Jun-Yuan Ji

YAP-dependent induction of amphiregulin identifies a non-cell-autonomous component of the Hippo pathway.

The Hippo signalling pathway regulates cellular proliferation and survival, thus has profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP (yes-associated protein), a transcriptional co-activator amplified in mouse and human cancers, where it promotes epithelial to mesenchymal transition (EMT) and malignant transformation. So far, studies of YAP target genes have focused on cell-autonomous mediators; here we show that YAP-expressing MCF10A breast epithelial cells enhance the proliferation of neighbouring untransfected cells, implicating a non-cell-autonomous mechanism. We identify the gene for the epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) as a transcriptional target of YAP, whose induction contributes to YAP-mediated cell proliferation and migration, but not EMT. Knockdown of AREG or addition of an EGFR kinase inhibitor abrogates the proliferative effects of YAP expression. Suppression of the negative YAP regulators LATS1 and 2 (large tumour suppressor 1 and 2) is sufficient to induce AREG expression, consistent with physiological regulation of AREG by the Hippo pathway. Genetic interaction between the Drosophila YAP orthologue Yorkie and Egfr signalling components supports the link between these two highly conserved signalling pathways. Thus, YAP-dependent secretion of AREG indicates that activation of EGFR signalling is an important non-cell-autonomous effector of the Hippo pathway, which has implications for the regulation of both physiological and malignant cell proliferation.

Conserved role of SIRT1 orthologs in fasting-dependent inhibition of the lipid/cholesterol regulator SREBP

The sterol regulatory element-binding protein (SREBP) transcription factor family is a critical regulator of lipid and sterol homeostasis in eukaryotes. In mammals, SREBPs are highly active in the fed state to promote the expression of lipogenic and cholesterogenic genes and facilitate fat storage. During fasting, SREBP-dependent lipid/cholesterol synthesis is rapidly diminished in the mouse liver; however, the mechanism has remained incompletely understood. Moreover, the evolutionary conservation of fasting regulation of SREBP-dependent programs of gene expression and control of lipid homeostasis has been unclear. We demonstrate here a conserved role for orthologs of the NAD(+)-dependent deacetylase SIRT1 in metazoans in down-regulation of SREBP orthologs during fasting, resulting in inhibition of lipid synthesis and fat storage. Our data reveal that SIRT1 can directly deacetylate SREBP, and modulation of SIRT1 activity results in changes in SREBP ubiquitination, protein stability, and target gene expression. In addition, chemical activators of SIRT1 inhibit SREBP target gene expression in vitro and in vivo, correlating with decreased hepatic lipid and cholesterol levels and attenuated liver steatosis in diet-induced and genetically obese mice. We conclude that SIRT1 orthologs play a critical role in controlling SREBP-dependent gene regulation governing lipid/cholesterol homeostasis in metazoans in response to fasting cues. These findings may have important biomedical implications for the treatment of metabolic disorders associated with aberrant lipid/cholesterol homeostasis, including metabolic syndrome and atherosclerosis.

E2F1 represses β-catenin transcription and is antagonized by both pRB and CDK8

The E2F1 transcription factor can promote proliferation or apoptosis when activated, and is a key downstream target of the retinoblastoma tumour suppressor protein (pRB). Here we show that E2F1 is a potent and specific inhibitor of β-catenin/T-cell factor (TCF)-dependent transcription, and that this function contributes to E2F1-induced apoptosis. E2F1 deregulation suppresses β-catenin activity in an adenomatous polyposis coli (APC)/glycogen synthase kinase-3 (GSK3)-independent manner, reducing the expression of key β-catenin targets including c-MYC. This interaction explains why colorectal tumours, which depend on β-catenin transcription for their abnormal proliferation, keep RB1 intact. Remarkably, E2F1 activity is also repressed by cyclin-dependent kinase-8 (CDK8), a colorectal oncoprotein. Elevated levels of CDK8 protect β-catenin/TCF-dependent transcription from inhibition by E2F1. Thus, by retaining RB1 and amplifying CDK8, colorectal tumour cells select conditions that collectively suppress E2F1 and enhance the activity of β-catenin.

Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9

Significance Using the recently introduced Cas9/sgRNA technique, we have developed a method for specifically targeting Drosophila germ-line cells to generate heritable mutant alleles. We have established transgenic lines that stably express Cas9 in the germ line and compared different promoters and scaffolds of sgRNA in terms of their efficiency of mutagenesis. An overall mutagenesis rate of 74.2% was achieved with this optimized system, as determined by the number of mutant progeny out of all progeny screened. We also evaluated the off-targets associated with the method and established a Web-based resource, as well as a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Our results demonstrate that this optimized Cas9/sgRNA system in Drosophila is efficient, specific, and cost-effective and can be readily applied in a semi-high-throughput manner. The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.

A SIRT1-LSD1 Corepressor Complex Regulates Notch Target Gene Expression and Development

Epigenetic regulation of gene expression by histone-modifying corepressor complexes is central to normal animal development. The NAD(+)-dependent deacetylase and gene repressor SIRT1 removes histone H4K16 acetylation marks and facilitates heterochromatin formation. However, the mechanistic contribution of SIRT1 to epigenetic regulation at euchromatic loci and whether it acts in concert with other chromatin-modifying activities to control developmental gene expression programs remain unclear. We describe here a SIRT1 corepressor complex containing the histone H3K4 demethylase LSD1/KDM1A and several other LSD1-associated proteins. SIRT1 and LSD1 interact directly and play conserved and concerted roles in H4K16 deacetylation and H3K4 demethylation to repress genes regulated by the Notch signaling pathway. Mutations in Drosophila SIRT1 and LSD1 orthologs result in similar developmental phenotypes and genetically interact with the Notch pathway in Drosophila. These findings offer new insights into conserved mechanisms of epigenetic gene repression and regulation of development by SIRT1 in metazoans.

Regulation of lipogenesis by cyclin-dependent kinase 8-mediated control of SREBP-1.

Altered lipid metabolism underlies several major human diseases, including obesity and type 2 diabetes. However, lipid metabolism pathophysiology remains poorly understood at the molecular level. Insulin is the primary stimulator of hepatic lipogenesis through activation of the SREBP-1c transcription factor. Here we identified cyclin-dependent kinase 8 (CDK8) and its regulatory partner cyclin C (CycC) as negative regulators of the lipogenic pathway in Drosophila, mammalian hepatocytes, and mouse liver. The inhibitory effect of CDK8 and CycC on de novo lipogenesis was mediated through CDK8 phosphorylation of nuclear SREBP-1c at a conserved threonine residue. Phosphorylation by CDK8 enhanced SREBP-1c ubiquitination and protein degradation. Importantly, consistent with the physiologic regulation of lipid biosynthesis, CDK8 and CycC proteins were rapidly downregulated by feeding and insulin, resulting in decreased SREBP-1c phosphorylation. Moreover, overexpression of CycC efficiently suppressed insulin and feeding-induced lipogenic gene expression. Taken together, these results demonstrate that CDK8 and CycC function as evolutionarily conserved components of the insulin signaling pathway in regulating lipid homeostasis.

RBF1 promotes chromatin condensation through a conserved interaction with the Condensin II protein dCAP-D3.

The Drosophila retinoblastoma family of proteins (RBF1 and RBF2) and their mammalian homologs (pRB, p130, and p107) are best known for their regulation of the G1/S transition via the repression of E2F-dependent transcription. However, RB family members also possess additional functions. Here, we report that rbf1 mutant larvae have extensive defects in chromatin condensation during mitosis. We describe a novel interaction between RBF1 and dCAP-D3, a non-SMC component of the Condensin II complex that links RBF1 to the regulation of chromosome structure. RBF1 physically interacts with dCAP-D3, RBF1 and dCAP-D3 partially colocalize on polytene chromosomes, and RBF1 is required for efficient association of dCAP-D3 with chromatin. dCap-D3 mutants also exhibit chromatin condensation defects, and mutant alleles of dCap-D3 suppress cellular and developmental phenotypes induced by the overexpression of RBF1. Interestingly, this interaction is conserved between flies and humans. The re-expression of pRB into a pRB-deficient human tumor cell line promotes chromatin association of hCAP-D3 in a manner that depends on the LXCXE-binding cleft of pRB. These results uncover an unexpected link between pRB/RBF1 and chromatin condensation, providing a mechanism by which the functional inactivation of RB family members in human tumor cells may contribute to genome instability.

Mutation of Drosophila Lsd1 Disrupts H3-K4 Methylation, Resulting in Tissue-Specific Defects during Development

Histone-tail modifications play a fundamental role in the processes that establish chromatin structure and determine gene expression. One such modification, histone methylation, was considered irreversible until the recent discovery of histone demethylases. Lsd1 was the first histone demethylase to be identified. Lsd1 is highly conserved in many species, from yeast to humans, but its function has primarily been studied through biochemical approaches. The mammalian ortholog has been shown to demethylate monomethyl- and dimethyl-K4 and -K9 residues of histone H3. Here we describe the effects of Lsd1 mutation in Drosophila. The inactivation of dLsd1 strongly affects the global level of monomethyl- and dimethyl-H3-K4 methylation and results in elevated expression of a subset of genes. dLsd1 is not an essential gene, but animal viability is strongly reduced in mutant animals in a gender-specific manner. Interestingly, dLsd1 mutants are sterile and possess defects in ovary development, indicating that dLsd1 has tissue-specific functions. Mutant alleles of dLsd1 suppress positional-effect variegation, suggesting a disruption of the balance between euchromatin and heterochromatin. Taken together, these results show that dLsd1-mediated H3-K4 demethylation has a significant and specific role in Drosophila development.

Both Cyclin B levels and DNA-replication checkpoint control the early embryonic mitoses in Drosophila

The earliest embryonic mitoses in Drosophila, as in other animals except mammals, are viewed as synchronous and of equal duration. However, we observed that total cell-cycle length steadily increases after cycle 7, solely owing to the extension of interphase. Between cycle 7 and cycle 10, this extension is DNA-replication checkpoint independent, but correlates with the onset of Cyclin B oscillation. In addition, nuclei in the middle of embryos have longer metaphase and shorter anaphase than nuclei at the two polar regions. Interestingly, sister chromatids move faster in anaphase in the middle than the posterior region. These regional differences correlate with local differences in Cyclin B concentration. After cycle 10, interphase and total cycle duration of nuclei in the middle of the embryo are longer than at the poles. Because interphase also extends in checkpoint mutant (grapes) embryo after cycle 10, although less dramatic than wild-type embryos, interphase extension after cycle 10 is probably controlled by both Cyclin B limitation and the DNA-replication checkpoint.

Retinoblastoma family 2 is required in vivo for the tissue-specific repression of dE2F2 target genes.

In higher eukaryotes, the Retinoblastoma and E2F families of proteins control the transcription of a large number of target genes. Here, we have mutated the second Drosophila Retinoblastoma family gene (Rbf2), and contrasted the in vivo molecular functions of RBF2 with dE2F2, the only E2F partner of RBF2. Previous studies failed to uncover a unique role for RBF2 in E2F regulation. Here we find that RBF2 functions in concert with dE2F2 in vivo to repress the expression of differentiation markers in ovaries and embryos where RBF2 is highly expressed. We have compared the profiles of transcripts that are mis-expressed in ovaries, embryos and S2 cells where RBF2 function has been ablated and find that RBF2 and dE2F2 control strikingly different transcriptional programs in each situation. In vivo promoter occupancy studies point to the redistribution of dE2F/RBF complexes to different promoters in different cell types as one mechanism governing the tissue-specific regulation of dE2F/RBF target genes. These results demonstrate that RBF2 has a unique function in repressing E2F-regulated differentiation markers and that dE2F2 and RBF2 are required to regulate different sets of target genes in different tissues.