimeric transcripts, we develop CICERO, a local assembly-based algorithm that integrates RNA-seq read support with extensive annotation for candidate ranking. CICERO outperforms commonly used methods, achieving a 95% detection rate for 184 independently validated driver fusions including internal tandem duplications and other non-canonical events in 170 pediatric cancer transcriptomes. Re-analysis of TCGA glioblastoma RNA-seq unveils previously unreported kinase fusions (KLHL7-BRAF) and a 13% prevalence of EGFR C-terminal truncation. Accessible via standard or cloud-based implementation, CICERO enhances driver fusion detection for research and precision oncology. The CICERO source code is available at https://github.com/stjude/Cicero.

Characterization of NRG1 gene fusion events in solid tumors.

3113 Background: NRG1 fusions are actionable genomic alterations detected across tumor types. The NRG1 gene encode for neuregulin, which serves as a ligand for ERBB3 and ERBB4 receptors and activates downstream signaling through the MAPK and PI3K pathways. Here, we update the detection of NRG1 gene fusions across tumor types and further describe fusion characteristics. Methods: Samples submitted for clinical molecular profiling that included RNA-sequencing (Archer Dx or Caris MI transcriptome) were retrospectively analyzed for NRG1 fusion events. All NRG1 fusions with ≥ 3 junction reads were identified for manual review and for characterization of fusion class, intact functional domains, domain prediction, breakpoints, frame retention and co-occurring alterations by NGS. Results: A total of 82 NRG1 fusion events (0.2% of 44,570) were identified. Among the fusions identified, the distribution across tumor types was as follows: non-small cell lung cancer (NSCLC, 54%), breast cancer (11%), ovarian cancer (7%), pancreatic cancer (7%), cholangiocarcinoma (6%), colorectal cancer (5%), and other (10%). Forty-two unique fusion partners were identified, the most common being CD74 (23%), ATP1B1 (9%), SLC3A2 (7%), RBPMS (6%) and SDC4 (4%). Almost half (47%) of all fusion events are expected to include the transmembrane domain contributed by the NRG1 fusion partner. Lung and pancreatobilliary cancers had the highest rates of transmembrane domain retention from their fusion partners (63.6% and 54.5%, respectively). In all other tumor groups, most fusion partners lacked transmembrane domains. In 15% of cases, the chimeric transcripts are predicted to lead to increased expression of NRG1. The most commonly reported breakpoints in NRG1 occur in exon 6 and exon 2. While fusions with the NRG1 breakpoint at exon 2 retain the immunoglobulin (Ig) domain and all downstream portions (including EGF-like domain), those at exon 6 do not contain the Ig portion and result in shorter chimeric proteins. The breakpoints in all CD74:NRG1 fusions, the most common fusions in NSCLC, occur at exon 5 or 6 and cause truncation of domains upstream of the EGF-like domain. In ATP1B1:NRG1 fusions, the most common fusions in pancreatobilliary cancers, the breakpoints are at exon 1 or 2 and retain the Ig domain. Conclusions: NRG1 fusion products are diverse across tumor types, but the significance of these variations is not clear. The biological and clinical implications of retaining certain domains of NRG1 (such as the Ig domain) and of fusion partners warrants further investigation.

Preferential expression of a HPV genotype in invasive cervical carcinomas infected by multiple genotypes

Multiple infections by HPV genotypes are frequently detected in HPV+ cervical lesions but the interaction between each viral genotype during carcinogenesis is poorly understood. Here we carried out a comprehensive study to characterize the multiple HPV expression and integration by RNA-seq analyses of 19 invasive cervical carcinomas coinfected by several HPV genotypes. Analysis of tumor DNA by a hybridization assay indicated multiple infections ranging from 2 to 6 different HPV genotypes. RNA-seq analysis showed that a single HPV genotype was preferentially expressed. Finally, the search for HPV/human chimeric transcripts indicated integration from preferentially expressed genotypes. In conclusion, the present study indicated that, in invasive cervical carcinomas infected by multiple HPV genotypes, one HPV was preferentially expressed, supporting the hypothesis that a single HPV genotype was associated with cancer development.

Tricking the devil with an engineered protein switch for ex vivo blood cell production

Background & Aim Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells (HSPCs) remains challenging due to induction of rapid cellular differentiation after detachment from the supporting bone marrow stem cell niche. However, a few leukemia-related chimeric transcription factors, including MLL fusion proteins, confer remarkable CD34+ ex vivo expansion capacity. Methods, Results & Conclusion In this study, we fused the coding sequence of a FKBP12-derived destabilization domain (DD) to an MLL-fusion gene (MLL-X) and then retrovirally expressed the protein switch in human CD34+ progenitors derived from healthy donors. In the presence of Shield1, a chemical inhibitor of DD fusion protein turnover, the cytokines FLT3-L and IL-3 were necessary and sufficient to yield massive and long-term expansion of DD-MLL-X engineered HSPC-derived late monocytic precursors, with normal karyotype preserved. Upon removal of Shield1, the cells completely lost self-renewal capacity, lost colony-forming properties and spontaneously differentiated, even after 150 days of ex vivo expansion. In the absence of Shield1, a five-day stimulation with IFNγ, LPS, and GM-CSF triggered robust monocytic differentiation. Immunophenotypic characterization revealed upregulation of the monocytic cell surface markers CD14, CD68, CD80, CCR2, and MHC class I and II, in well-accordance with cellular morphology as judged by cytospin preparations. Upregulation of inflammatory markers such as IL-6, IL-10, and CCL2 on mRNA level was detected by qRT-PCR. In functional assays, we finally demonstrated the propensity of the obtained cells to migrate towards the chemokine CCL2, attach to tissue-culture treated plastic surfaces and VCAM-1 under flow and shear stress, to produce reactive oxygen species and engulf both bacterial and cellular particles. Moreover, we demonstrated Fc receptor recognition and phagocytosis of Raji lymphoblastic tumor cells in a daratumumab antibody-dependent manner, proving their identity as monocytes. Taken together, we demonstrate the conversion of a harmful transcription factor to a useful tool for ex vivo blood cell production. Using this engineered protein switch, we were able to produce HSPC-derived late monocytic progenitors in large-scale and differentiate those towards functional monocytes under well-defined ex vivo conditions, both efficiently and quantitatively. This controllable system could set the stage for directed generation of patient-derived monocytes for cell-based immunotherapeutic approaches.

KDM4A regulates the maternal-to-zygotic transition by protecting broad H3K4me3 domains from H3K9me3 invasion in oocytes.

The importance of germline-inherited posttranslational histone modifications on priming early mammalian development is just emerging1–4. Histone H3 lysine 9 (H3K9) trimethylation is associated with heterochromatin and gene repression during cell-fate change5, while histone H3 lysine 4 (H3K4) trimethylation marks active gene promoters6. Mature oocytes are transcriptionally quiescent and possess remarkably broad domains of H3K4me3 (bdH3K4me3)1,2. It remains unknown as to which factors contribute to the maintenance of the bdH3K4me3 landscape. Lysine-specific demethylase 4A (KDM4A) demethylates H3K9me3 at promoters marked by H3K4me3 in actively transcribing somatic cells7. Here, we report that KDM4A-mediated H3K9me3 demethylation at bdH3K4me3 in oocytes is crucial for normal preimplantation development and zygotic genome activation (ZGA) after fertilization. Loss of KDM4A in oocytes causes aberrant H3K9me3 spreading over bdH3K4me3, resulting in insufficient transcriptional activation of genes, endogenous retroviral elements and long terminal repeat initiated chimeric transcripts during ZGA. The catalytic activity of KDM4A is essential for normal epigenetic reprogramming and preimplantation development. Hence, KDM4A plays a crucial role in preserving maternal epigenome integrity required for proper ZGA and transfer of developmental control to the embryo.

Read-through transcripts in lung: germline genetic regulation and correlation with the expression of other genes.

Transcripts originating from the transcriptional read through of two adjacent, similarly oriented genes have been identified in normal and neoplastic tissues, but their functional role and the mechanisms that regulate their expression are mostly unknown. Here, we investigated whether the expression of read-through transcripts previously identified in the non-involved lung tissue of lung adenocarcinoma patients was genetically regulated. Data on genome-wide single nucleotide variant genotypes and expression levels of 10 read-through transcripts in 201 samples of lung tissue were combined to identify expression quantitative trait loci (eQTLs). Then, to identify genes whose expression levels correlated with the 10 read-through transcripts, we used whole transcriptome profiles available for 154 patients. For 8 read-though transcripts, we identified 60 eQTLs (false discovery rate <0.05), including 17 cis-eQTLs and 43 trans-eQTLs. These eQTLs did not maintain their behavior on the 'parental' genes involved in the read-through transcriptional event. The expression levels of 7 read-through transcripts were found to correlate with the expression of other genes: CHIA-PIFO and CTSC-RAB38 correlated with CHIA and RAB38, respectively, while 5 other read-through transcripts correlated with 43 unique non-parental transcripts; thus offering indications about the molecular processes in which these chimeric transcripts may be involved. We confirmed 9 eQTLs (for 4 transcripts) in the non-involved lung tissue from an independent series of 188 lung adenocarcinoma patients. Therefore, this study indicates that the expression of four read-through transcripts in normal lung tissue is under germline genetic regulation, and that this regulation is independent of that of the genes involved in the read-through event.

Analysis of fusion transcripts indicates widespread deregulation of snoRNAs and their host genes in breast cancer.

Genomic rearrangements in cancer can join the sequences of two separate genes. Studies of such gene fusion events have mainly focused on identification of fusion proteins from the chimeric transcripts. We have previously investigated how fusions instead can affect the expression of intronic microRNA (miRNA) genes that are encoded within fusion gene partners. Here, we extend our analysis to small nucleolar RNAs (snoRNAs) that also are embedded within protein-coding or noncoding host genes. We found that snoRNA hosts are selectively enriched in fusion transcripts, like miRNA host genes, and that this enrichment is associated with all snoRNA classes. These structural changes may have functional consequences for the cell; proteins involved in the protein translation machinery are overrepresented among snoRNA host genes, a gene architecture assumed to be needed for closely coordinated expression of snoRNAs and host proteins. Our data indicate that this structure is frequently disrupted in cancer. We furthermore observed that snoRNA genes involved in fusions tend to associate with stronger promoters than the natural host, suggesting a mechanism that selects for snoRNA overexpression. In summary, we highlight a previously unexplored frequent structural change in cancer that affects important components of cellular physiology.