Courtney Onodera

An RNA gene expressed during cortical development evolved rapidly in humans

The developmental and evolutionary mechanisms behind the emergence of human-specific brain features remain largely unknown. However, the recent ability to compare our genome to that of our closest relative, the chimpanzee, provides new avenues to link genetic and phenotypic changes in the evolution of the human brain. We devised a ranking of regions in the human genome that show significant evolutionary acceleration. Here we report that the most dramatic of these ‘human accelerated regions’, HAR1, is part of a novel RNA gene (HAR1F) that is expressed specifically in Cajal–Retzius neurons in the developing human neocortex from 7 to 19 gestational weeks, a crucial period for cortical neuron specification and migration. HAR1F is co-expressed with reelin, a product of Cajal–Retzius neurons that is of fundamental importance in specifying the six-layer structure of the human cortex. HAR1 and the other human accelerated regions provide new candidates in the search for uniquely human biology.

Functional Genomic Analysis of the AUXIN RESPONSE FACTOR Gene Family Members in Arabidopsis thaliana: Unique and Overlapping Functions of ARF7 and ARF19

The AUXIN RESPONSE FACTOR (ARF) gene family products, together with the AUXIN/INDOLE-3-ACETIC ACID proteins, regulate auxin-mediated transcriptional activation/repression. The biological function(s) of most ARFs is poorly understood. Here, we report the identification and characterization of T-DNA insertion lines for 18 of the 23 ARF gene family members in Arabidopsis thaliana. Most of the lines fail to show an obvious growth phenotype except of the previously identified arf2/hss, arf3/ett, arf5/mp, and arf7/nph4 mutants, suggesting that there are functional redundancies among the ARF proteins. Subsequently, we generated double mutants. arf7 arf19 has a strong auxin-related phenotype not observed in the arf7 and arf19 single mutants, including severely impaired lateral root formation and abnormal gravitropism in both hypocotyl and root. Global gene expression analysis revealed that auxin-induced gene expression is severely impaired in the arf7 single and arf7 arf19 double mutants. For example, the expression of several genes, such as those encoding members of LATERAL ORGAN BOUNDARIES domain proteins and AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE, are disrupted in the double mutant. The data suggest that the ARF7 and ARF19 proteins play essential roles in auxin-mediated plant development by regulating both unique and partially overlapping sets of target genes. These observations provide molecular insight into the unique and overlapping functions of ARF gene family members in Arabidopsis.

Functional genomic analysis of the AUXIN/INDOLE-3-ACETIC ACID gene family members in Arabidopsis thaliana

Auxin regulates various aspects of plant growth and development. The AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) genes encode short-lived transcriptional repressors that are targeted by the TRANSPORT INHIBITOR RESPONSE1/AUXIN RECEPTOR F-BOX proteins. The Aux/IAA proteins regulate auxin-mediated gene expression by interacting with members of the AUXIN RESPONSE FACTOR protein family. Aux/IAA function is poorly understood; herein, we report the identification and characterization of insertion mutants in 12 of the 29 Aux/IAA family members. The mutants show no visible developmental defects compared with the wild type. Double or triple mutants of closely related Aux/IAA genes, such as iaa8-1 iaa9-1 or iaa5-1 iaa6-1 iaa19-1, also exhibit wild-type phenotypes. Global gene expression analysis reveals that the molecular phenotypes of auxin-treated and untreated light-grown seedlings are unaffected in the iaa17-6 and iaa5-1 iaa6-1 iaa19-1 mutants. By contrast, similar analysis with the gain-of-function axr3-1/iaa17-1 mutant seedlings reveals dramatic changes in basal and auxin-induced gene expression compared with the wild type. Expression of several type-A ARABIDOPSIS RESPONSE REGULATOR genes and a number of genes involved in cell wall biosynthesis and degradation is repressed in axr3-1/iaa17-1. The data suggest extensive functional redundancy among Aux/IAA gene family members and that enhanced stability of the AXR3/IAA17 protein severely alters the molecular phenotype, resulting in developmental defects.

FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing

Classical approaches to determine structures of noncoding RNA (ncRNA) probed only one RNA at a time with enzymes and chemicals, using gel electrophoresis to identify reactive positions. To accelerate RNA structure inference, we developed fragmentation sequencing (FragSeq), a high-throughput RNA structure probing method that uses high-throughput RNA sequencing of fragments generated by digestion with nuclease P1, which specifically cleaves single-stranded nucleic acids. In experiments probing the entire mouse nuclear transcriptome, we accurately and simultaneously mapped single-stranded RNA regions in multiple ncRNAs with known structure. We probed in two cell types to verify reproducibility. We also identified and experimentally validated structured regions in ncRNAs with, to our knowledge, no previously reported probing data.

Somatic cells regulate maternal mRNA translation and developmental competence of mouse oocytes

Germ cells divide and differentiate in a unique local microenvironment under the control of somatic cells. Signals released in this niche instruct oocyte reentry into the meiotic cell cycle. Once initiated, the progression through meiosis and the associated programme of maternal messenger RNA translation are thought to be cell autonomous. Here we show that translation of a subset of maternal mRNAs critical for embryo development is under the control of somatic cell inputs. Translation of specific maternal transcripts increases in oocytes cultured in association with somatic cells and is sensitive to EGF-like growth factors that act only on the somatic compartment. In mice deficient in amphiregulin, decreased fecundity and oocyte developmental competence is associated with defective translation of a subset of maternal mRNAs. These somatic cell signals that affect translation require activation of the PI(3)K–AKT–mTOR pathway. Thus, mRNA translation depends on somatic cell cues that are essential to reprogramme the oocyte for embryo development.

Targeted next-generation sequencing of pediatric neuro-oncology patients improves diagnosis, identifies pathogenic germline mutations, and directs targeted therapy

Background Molecular profiling is revolutionizing cancer diagnostics and leading to personalized therapeutic approaches. Herein we describe our clinical experience performing targeted sequencing for 31 pediatric neuro-oncology patients. Methods We sequenced 510 cancer-associated genes from tumor and peripheral blood to identify germline and somatic mutations, structural variants, and copy number changes. Results Genomic profiling was performed on 31 patients with tumors including 11 high-grade gliomas, 8 medulloblastomas, 6 low-grade gliomas, 1 embryonal tumor with multilayered rosettes, 1 pineoblastoma, 1 uveal ganglioneuroma, 1 choroid plexus carcinoma, 1 chordoma, and 1 high-grade neuroepithelial tumor. In 25 cases (81%), results impacted patient management by: (i) clarifying diagnosis, (ii) identifying pathogenic germline mutations, or (iii) detecting potentially targetable alterations. The pathologic diagnosis was amended after genomic profiling for 6 patients (19%), including a high-grade glioma to pilocytic astrocytoma, medulloblastoma to pineoblastoma, ependymoma to high-grade glioma, and medulloblastoma to CNS high-grade neuroepithelial tumor with BCOR alteration. Multiple patients had pathogenic germline mutations, many of which were previously unsuspected. Potentially targetable alterations were identified in 19 patients (61%). Additionally, novel likely pathogenic alterations were identified in 3 cases: an in-frame RAF1 fusion in a BRAF wild-type pleomorphic xanthoastrocytoma, an inactivating ASXL1 mutation in a histone H3 wild-type diffuse pontine glioma, and an in-frame deletion within exon 2 of MAP2K1 in a low-grade astrocytic neoplasm. Conclusions Our experience demonstrates the significant impact of molecular profiling on diagnosis and treatment of pediatric brain tumors and confirms its feasibility for use at the time of diagnosis or recurrence.

Chd1 is essential for the high transcriptional output and rapid growth of the mouse epiblast

The pluripotent mammalian epiblast undergoes unusually fast cell proliferation. This rapid growth is expected to generate a high transcriptional demand, but the underlying mechanisms remain unknown. We show here that the chromatin remodeler Chd1 is required for transcriptional output and development of the mouse epiblast. Chd1−/− embryos exhibit proliferation defects and increased apoptosis, are smaller than controls by E5.5 and fail to grow, to become patterned or to gastrulate. Removal of p53 allows progression of Chd1−/− mutants only to E7.0-8.0, highlighting the crucial requirement for Chd1 during early post-implantation development. Chd1−/− embryonic stem cells (ESCs) have a self-renewal defect and a genome-wide reduction in transcriptional output at both known mRNAs and intergenic transcripts. These transcriptional defects were only uncovered when cell number-normalized approaches were used, and correlate with a lower engagement of RNAP II with transcribed genes in Chd1−/− ESCs. We further show that Chd1 directly binds to ribosomal DNA, and that both Chd1−/− epiblast cells in vivo and ESCs in vitro express significantly lower levels of ribosomal RNA. In agreement with these findings, mutant cells in vivo and in vitro exhibit smaller and more elongated nucleoli. Thus, the RNA output by both Pol I and II is reduced in Chd1−/− cells. Our data indicate that Chd1 promotes a globally elevated transcriptional output required to sustain the distinctly rapid growth of the mouse epiblast. Highlighted article: The chromatin remodeler Chd1 promotes a global increase in transcriptional output by both RNA polymerase I and II that sustains the rapid growth of the mouse epiblast.

Genomic profiling of malignant phyllodes tumors reveals aberrations in FGFR1 and PI-3 kinase/RAS signaling pathways and provides insights into intratumoral heterogeneity

Malignant phyllodes tumors of the breast are poorly understood rare neoplasms with potential for aggressive behavior. Few efficacious treatment options exist for progressed or metastatic disease. The molecular features of malignant phyllodes tumors are poorly defined, and a deeper understanding of the genetics of these tumors may shed light on pathogenesis and progression and potentially identify novel treatment approaches. We sequenced 510 cancer-related genes in 10 malignant phyllodes tumors, including 5 tumors with liposarcomatous differentiation and 1 with myxoid chondrosarcoma-like differentiation. Intratumoral heterogeneity was assessed by sequencing two separate areas in 7 tumors, including non-heterologous and heterologous components of tumors with heterologous differentiation. Activating hotspot mutations in FGFR1 were identified in 2 tumors. Additional recurrently mutated genes included TERT promoter (6/10), TP53 (4/10), PIK3CA (3/10), MED12 (3/10), SETD2 (2/10) and KMT2D (2/10). Together, genomic aberrations in FGFR/EGFR PI-3 kinase and RAS pathways were identified in 8 (80%) tumors and included mutually exclusive and potentially actionable activating FGFR1, PIK3CA and BRAF V600E mutations, inactivating TSC2 mutation, EGFR amplification and PTEN loss. Seven (70%) malignant phyllodes tumors harbored TERT aberrations (six promoter mutations, one amplification). For comparison, TERT promoter mutations were identified by Sanger sequencing in 33% borderline (n=12) and no (0%, n=8) benign phyllodes tumors (P=0.391 and P=0.013 vs malignant tumors, respectively). Genetic features specific to liposarcoma, including CDK4/MDM2 amplification, were not identified. Copy number analysis revealed intratumoral heterogeneity and evidence for divergent tumor evolution in malignant phyllodes tumors with and without heterologous differentiation. Tumors with liposarcomatous differentiation revealed more chromosomal aberrations in non-heterologous components compared with liposarcomatous components. EGFR amplification was heterogeneous and present only in the non-heterologous component of one tumor with liposarcomatous differentiation. The results identify novel pathways involved in the pathogenesis of malignant phyllodes tumors, which significantly increase our understanding of tumor biology and have potential clinical impact.

Genomic profiling of breast secretory carcinomas reveals distinct genetics from other breast cancers and similarity to mammary analog secretory carcinomas

Secretory carcinomas of the breast are rare tumors with distinct histologic features, recurrent t(12;15)(p13;q25) translocation resulting in ETV6–NTRK3 gene fusion and indolent clinical behavior. Mammary analog secretory carcinomas arising in other sites are histopathologically similar to the breast tumors and also harbor ETV6–NTRK3 fusions. Breast secretory carcinomas are often triple (estrogen and progesterone receptor, HER2) negative with a basal-like immunophenotype. However, genomic studies are lacking, and whether these tumors share genetic features with other basal and/or triple negative breast cancers is unknown. Aside from shared ETV6–NTRK3 fusions, the genetic relatedness of secretory carcinomas arising in different sites is also uncertain. We immunoprofiled and sequenced 510 cancer-related genes in nine breast secretory carcinomas and six salivary gland mammary analog secretory carcinomas. Immunoprofiles of breast and salivary gland secretory carcinomas were similar. All the tumors showed strong diffuse MUC4 expression (n=15), and SOX10 was positive in all nine breast and in five out of six salivary gland tumors. All breast secretory carcinomas were triple negative or weakly ER-positive, and all tumors at both the sites expressed CK5/6 and/or EGFR, consistent with a basal-like phenotype. Sequencing revealed classic ETV6–NTRK3 fusion genes in all cases, including in carcinoma in situ of one breast tumor. Translocations were reciprocal and balanced in six out of nine breast and three out of six salivary gland tumors and were complex in three others. In contrast to most breast basal carcinomas, the mutational burden of secretory carcinomas was very low, and no additional pathogenic aberrations were identified in genes typically mutated in breast cancer. Five (56%) breast and two (33%) salivary gland tumors had simple genomes without copy number changes; the remainder had very few changes, averaging 1.3 per tumor. The ETV6–NTRK3 derivative chromosome was duplicated in one breast and one salivary gland tumor, and was the only copy number change in the latter. The findings highlight breast secretory carcinoma as a subtype more closely related to mammary analog secretory carcinoma than to basal/triple negative breast cancers of no special type. Lack of pathogenic mutations in common cancer-related genes suggests that ETV6–NTRK3 alone may suffice to drive these tumors and likely helps explain their indolent behavior.

Genomic profiling of malignant peritoneal mesothelioma reveals recurrent alterations in epigenetic regulatory genes BAP1 , SETD2 , and DDX3X

Malignant mesothelioma is a rare cancer that arises from the mesothelial cells that line the pleural cavity and less commonly from the peritoneal lining of the abdomen and pelvis. Most pleural mesotheliomas arise in patients with a history of asbestos exposure, whereas the association of peritoneal mesotheliomas with exposure to asbestos and other potential carcinogens is less clear, suggesting that the genetic alterations that drive malignant peritoneal mesothelioma may be unique from those in pleural mesothelioma. Treatment options for all malignant mesotheliomas are currently limited, with no known targeted therapies available. To better understand the molecular pathogenesis of malignant peritoneal mesothelioma, we sequenced 510 cancer-related genes in 13 patients with malignant mesothelioma arising in the peritoneal cavity. The most frequent genetic alteration was biallelic inactivation of the BAP1 gene, which occurred in 9/13 cases, with an additional two cases demonstrating monoallelic loss of BAP1. All 11 of these cases demonstrated loss of BAP1 nuclear staining by immunohistochemistry, whereas two tumors without BAP1 alteration and all 42 cases of histologic mimics in peritoneum (8 multilocular peritoneal inclusion cyst, 6 well-differentiated papillary mesothelioma of the peritoneum, 16 adenomatoid tumor, and 12 low-grade serous carcinoma of the ovary) demonstrated intact BAP1 nuclear staining. Additional recurrently mutated genes in this cohort of malignant peritoneal mesotheliomas included NF2 (3/13), SETD2 (2/13), and DDX3X (2/13). While these genes are known to be recurrently mutated in pleural mesotheliomas, the frequencies are distinct in peritoneal mesotheliomas, with nearly 85% of peritoneal tumors harboring BAP1 alterations versus only 20–30% of pleural tumors. Together, these findings demonstrate the importance of epigenetic modifiers including BAP1, SETD2, and DDX3X in mesothelial tumorigenesis and suggest opportunities for targeted therapies.