Jianhong Ou

The BRAF Oncoprotein Functions through the Transcriptional Repressor MAFG to Mediate the CpG Island Methylator Phenotype

Most colorectal cancers (CRCs) containing activated BRAF (BRAF[V600E]) have a CpG island methylator phenotype (CIMP) characterized by aberrant hypermethylation of many genes, including the mismatch repair gene MLH1. MLH1 silencing results in microsatellite instability and a hypermutable phenotype. Through an RNAi screen, here we identify the transcriptional repressor MAFG as the pivotal factor required for MLH1 silencing and CIMP in CRCs containing BRAF(V600E). In BRAF-positive human CRC cell lines and tumors, MAFG is bound at the promoters of MLH1 and other CIMP genes, and recruits a corepressor complex that includes its heterodimeric partner BACH1, the chromatin remodeling factor CHD8, and the DNA methyltransferase DNMT3B, resulting in hypermethylation and transcriptional silencing. BRAF(V600E) increases BRAF/MEK/ERK signaling resulting in phosphorylation and elevated levels of MAFG, which drives DNA binding. Analysis of transcriptionally silenced CIMP genes in KRAS-positive CRCs indicates that different oncoproteins direct the assembly of distinct repressor complexes on common promoters.

Tead and AP1 Coordinate Transcription and Motility

The Tead family transcription factors are the major intracellular mediators of the Hippo-Yap pathway. Despite the importance of Hippo signaling in tumorigenesis, Tead-dependent downstream oncogenic programs and target genes in cancer cells remain poorly understood. Here, we characterize Tead4-mediated transcriptional networks in a diverse range of cancer cells, including neuroblastoma, colorectal, lung, and endometrial carcinomas. By intersecting genome-wide chromatin occupancy analyses of Tead4, JunD, and Fra1/2, we find that Tead4 cooperates with AP1 transcription factors to coordinate target gene transcription. We find that Tead-AP1 interaction is JNK independent but engages the SRC1-3 co-activators to promote downstream transcription. Furthermore, we show that Tead-AP1 cooperation regulates the activity of the Dock-Rac/CDC42 module and drives the expression of a unique core set of target genes, thereby directing cell migration and invasion. Together, our data unveil a critical regulatory mechanism underlying Tead- and AP1-controlled transcriptional and functional outputs in cancer cells.

miR-146a promotes the initiation and progression of melanoma by activating Notch signaling

Oncogenic mutations in BRAF and NRAS occur in 70% of melanomas. In this study, we identify a microRNA, miR-146a, that is highly upregulated by oncogenic BRAF and NRAS. Expression of miR-146a increases the ability of human melanoma cells to proliferate in culture and form tumors in mice, whereas knockdown of miR-146a has the opposite effects. We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling. Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164). We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C. Analysis of melanoma cell lines and matched patient samples indicates that during melanoma progression pre-miR-146a/G is enriched relative to pre-miR-146a/C, resulting from a C-to-G somatic mutation in pre-miR-146a/C. Collectively, our results reveal a central role for miR-146a in the initiation and progression of melanoma. DOI: http://dx.doi.org/10.7554/eLife.01460.001

Global analysis of Drosophila Cys2-His2 zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants

Cys2-His2 zinc finger proteins (ZFPs) are the largest group of transcription factors in higher metazoans. A complete characterization of these ZFPs and their associated target sequences is pivotal to fully annotate transcriptional regulatory networks in metazoan genomes. As a first step in this process, we have characterized the DNA-binding specificities of 129 zinc finger sets from Drosophila using a bacterial one-hybrid system. This data set contains the DNA-binding specificities for at least one encoded ZFP from 70 unique genes and 23 alternate splice isoforms representing the largest set of characterized ZFPs from any organism described to date. These recognition motifs can be used to predict genomic binding sites for these factors within the fruit fly genome. Subsets of fingers from these ZFPs were characterized to define their orientation and register on their recognition sequences, thereby allowing us to define the recognition diversity within this finger set. We find that the characterized fingers can specify 47 of the 64 possible DNA triplets. To confirm the utility of our finger recognition models, we employed subsets of Drosophila fingers in combination with an existing archive of artificial zinc finger modules to create ZFPs with novel DNA-binding specificity. These hybrids of natural and artificial fingers can be used to create functional zinc finger nucleases for editing vertebrate genomes.

TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein

The TRIM37 (also known as MUL) gene is located in the 17q23 chromosomal region, which is amplified in up to ∼40% of breast cancers. TRIM37 contains a RING finger domain, a hallmark of E3 ubiquitin ligases, but its protein substrate(s) is unknown. Here we report that TRIM37 mono-ubiquitinates histone H2A, a chromatin modification associated with transcriptional repression. We find that in human breast cancer cell lines containing amplified 17q23, TRIM37 is upregulated and, reciprocally, the major H2A ubiquitin ligase RNF2 (also known as RING1B) is downregulated. Genome-wide chromatin immunoprecipitation (ChIP)-chip experiments in 17q23-amplified breast cancer cells identified many genes, including multiple tumour suppressors, whose promoters were bound by TRIM37 and enriched for ubiquitinated H2A. However, unlike RNF2, which is a subunit of polycomb repressive complex 1 (PRC1), we find that TRIM37 associates with polycomb repressive complex 2 (PRC2). TRIM37, PRC2 and PRC1 are co-bound to specific target genes, resulting in their transcriptional silencing. RNA-interference-mediated knockdown of TRIM37 results in loss of ubiquitinated H2A, dissociation of PRC1 and PRC2 from target promoters, and transcriptional reactivation of silenced genes. Knockdown of TRIM37 in human breast cancer cells containing amplified 17q23 substantially decreases tumour growth in mouse xenografts. Conversely, ectopic expression of TRIM37 renders non-transformed cells tumorigenic. Collectively, our results reveal TRIM37 as an oncogenic H2A ubiquitin ligase that is overexpressed in a subset of breast cancers and promotes transformation by facilitating silencing of tumour suppressors and other genes.

A therapeutically targetable mechanism of BCR-ABL–independent imatinib resistance in chronic myeloid leukemia

A large-scale RNA interference screen reveals a new mechanism of imatinib resistance in chronic myeloid leukemia that can be therapeutically targeted. An Irresistible Combination Unlike most cancers, which have variable mutation patterns, chronic myeloid leukemia is normally associated with a specific genetic alteration, which produces a fusion protein called BCR-ABL. Famously, this protein was targeted with the first cancer-specific drug, known as imatinib (Gleevec), which is still the standard therapy for this cancer. Unfortunately, leukemia cells can develop resistance to imatinib, which does not always require mutations in BCR-ABL. Now, Ma et al. have identified a mechanism for imatinib resistance in chronic myeloid leukemia cells that lack mutations in BCR-ABL. The authors also demonstrated that a U.S. Food and Drug Administration–approved drug called trametinib can overcome this resistance and kill leukemia stem cells without harming the nonmalignant precursors that give rise to normal blood cells. Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) poses a major problem for the treatment of chronic myeloid leukemia (CML). IM resistance often results from a secondary mutation in BCR-ABL that interferes with drug binding. However, in many instances, there is no mutation in BCR-ABL, and the basis of such BCR-ABL–independent IM resistance remains to be elucidated. To gain insight into BCR-ABL–independent IM resistance mechanisms, we performed a large-scale RNA interference screen and identified IM-sensitizing genes (IMSGs) whose knockdown renders BCR-ABL+ cells IM-resistant. In these IMSG knockdown cells, RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) signaling is sustained after IM treatment because of up-regulation of PRKCH, which encodes the protein kinase C (PKC) family member PKCη, an activator of CRAF. PRKCH is also up-regulated in samples from CML patients with BCR-ABL–independent IM resistance. Combined treatment with IM and trametinib, a U.S. Food and Drug Administration–approved MEK inhibitor, synergistically kills BCR-ABL+ IMSG knockdown cells and prolongs survival in mouse models of BCR-ABL–independent IM-resistant CML. Finally, we showed that CML stem cells contain high levels of PRKCH, and this contributes to their intrinsic IM resistance. Combined treatment with IM and trametinib synergistically kills CML stem cells with negligible effect on normal hematopoietic stem cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL–independent IM resistance in CML and CML stem cells.

Genetic and pharmacological reactivation of the mammalian inactive X chromosome

Significance In somatic cells of female mammals, one of the two X chromosomes is randomly silenced, a phenomenon called X-chromosome inactivation (XCI). XCI is initiated in cis by a noncoding RNA called Xist, but trans-acting factors that mediate XCI remain largely unknown. In this study, we perform a large-scale RNA interference screen and identify new trans-acting factors that are required for mammalian XCI. Chemical inhibitors of some of these factors can reversibly reactivate the inactive X chromosome. Our results have therapeutic implications for certain human diseases, in particular the neurodevelopmental disorder Rett syndrome, which is caused by loss-of-function mutations in the X-linked MECP2 gene. Reactivation of the silenced wild-type MECP2 allele is a potential strategy for treating the disease. X-chromosome inactivation (XCI), the random transcriptional silencing of one X chromosome in somatic cells of female mammals, is a mechanism that ensures equal expression of X-linked genes in both sexes. XCI is initiated in cis by the noncoding Xist RNA, which coats the inactive X chromosome (Xi) from which it is produced. However, trans-acting factors that mediate XCI remain largely unknown. Here, we perform a large-scale RNA interference screen to identify trans-acting XCI factors (XCIFs) that comprise regulators of cell signaling and transcription, including the DNA methyltransferase, DNMT1. The expression pattern of the XCIFs explains the selective onset of XCI following differentiation. The XCIFs function, at least in part, by promoting expression and/or localization of Xist to the Xi. Surprisingly, we find that DNMT1, which is generally a transcriptional repressor, is an activator of Xist transcription. Small-molecule inhibitors of two of the XCIFs can reversibly reactivate the Xi, which has implications for treatment of Rett syndrome and other dominant X-linked diseases. A homozygous mouse knockout of one of the XCIFs, stanniocalcin 1 (STC1), has an expected XCI defect but surprisingly is phenotypically normal. Remarkably, X-linked genes are not overexpressed in female Stc1−/− mice, revealing the existence of a mechanism(s) that can compensate for a persistent XCI deficiency to regulate X-linked gene expression.

Transcription factor ATF5 is required for terminal differentiation and survival of olfactory sensory neurons

Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element-binding family of transcription factors, which compose a large group of basic region leucine zipper proteins whose members mediate diverse transcriptional regulatory functions. ATF5 has a well-established prosurvival activity and has been found to be overexpressed in several human cancers, in particular glioblastoma. However, the role(s) of ATF5 in development and normal physiology are unknown. Here we address this issue by deriving and characterizing homozygous Atf5 knockout mice. We find that Atf5−/− pups die neonatally, which, as explained below, is consistent with an olfactory defect resulting in a competitive suckling deficit. We show that Atf5 is highly expressed in olfactory sensory neurons (OSNs) in the main olfactory epithelium starting from embryonic stage 11.5 through adulthood. Immunostaining experiments with OSN-specific markers reveal that ATF5 is expressed in some immature OSNs and in all mature OSNs. Expression profiling and immunostaining experiments indicate that loss of Atf5 leads to a massive reduction in mature OSNs resulting from a differentiation defect and the induction of apoptosis. Ectopic expression of Atf5 in neural progenitor cells induces expression of multiple OSN-specific genes. Collectively, our results suggest a model in which Atf5 is first expressed in immature OSNs and the resultant ATF5 functions to promote differentiation into mature OSNs. Thus, ATF5 is required for terminal differentiation and survival of OSNs.

Jmjd3-Mediated H3K27me3 Dynamics Orchestrate Brown Fat Development and Regulate White Fat Plasticity

Progression from brown preadipocytes to adipocytes engages two transcriptional programs: the expression of adipogenic genes common to both brown fat (BAT) and white fat (WAT), and the expression of BAT-selective genes. However, the dynamics of chromatin states and epigenetic enzymes involved remain poorly understood. Here we show that BAT development is selectively marked and guided by repressive H3K27me3 and is executed by its demethylase Jmjd3. We find that a significant subset of BAT-selective genes, but not common fat genes or WAT-selective genes, are demarcated by H3K27me3 in both brown and white preadipocytes. Jmjd3-catalyzed removal of H3K27me3, in part through Rreb1-mediated recruitment, is required for expression of BAT-selective genes and for development of beige adipocytes both in vitro and in vivo. Moreover, gain- and loss-of-function Jmjd3 transgenic mice show age-dependent body weight reduction and cold intolerance, respectively. Together, we identify an epigenetic mechanism governing BAT fate determination and WAT plasticity.

CRISPR-Cas9-mediated saturated mutagenesis screen predicts clinical drug resistance with improved accuracy

Significance Many therapeutic strategies are hampered by the development of drug resistance. High-throughput random mutagenesis screens represent a promising approach for identifying mutations that lead to drug resistance, but have often identified more resistant mutations than are observed in patients, raising questions of their clinical significance. We developed an improved high-throughput mutagenesis screening approach that uses CRISPR-Cas9–based genome editing to generate comprehensive libraries of point mutations at a defined genomic location and systematically study their effect on cell growth. As proof-of-concept, we show our approach accurately predicts the clinical prevalence of drug-resistant mutations in the oncogene BCR-ABL. Our approach can be broadly applied to a variety of oncogenes and represents a new strategy for evaluating resistance susceptibility during drug development. Developing tools to accurately predict the clinical prevalence of drug-resistant mutations is a key step toward generating more effective therapeutics. Here we describe a high-throughput CRISPR-Cas9–based saturated mutagenesis approach to generate comprehensive libraries of point mutations at a defined genomic location and systematically study their effect on cell growth. As proof of concept, we mutagenized a selected region within the leukemic oncogene BCR-ABL1. Using bulk competitions with a deep-sequencing readout, we analyzed hundreds of mutations under multiple drug conditions and found that the effects of mutations on growth in the presence or absence of drug were critical for predicting clinically relevant resistant mutations, many of which were cancer adaptive in the absence of drug pressure. Using this approach, we identified all clinically isolated BCR-ABL1 mutations and achieved a prediction score that correlated highly with their clinical prevalence. The strategy described here can be broadly applied to a variety of oncogenes to predict patient mutations and evaluate resistance susceptibility in the development of new therapeutics.