Xiang-Dong Fu

Regulation of the Hippo-YAP pathway by G-protein coupled receptor signaling

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.

9p21 DNA variants associated with coronary artery disease impair interferon-γ signalling response.

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the 9p21 gene desert associated with coronary artery disease (CAD) and type 2 diabetes. Despite evidence for a role of the associated interval in neighbouring gene regulation, the biological underpinnings of these genetic associations with CAD or type 2 diabetes have not yet been explained. Here we identify 33 enhancers in 9p21; the interval is the second densest gene desert for predicted enhancers and six times denser than the whole genome (P < 6.55 × 10−33). The CAD risk alleles of SNPs rs10811656 and rs10757278 are located in one of these enhancers and disrupt a binding site for STAT1. Lymphoblastoid cell lines homozygous for the CAD risk haplotype show no binding of STAT1, and in lymphoblastoid cell lines homozygous for the CAD non-risk haplotype, binding of STAT1 inhibits CDKN2BAS (also known as CDKN2B-AS1) expression, which is reversed by short interfering RNA knockdown of STAT1. Using a new, open-ended approach to detect long-distance interactions, we find that in human vascular endothelial cells the enhancer interval containing the CAD locus physically interacts with the CDKN2A/B locus, the MTAP gene and an interval downstream of IFNA21. In human vascular endothelial cells, interferon-γ activation strongly affects the structure of the chromatin and the transcriptional regulation in the 9p21 locus, including STAT1-binding, long-range enhancer interactions and altered expression of neighbouring genes. Our findings establish a link between CAD genetic susceptibility and the response to inflammatory signalling in a vascular cell type and thus demonstrate the utility of genome-wide association study findings in directing studies to novel genomic loci and biological processes important for disease aetiology.

Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA

Mammalian genomes are populated with thousands of transcriptional enhancers that orchestrate cell-type-specific gene expression programs, but how those enhancers are exploited to institute alternative, signal-dependent transcriptional responses remains poorly understood. Here we present evidence that cell-lineage-specific factors, such as FoxA1, can simultaneously facilitate and restrict key regulated transcription factors, exemplified by the androgen receptor (AR), to act on structurally and functionally distinct classes of enhancer. Consequently, FoxA1 downregulation, an unfavourable prognostic sign in certain advanced prostate tumours, triggers dramatic reprogramming of the hormonal response by causing a massive switch in AR binding to a distinct cohort of pre-established enhancers. These enhancers are functional, as evidenced by the production of enhancer-templated non-coding RNA (eRNA) based on global nuclear run-on sequencing (GRO-seq) analysis, with a unique class apparently requiring no nucleosome remodelling to induce specific enhancer–promoter looping and gene activation. GRO-seq data also suggest that liganded AR induces both transcription initiation and elongation. Together, these findings reveal a large repository of active enhancers that can be dynamically tuned to elicit alternative gene expression programs, which may underlie many sequential gene expression events in development, cell differentiation and disease progression.

Nuclear receptor-induced chromosomal proximity and DNA breaks underlie specific translocations in cancer.

Chromosomal translocations are a hallmark of leukemia/lymphoma and also appear in solid tumors, but the underlying mechanism remains elusive. By establishing a cellular model that mimics the relative frequency of authentic translocation events without proliferation selection, we report mechanisms of nuclear receptor-dependent tumor translocations. Intronic binding of liganded androgen receptor (AR) first juxtaposes translocation loci by triggering intra- and interchromosomal interactions. AR then promotes site-specific DNA double-stranded breaks (DSBs) at translocation loci by recruiting two types of enzymatic activities induced by genotoxic stress and liganded AR, including activation-induced cytidine deaminase and the LINE-1 repeat-encoded ORF2 endonuclease. These enzymes synergistically generate site-selective DSBs at juxtaposed translocation loci that are ligated by nonhomologous end joining pathway for specific translocations. Our data suggest that the confluence of two parallel pathways initiated by liganded nuclear receptor and genotoxic stress underlies nonrandom tumor translocations, which may function in many types of tumors and pathological processes.

Associations between distinct pre-mRNA splicing components and the cell nucleus

SC‐35 is a non‐snRNP spliceosome component that is specifically recognized by the anti‐spliceosome monoclonal antibody alpha SC‐35. In this paper we provide direct evidence that SC‐35 is an essential splicing factor and we examine the immunolocalization of SC‐35 by confocal laser scanning microscopy and by electron microscopy. We have found that the speckled staining pattern observed by fluorescence microscopy corresponds to structures previously designated as interchromatin granules and perichromatin fibrils. Although snRNP antigens are also concentrated in these nuclear regions, we show that the two types of spliceosome components are localized through different molecular interactions: The distribution of SC‐35 was not affected by treatment with DNase I or RNase A, or when the cells were heat shocked. In contrast, snRNP antigens become diffusely distributed after RNase A digestion or heat shock. Examination of cells at different stages of mitosis revealed that the SC‐35 speckled staining pattern is lost during prophase and speckles containing SC‐35 begin to reform in the cytoplasm of anaphase cells. In contrast, snRNP antigens do not associate with speckled regions until late in telophase. These studies reveal a dynamic pattern of assembly and disassembly of the splicing factor SC‐35 into discrete nuclear structures that colocalize with interchromatin granules and perichromatin fibrils. These subnuclear regions may therefore be nuclear organelles involved in the assembly of spliceosomes, or splicing itself.

An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells

The elucidation of a code for regulated splicing has been a long-standing goal in understanding the control of post-transcriptional gene expression events that are crucial for cell survival, differentiation and development. We decoded functional RNA elements in vivo by constructing an RNA map for the cell type–specific splicing regulator FOX2 (also known as RBM9) via cross-linking immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells. The map identified a large cohort of specific FOX2 targets, many of which are themselves splicing regulators, and comparison between the FOX2 binding profile and validated splicing events revealed a general rule for FOX2-regulated exon inclusion or skipping in a position-dependent manner. These findings suggest that FOX2 functions as a critical regulator of a splicing network, and we further show that FOX2 is important for the survival of human embryonic stem cells.

Opposing LSD1 complexes function in developmental gene activation and repression programmes

Precise control of transcriptional programmes underlying metazoan development is modulated by enzymatically active co-regulatory complexes, coupled with epigenetic strategies. One thing that remains unclear is how specific members of histone modification enzyme families, such as histone methyltransferases and demethylases, are used in vivo to simultaneously orchestrate distinct developmental gene activation and repression programmes. Here, we report that the histone lysine demethylase, LSD1—a component of the CoREST-CtBP co-repressor complex—is required for late cell-lineage determination and differentiation during pituitary organogenesis. LSD1 seems to act primarily on target gene activation programmes, as well as in gene repression programmes, on the basis of recruitment of distinct LSD1-containing co-activator or co-repressor complexes. LSD1-dependent gene repression programmes can be extended late in development with the induced expression of ZEB1, a Krüppel-like repressor that can act as a molecular beacon for recruitment of the LSD1-containing CoREST-CtBP co-repressor complex, causing repression of an additional cohort of genes, such as Gh, which previously required LSD1 for activation. These findings suggest that temporal patterns of expression of specific components of LSD1 complexes modulate gene regulatory programmes in many mammalian organs.

Histone Methylation-Dependent Mechanisms Impose Ligand Dependency for Gene Activation by Nuclear Receptors

Nuclear receptors undergo ligand-dependent conformational changes that are required for corepressor-coactivator exchange, but whether there is an actual requirement for specific epigenetic landmarks to impose ligand dependency for gene activation remains unknown. Here we report an unexpected and general strategy that is based on the requirement for specific cohorts of inhibitory histone methyltransferases (HMTs) to impose gene-specific gatekeeper functions that prevent unliganded nuclear receptors and other classes of regulated transcription factors from binding to their target gene promoters and causing constitutive gene activation in the absence of stimulating signals. This strategy, based at least in part on an HMT-dependent inhibitory histone code, imposes a requirement for specific histone demethylases, including LSD1, to permit ligand- and signal-dependent activation of regulated gene expression. These events link an inhibitory methylation component of the histone code to a broadly used strategy that circumvents pathological constitutive gene induction by physiologically regulated transcription factors.

Genome-wide Analysis of PTB-RNA Interactions Reveals a Strategy Used by the General Splicing Repressor to Modulate Exon Inclusion or Skipping

Recent transcriptome analysis indicates that > 90% of human genes undergo alternative splicing, underscoring the contribution of differential RNA processing to diverse proteomes in higher eukaryotic cells. The polypyrimidine tract-binding protein PTB is a well-characterized splicing repressor, but PTB knockdown causes both exon inclusion and skipping. Genome-wide mapping of PTB-RNA interactions and construction of a functional RNA map now reveal that dominant PTB binding near a competing constitutive splice site generally induces exon inclusion, whereas prevalent binding close to an alternative site often causes exon skipping. This positional effect was further demonstrated by disrupting or creating a PTB-binding site on minigene constructs and testing their responses to PTB knockdown or overexpression. These findings suggest a mechanism for PTB to modulate splice site competition to produce opposite functional consequences, which may be generally applicable to RNA-binding splicing factors to positively or negatively regulate alternative splicing in mammalian cells.

Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration.

Significance The most frequent genetic cause of ALS and frontotemporal degeneration is a hexanucleotide expansion in a noncoding region of the C9orf72 gene. Similar to other repeat expansion diseases, we characterize the hallmark feature of repeat expansion RNA-mediated toxicity: nuclear RNA foci. Remarkably, two distinct sets of foci are found, one containing RNAs transcribed in the sense direction and the other containing antisense RNAs. Antisense oligonucleotides (ASOs) are developed that selectively target sense strand repeat-containing RNAs and reduce sense-oriented foci without affecting overall C9orf72 expression. Importantly, reducing C9orf72 expression does not cause behavioral or pathological changes in mice and induces only a few genome-wide mRNA alterations. These findings establish ASO-mediated degradation of repeat-containing RNAs as a significant therapeutic approach. Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions.