Li-Wei Chang

Target Selectivity of Vertebrate Notch Proteins COLLABORATION BETWEEN DISCRETE DOMAINS AND CSL-BINDING SITE ARCHITECTURE DETERMINES ACTIVATION PROBABILITY

All four mammalian Notch proteins interact with a single DNA-binding protein (RBP-jκ), yet they are not equivalent in activating target genes. Parallel assays of three Notch-responsive promoters in several cell lines revealed that relative activation strength is dependent on protein module and promoter context more than the cellular context. Each Notch protein reads binding site orientation and distribution on the promoter differently; Notch1 performs extremely well on paired sites, and Notch3 prefers single sites in conjunction with a proximal zinc finger transcription factor. Although head-head sites can elicit a Notch response on their own, use of CBS (CSL binding site) in tail-tail orientation is context-dependent. Bias for specific DNA elements is achieved by interplay between the N-terminal RAM (RBP-jκ-associated molecule/ankyrin region), which interprets CBS proximity and orientation, and the C-terminal transactivation domain that interacts specifically with the transcription machinery or nearby factors. To confirm the prediction that modular design underscores the evolution of functional divergence between Notch proteins, we generated a synthetic Notch protein (Notch1 ankyrin with Notch3 transactivation domain) that displayed superior signaling strength on the hes5 promoter. Consistent with the prediction that “preferred” targets (Hes1) should respond faster and at lower Notch concentration than other targets, we showed that Hes5-GFP was extinguished fast and recovered slowly, whereas Hes1-GFP was inhibited late and recovered quickly after a pulse of DAPT in metanephroi cultures.

High throughput digital quantification of mRNA abundance in primary human acute myeloid leukemia samples

Acute promyelocytic leukemia (APL) is characterized by the t(15;17) chromosomal translocation, which results in fusion of the retinoic acid receptor alpha (RARA) gene to another gene, most commonly promyelocytic leukemia (PML). The resulting fusion protein, PML-RARA, initiates APL, which is a subtype (M3) of acute myeloid leukemia (AML). In this report, we identify a gene expression signature that is specific to M3 samples; it was not found in other AML subtypes and did not simply represent the normal gene expression pattern of primary promyelocytes. To validate this signature for a large number of genes, we tested a recently developed high throughput digital technology (NanoString nCounter). Nearly all of the genes tested demonstrated highly significant concordance with our microarray data (P < 0.05). The validated gene signature reliably identified M3 samples in 2 other AML datasets, and the validated genes were substantially enriched in our mouse model of APL, but not in a cell line that inducibly expressed PML-RARA. These results demonstrate that nCounter is a highly reproducible, customizable system for mRNA quantification using limited amounts of clinical material, which provides a valuable tool for biomarker measurement in low-abundance patient samples.

MicroRNAs Modulate Schwann Cell Response to Nerve Injury by Reinforcing Transcriptional Silencing of Dedifferentiation-Related Genes

In the peripheral nervous system, Schwann cells (SCs) surrounding damaged axons undergo an injury response that is driven by an intricate transcriptional program and is critical for nerve regeneration. To examine whether these injury-induced changes in SCs are also regulated posttranscriptionally by miRNAs, we performed miRNA expression profiling of mouse sciatic nerve distal segment after crush injury. We also characterized the SC injury response in mice containing SCs with disrupted miRNA processing due to loss of Dicer. We identified 87 miRNAs that were expressed in mouse adult peripheral nerve, 48 of which were dynamically regulated after nerve injury. Most of these injury-regulated SC miRNAs were computationally predicted to inhibit drivers of SC dedifferentiation/proliferation and thereby re-enforce the transcriptional program driving SC remyelination. SCs deficient in miRNAs manifested a delay in the transition between the distinct differentiation states required to support peripheral nerve regeneration. Among the miRNAs expressed in adult mouse SCs, miR-34a and miR-140 were identified as functional regulators of SC dedifferentiation/proliferation and remyelination, respectively. We found that miR-34a interacted with positive regulators of dedifferentiation and proliferation such as Notch1 and Ccnd1 to control cell cycle dynamics in SCs. miR-140 targeted the transcription factor Egr2, a master regulator of myelination, and modulated myelination in DRG/SC cocultures. Together, these results demonstrate that SC miRNAs are important modulators of the SC regenerative response after nerve damage.

Interactions of Sox10 and Egr2 in Myelin Gene Regulation

Myelination in the PNS is accompanied by a large induction of the myelin protein zero (Mpz) gene to produce the most abundant component in peripheral myelin. Analyses of knockout mice have shown that the EGR2/Krox20 and SOX10 transcription factors are required for Mpz expression. Our recent work has shown that the dominant EGR2 mutations associated with human peripheral neuropathies cause disruption of EGR2/SOX10 synergy at specific sites, including a conserved enhancer element in the first intron of the Mpz gene. Further investigation of Egr2/Sox10 interactions reveals that activation of the Mpz intron element by Egr2 requires both Sox10-binding sites. In addition, both Egr1 and Egr3 cooperate with Sox10 to activate this element, which indicates that this capacity is conserved among Egr family members. Finally, a conserved composite structure of Egr2/Sox10-binding sites in the genes encoding Mpz, myelin-associated glycoprotein and myelin basic protein genes was used to screen for similar modules in other myelin genes, revealing a potential regulatory element in the periaxin gene. Overall, these results elucidate a working model for developmental regulation of Mpz expression, several facets of which extend to regulation of other peripheral myelin genes.

Regulation of the PMP22 Gene through an Intronic Enhancer

Successful myelination of the peripheral nervous system depends upon induction of major protein components of myelin, such as peripheral myelin protein 22 (PMP22). Myelin stability is also sensitive to levels of PMP22, as a 1.4 Mb duplication on human chromosome 17, resulting in three copies of PMP22, is the most common cause of the peripheral neuropathy Charcot-Marie-Tooth disease. The transcription factor Egr2/Krox20 is required for induction of high level expression of Pmp22 in Schwann cells but its activation elements have not yet been determined. Using chromatin immunoprecipitation analysis of the rat Pmp22 locus, we found a major peak of Egr2 binding within the large intron of the Pmp22 gene. Analysis of a 250 bp region within the largest intron showed that it is strongly activated by Egr2 expression in reporter assays. Moreover, this region contains conserved binding sites not only for Egr2 but also for Sox10, which is also required for Schwann cell development. Our analysis shows that Sox10 is required for optimal activity of the intronic site as well as PMP22 expression. Finally, mouse transgenic analysis revealed tissue-specific expression of this intronic sequence in peripheral nerve. Overall, these data show that Egr2 and Sox10 activity are directly involved in mediating the developmental induction of Pmp22 expression.

PAP: a comprehensive workbench for mammalian transcriptional regulatory sequence analysis

Given the recent explosion of publications that employ microarray technology to monitor genome-wide expression and that correlate these expression changes to biological processes or to disease states, the determination of the transcriptional regulation of these co-expressed genes is the next major step toward deciphering the genetic network governing the pathway or disease under study. Although computational approaches have been proposed for this purpose, there is no integrated and user-friendly software application that allows experimental biologists to tackle this problem in higher eukaryotes. We have previously reported a systematic, statistical model of mammalian transcriptional regulatory sequence analysis. We have now made crucial extensions to this model and have developed a comprehensive, user-friendly web application suite termed the Promoter Analysis Pipeline (PAP). PAP is available at: http://bioinformatics.wustl.edu/webTools/portalModule/PromoterSearch.do

Differentially Expressed MicroRNAs in Chondrocytes from Distinct Regions of Developing Human Cartilage

There is compelling in vivo evidence from reports on human genetic mutations and transgenic mice that some microRNAs (miRNAs) play an important functional role in regulating skeletal development and growth. A number of published in vitro studies also point toward a role for miRNAs in controlling chondrocyte gene expression and differentiation. However, information on miRNAs that may regulate a specific phase of chondrocyte differentiation (i.e. production of progenitor, differentiated or hypertrophic chondrocytes) is lacking. To attempt to bridge this knowledge gap, we have investigated miRNA expression patterns in human embryonic cartilage tissue. Specifically, a developmental time point was selected, prior to endochondral ossification in the embryonic limb, to permit analysis of three distinct populations of chondrocytes. The location of chondroprogenitor cells, differentiated chondrocytes and hypertrophic chondrocytes in gestational day 54–56 human embryonic limb tissue sections was confirmed both histologically and by specific collagen expression patterns. Laser capture microdissection was utilized to separate the three chondrocyte populations and a miRNA profiling study was carried out using TaqMan® OpenArray® Human MicroRNA Panels (Applied Biosystems®). Here we report on abundantly expressed miRNAs in human embryonic cartilage tissue and, more importantly, we have identified miRNAs that are significantly differentially expressed between precursor, differentiated and hypertrophic chondrocytes by 2-fold or more. Some of the miRNAs identified in this study have been described in other aspects of cartilage or bone biology, while others have not yet been reported in chondrocytes. Finally, a bioinformatics approach was applied to begin to decipher developmental cellular pathways that may be regulated by groups of differentially expressed miRNAs during distinct stages of chondrogenesis. Data obtained from this work will serve as an important resource of information for the field of cartilage biology and will enhance our understanding of miRNA-driven mechanisms regulating cartilage and endochondral bone development, regeneration and repair.

Locus-wide identification of Egr2/Krox20 regulatory targets in myelin genes.

J. Neurochem. (2010) 115, 1409–1420.

An integrated approach to characterize transcription factor and microRNA regulatory networks involved in Schwann cell response to peripheral nerve injury

BackgroundThe regenerative response of Schwann cells after peripheral nerve injury is a critical process directly related to the pathophysiology of a number of neurodegenerative diseases. This SC injury response is dependent on an intricate gene regulatory program coordinated by a number of transcription factors and microRNAs, but the interactions among them remain largely unknown. Uncovering the transcriptional and post-transcriptional regulatory networks governing the Schwann cell injury response is a key step towards a better understanding of Schwann cell biology and may help develop novel therapies for related diseases. Performing such comprehensive network analysis requires systematic bioinformatics methods to integrate multiple genomic datasets.ResultsIn this study we present a computational pipeline to infer transcription factor and microRNA regulatory networks. Our approach combined mRNA and microRNA expression profiling data, ChIP-Seq data of transcription factors, and computational transcription factor and microRNA target prediction. Using mRNA and microRNA expression data collected in a Schwann cell injury model, we constructed a regulatory network and studied regulatory pathways involved in Schwann cell response to injury. Furthermore, we analyzed network motifs and obtained insights on cooperative regulation of transcription factors and microRNAs in Schwann cell injury recovery.ConclusionsThis work demonstrates a systematic method for gene regulatory network inference that may be used to gain new information on gene regulation by transcription factors and microRNAs.

Abstract P5-10-02: A copy number aberration driven endocrine response gene signature stratifies risk in estrogen receptor positive breast cancer

Background: Many prognostic gene signatures have been developed for estrogen receptor positive (ER+) breast cancer (BC); however, most have been solely based on mRNA expression data without integrated information on underlying primary drivers such as genomic aberrations. We therefore coupled gene expression and copy number aberration (CNA) in an attempt to improve upon prognostic signatures for ER+ BC. Methods: mRNA expression based discovery was conducted between 172/59 ER+ BC with low/high Ki67 levels after neoadjuvant aromatase inhibition and significant genes (significance analysis of microarray, q-value less than 0.05) were screened by correlation (Mann-Whitney-Wilcoxon test P less than 0.05) with CNA using Agilent comparative genomic hybridization array. Further interrogation on prognosis of relapse-free survival (RFS) by univariate survival analysis (P less than 0.05) in patients treated with adjuvant endocrine monotherapy from a public data set produced a Copy Number Aberration Driven Endocrine Response (CADER) signature consisting of treatment sensitive/resistant genes. MetaCore (GeneGo Inc) pathway analysis was conducted for enriched pathways. We subsequently applied Nanostring nCounter technology to formalin fixed archival tumor RNA from 620 ER+ adjuvant tamoxifen treated BC (UBC TAM-series) for CADER gene profiling. Patients in multiple independent public data sets and the TAM-series were classified into treatment sensitive defined by up-regulated sensitive gene centroid and down-regulated resistant gene centroid by the median cutoffs, treatment resistant defined with the reverse pattern and indeterminate otherwise. CADER risk stratifications were associated with patient survival outcomes in public cohorts and the TAM-series. The Kaplan-Meier (KM) analysis and Cox models were used for survival analysis. Published PAM50 intrinsic subtypes and subtype based risk of relapse (ROR-S) assignments were used (Nielsen CCR 16:5222, 2010). Results: A 54-gene CADER signature, 27 resistant/27 sensitive genes, was derived. Pathway analysis indicated that CADER was enriched with sensitive genes of cell survival functions while resistant genes were largely drivers of cell cycle progression. CADER stratifications were significantly prognostic of relapse free survival (RFS) in all public cohorts (log rank test P=0.05 for all) and in the UBC TMA-series (P=0.0001, BC specific survival and RFS). CADER showed an additional value (likelihood ratio test P=0.05) in all cohorts when both standard clinical variables and ROR-S were incorporated in multivariate Cox models. CADER were highly concordant with intrinsic subtypes and ROR-S (p=0.0001) in all data sets. However, CADER may stratify risk within ROR-S medium risk patients (P=0.002 METABRIC; P=0.003 and 0.036 in TAM-series for BC specific survival and RFS). Conclusions: We have developed a signature that is prognostic of long-term survival in postmenopausal BC, further splits risk within ROR-S medium risk group and identifies some highly resistant BC in presence of ROR-S and clinical variables (see Ellis et. al. abstract for evaluation of a CADER single sample predictor in the MA12 Phase 3 clinical trial). Citation Format: Jingqin Luo, Li-Wei Chang, Yu Tao, Jeremy Hoog, Samuel Leung, Torsten O Nielsen, Matthew J Ellis. A copy number aberration driven endocrine response gene signature stratifies risk in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-10-02.