Xuanming Shi

Dual role of Brg chromatin remodeling factor in Sonic hedgehog signaling during neural development

Sonic hedgehog (Shh) signaling plays diverse roles during animal development and adult tissue homeostasis through differential regulation of Gli family transcription factors. Dysregulated Shh signaling activities have been linked to birth defects and tumorigenesis. Here we report that Brg, an ATP-dependent chromatin remodeling factor, has dual functions in regulating Shh target gene expression. Using a Brg conditional deletion in Shh-responding neural progenitors and fibroblasts, we demonstrate that Brg is required both for repression of the basal expression and for the activation of signal-induced transcription of Shh target genes. In developing telencephalons deficient for Brg, Shh target genes were derepressed, whereas Brg-deleted cerebellar granule neuron precursors failed to respond to Shh to increase their proliferation. The repressor function of Brg was mediated through Gli3 and both the repressor and activator functions of Brg appeared to be independent of its ATPase activity. Furthermore, Brg facilitates Gli coactivator histone deacetylase (HDAC) binding to the regulatory regions of Shh target genes, providing a possible mechanism for its positive role in Shh signaling. Our results thus reveal that a complex chromatin regulation mechanism underlies the precise transcription outcomes of Shh signaling and its diverse roles during development.

An epigenetic switch induced by Shh signalling regulates gene activation during development and medulloblastoma growth

The Sonic hedgehog (Shh) signalling pathway plays important roles during development and in cancer. Here we report a Shh-induced epigenetic switch that cooperates with Gli to control transcription outcomes. Before induction, poised Shh target genes are marked by a bivalent chromatin domain containing a repressive histone H3K27me3 mark and an active H3K4me3 mark. Shh activation induces a local switch of epigenetic cofactors from the H3K27 methyltransferase polycomb repressive complex 2 (PRC2) to an H3K27me3 demethylase Jmjd3/Kdm6b-centred coactivator complex. We also find that non-enzymatic activities of Jmjd3 are important and that Jmjd3 recruits the Set1/MLL H3K4 methyltransferase complexes in a Shh-dependent manner to resolve the bivalent domain. In vivo, changes of the bivalent domain accompanied Shh-activated cerebellar progenitor proliferation. Overall, our results reveal a regulatory mechanism that underlies the activation of Shh target genes and provides insight into the causes of various diseases and cancers exhibiting altered Shh signalling.

Lingual antimicrobial peptide and IL-8 expression are oppositely regulated by the antagonistic effects of NF-κB p65 and C/EBPβ in mammary epithelial cells

Pathogen contact induces quickly in Mammary Epithelial Cells (MEC) the expression of the proinflammatory cytokine IL-8 and delayed that of the bactericidal β-defensin LAP. Both genes encoding these factors feature on their proximal promoter a composite NF-κB/CEBP binding site. We compare here in MEC the role of NF-κB and C/EBP factors in regulating basal and pathogen-induced expression of both genes from cattle. Abrogating NF-κB binding to that site by introduction of a single point mutation blocks promoter activity of both genes in reporter gene assays. Chromatin accessibility PCR and Chromatin immunoprecipitation reveal that the chromatin of the resting LAP promoter is tightly packed and NF-κB p50 homodimer binding prevails. Infection results in chromatin decompaction accompanied by predominant recruitment of NF-κB p65 for promoter activation. Overexpression of transcription factors confirms a stimulatory role of NF-κB p65 but also a repressive function of C/EBPβ for LAP promoter activity. These factors reverse roles to control IL-8 expression. NF-κB p65 homodimers already reside on the resting IL-8 promoter and induction recruits NF-κB p50. Overexpression of both NF-κB factors represses the promoter in MEC, but not in HEK293 cells. Inhibitors of NF-κB activation and nuclear recruitment both tremendously increase basal and pathogen stimulated IL-8 mRNA concentrations in MEC. Mutation of the C/EBP-binding site blocks and overexpression of C/EBPβ stimulates IL-8-promoter activity. Thus, the pathogen-induced fast activation of diverse transcription factors acting through a common promoter binding site is gene specifically differentiated into opposite functional significance for swiftly (IL-8) or slowly (LAP) induced genes in MEC.

C/EBP-beta drives expression of the nutritionally regulated promoter IA of the acetyl-CoA carboxylase-alpha gene in cattle

Acetyl-CoA carboxylase-alpha (ACC-alpha) is the rate-limiting enzyme for de novo fatty acid synthesis. Among the four promoters expressing the bovine gene, promoter IA (PIA) is dominantly active and nutritionally regulated in lipogenic tissues. CCAAT/enhancer binding proteins are crucially involved in regulating the activity of this promoter. We examine here, which member of this family of transcription factors is most important for promoter activation. To differentiate the individual contribution of different members of the C/EBP family transcription factors controlling the ACC-alpha gene expression in cattle, we established vectors expressing full length (FL) or N-terminally truncated (DeltaN) variants of the C/EBP factors (alpha, -beta, -delta, and -epsilon) in mammalian cells. Using nuclear extracts of cells expressing the DeltaN-C/EBP factors we determined in electrophoretic mobility shift assays that C/EBPalpha, -beta and -epsilon, but not C/EBPdelta may directly bind to the cognate C/EBP-binding site in the immediate ACC-alpha PIA. Co-transfection analyses of the various FL-C/EBP expression vectors together with a reporter gene driven by the ACC-alpha-PIA promoter demonstrated that C/EBPbeta has the strongest activation potential. Hence, activity of this factor may be a key regulator of ACC-alpha-expression in lipogenic tissues.

Interaction of C/EBP-beta and NF-Y factors constrains activity levels of the nutritionally controlled promoter IA expressing the acetyl-CoA carboxylase-alpha gene in cattle

BackgroundThe enzyme acetyl-CoA carboxylase-alpha (ACC-α) is rate limiting for de novo fatty acid synthesis. Among the four promoters expressing the bovine gene, promoter IA (PIA) is dominantly active in lipogenic tissues. This promoter is in principal repressed but activated under favorable nutritional conditions. Previous analyses already coarsely delineated the repressive elements on the distal promoter but did not resolve the molecular nature of the repressor. Knowledge about the molecular functioning of this repressor is fundamental to understanding the nutrition mediated regulation of PIA activity. We analyzed here the molecular mechanism calibrating PIA activity.ResultsWe finely mapped the repressor binding sites in reporter gene assays and demonstrate together with Electrophoretic Mobility Shift Assays that nuclear factor-Y (NF-Y) and CCAAT/enhancer binding protein-β(C/EBPβ) each separately repress PIA activity by binding to their cognate low affinity sites, located on distal elements of the promoter. Simultaneous binding of both factors results in strongest repression. Paradoxically, over expression of NFY factors, but also - and even more so - of C/EBPβ significantly activated the promoter when bound to high affinity sites on the proximal promoter. However, co-transfection experiments revealed that NF-Y may eventually diminish the strong stimulatory effect of C/EBPβ at the proximal PIA in a dose dependent fashion. We validated by chromatin immunoprecipitation, that NF-Y and C/EBP factors may physically interact.ConclusionThe proximal promoter segment of PIA appears to be principally in an active state, since even minute concentrations of both, NF-Y and C/EBPβ factors can saturate the high affinity activator sites. Higher factor concentrations will saturate the low affinity repressive sites on the distal promoter resulting in reduced and calibrated promoter activity. Based on measurements of the mRNA concentrations of those factors in different tissues we propose that the interplay of both factors may set tissue-specific limits for PIA activity.

Autism-Associated Chromatin Regulator Brg1/SmarcA4 Is Required for Synapse Development and Myocyte Enhancer Factor 2-Mediated Synapse Remodeling

ABSTRACT Synapse development requires normal neuronal activities and the precise expression of synapse-related genes. Dysregulation of synaptic genes results in neurological diseases such as autism spectrum disorders (ASD). Mutations in genes encoding chromatin-remodeling factor Brg1/SmarcA4 and its associated proteins are the genetic causes of several developmental diseases with neurological defects and autistic symptoms. Recent large-scale genomic studies predicted Brg1/SmarcA4 as one of the key nodes of the ASD gene network. We report that Brg1 deletion in early postnatal hippocampal neurons led to reduced dendritic spine density and maturation and impaired synapse activities. In developing mice, neuronal Brg1 deletion caused severe neurological defects. Gene expression analyses indicated that Brg1 regulates a significant number of genes known to be involved in synapse function and implicated in ASD. We found that Brg1 is required for dendritic spine/synapse elimination mediated by the ASD-associated transcription factor myocyte enhancer factor 2 (MEF2) and that Brg1 regulates the activity-induced expression of a specific subset of genes that overlap significantly with the targets of MEF2. Our analyses showed that Brg1 interacts with MEF2 and that MEF2 is required for Brg1 recruitment to target genes in response to neuron activation. Thus, Brg1 plays important roles in both synapse development/maturation and MEF2-mediated synapse remodeling. Our study reveals specific functions of the epigenetic regulator Brg1 in synapse development and provides insights into its role in neurological diseases such as ASD.

SMARCA4/Brg1 coordinates genetic and epigenetic networks underlying Shh-type medulloblastoma development

Recent large-scale genomic studies have classified medulloblastoma into four subtypes: Wnt, Shh, Group 3 and Group 4. Each is characterized by specific mutations and distinct epigenetic states. Previously, we showed that a chromatin regulator SMARCA4/Brg1 is required for Gli-mediated transcription activation in Sonic hedgehog (Shh) signaling. We report here that Brg1 controls a transcriptional program that specifically regulates Shh-type medulloblastoma growth. Using a mouse model of Shh-type medulloblastoma, we deleted Brg1 in precancerous progenitors and primary or transplanted tumors. Brg1 deletion significantly inhibited tumor formation and progression. Genome-wide expression analyses and binding experiments indicate that Brg1 specifically coordinates with key transcription factors including Gli1, Atoh1 and REST to regulate the expression of both oncogenes and tumor suppressors that are required for medulloblastoma identity and proliferation. Shh-type medulloblastoma displays distinct H3K27me3 properties. We demonstrate that Brg1 modulates activities of H3K27me3 modifiers to regulate the expression of medulloblastoma genes. Brg1-regulated pathways are conserved in human Shh-type medulloblastoma, and Brg1 is important for the growth of a human medulloblastoma cell line. Thus, Brg1 coordinates a genetic and epigenetic network that regulates the transcriptional program underlying the Shh-type medulloblastoma development.

Regulation of Gli ciliary localization and Hedgehog signaling by the PY-NLS/karyopherin-β2 nuclear import system

Hedgehog (Hh) signaling in vertebrates depends on primary cilia. Upon stimulation, Hh pathway components, including Gli transcription factors, accumulate at primary cilia to transduce the Hh signal, but the mechanisms underlying their ciliary targeting remains largely unknown. Here, we show that the PY-type nuclear localization signal (PY-NLS)/karyopherinβ2 (Kapβ2) nuclear import system regulates Gli ciliary localization and Hh pathway activation. Mutating the PY-NLS in Gli or knockdown of Kapβ2 diminished Gli ciliary localization. Kapβ2 is required for the formation of Gli activator (GliA) in wild-type but not in Sufu mutant cells. Knockdown of Kapβ2 affected Hh signaling in zebrafish embryos, as well as in vitro cultured cerebellum granule neuron progenitors (CGNPs) and SmoM2-driven medulloblastoma cells. Furthermore, Kapβ2 depletion impaired the growth of cultured medulloblastoma cells, which was rescued by Gli overexpression. Interestingly, Kapβ2 is a transcriptional target of the Hh pathway, thus forming a positive feedback loop for Gli activation. Our study unravels the molecular mechanism and cellular machinery regulating Gli ciliary localization and identifies Kapβ2 as a critical regulator of the Hh pathway and a potential drug target for Hh-driven cancers.

SMARCA4/Brg1 coordinates genetic and epigenetic networks underlying Shh type medulloblastoma development

T classification of B-cell and T-cell non-Hodgkin lymphoma has changed considerably over the last several decades. The currently used World Health Organization (WHO) classification system has a broader consensus among the clinical and biomedical community. However, there are still several challenges in regards to the understanding of tumor biology, clinical outcome and diagnostic accuracy in certain subtypes of lymphomas such as peripheral T-cell lymphoma (PTCL), where the diagnosis is frequently challenging even among expert hematopathologists and often time’s assessment requires additional molecular testing. Recently genome-wide high throughput techniques have greatly improved our understanding of B and T-cell lymphomas. This novel genetic information has not only aided in diagnosis, but has also revealed a landscape of critical molecular events that determine the biological and clinical behavior of a lymphoma. In this presentation, I will summarize the genetic characteristics of major subtypes of B-cell and T-cell lymphomas including diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt lymphoma (BL), and mantle cell lymphoma (MCL) and common subtypes of PTCL including angioimmunoblastic T-cell lymphoma (AITL), anaplastic T-cell lymphoma (ALCL), adult T-cell leukemia/lymphoma (ATLL) and extra-nodal NK/T cell lymphoma (ENKTL), and how can these improve precision in diagnosis and inform prognosis.Session Chair: Yet to be finalized Title: Identifying chromatin structures and the underlying novel mutations that predict therapeutic responses to epigenetic drugs in MDS and AML Jason X Cheng, University of Chicago, USA Title: Clinical applications of immunoglobulin expression in acute myeloid leukemia C Cameron Yin, University of Texas MD Anderson Cancer Center, USA Title: MYC/BCL2 double hit lymphoma Shaoying Li, University of Texas MD Anderson Cancer Center, USA Day 1 May 09, 2016C laboratories are rapidly implementing next-generation sequencing (NGS) tests for mutation analysis, however there are few guidelines regarding sample quality for successful results. We aimed to establish tissue quality parameters for successful NGS in solid tumors and to improve NGS performance. Using a 50-gene hotspot mutation panel we identified the major cause for unsuccessful NGS analysis being DNA 10 mm2. Independent factors leading to lower NGS success were cellular tumor areas and decalcification procedures. Tumor type and paraffin block age did not affect success. We optimized workflow and showed improved NGS success rates with pronounced improvement among tiny samples and cytology samples. Identifying preanalytical tissue factors allows us to improve NGS performance and to successfully test tumors obtained from minimally invasive procedures.

A positive feedback loop between Gli1 and tyrosine kinase Hck amplifies shh signaling activities in medulloblastoma

Sonic hedgehog (Shh) signaling is critical during normal development, and the abnormal activation of the Shh pathway is involved in many human cancers. As a target gene of the Shh pathway and as a transcription activator downstream of Shh signaling, Gli1 autoregulates and increases Shh signaling output. Gli1 is one of the key oncogenic factors in Shh-induced tumors such as medulloblastoma. Gli1 is posttranslationally modified, but the nature of the active form of Gli1 was unclear. Here we identified a Src family kinase Hck as a novel activator of Gli1. In Shh-responsive NIH3T3 cells, Hck interacts with Gli1 and phosphorylates multiple tyrosine residues in Gli1. Gli1-mediated target gene activation was significantly enhanced by Hck with both kinase activity-dependent and -independent mechanisms. We provide evidence showing that Hck disrupts the interaction between Gli1 and its inhibitor Sufu. In both NIH3T3 cells and cerebellum granule neuron precursors, the Hck gene is also a direct target of Gli1. Therefore, Gli1 and Hck form a positive feedback loop that amplifies Shh signaling transcription outcomes. In Shh-induced medulloblastoma, Hck is highly expressed and Gli1 is tyrosine phosphorylated, which may enhance the tumorigenic effects of the Gli1 oncogene. RNAi-mediated inhibition of Hck expression significantly repressed medulloblastoma cell growth. In summary, a novel positive feedback loop contributes to maximal Gli1 oncogenic activities in Shh-induced tumors such as medulloblastoma.