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Supplementary Figure A from Coordinated Regulation of Cell Cycle Transcripts by p53-Inducible microRNAs, miR-192 and miR-215

Supplementary Figure and Table Legends from Coordinated Regulation of Cell Cycle Transcripts by p53-Inducible microRNAs, miR-192 and miR-215

Supplementary Figure 1 from Identification of SULF2 as a Novel Transcriptional Target of p53 by Use of Integrated Genomic Analyses

Supplementary Table C from Coordinated Regulation of Cell Cycle Transcripts by p53-Inducible microRNAs, miR-192 and miR-215

Microarray analysis has been useful for identifying the targets of many transcription factors. However, gene expression changes in response to transcription factor perturbation reveal both direct transcriptional targets and secondary gene regulation. By integrating RNA interference, gene expression profiling, and chromatin immunoprecipitation technologies, we identified a set of 32 direct transcriptional targets of the tumor suppressor p53. Of these 32 genes, 11 are not currently associated with the core p53 pathway. From among these novel pathway members, we focused on understanding the connection between p53 and SULF2, which encodes an extracellular heparan sulfate 6-O-endosulfatase that modulates the binding of growth factors to their cognate receptors and that has been shown to function as a tumor suppressor. Genetic and pharmacologic perturbation of p53 directly influences SULF2 expression, and similar to silencing of TP53, RNA interference–mediated suppression of SULF2 results in an impaired senescence response of cells to genotoxic stress. Thus, our integrated genomic approach has led to the identification of a novel mediator of p53 network biology. [Cancer Res 2009;69(4):1368–74]

Supplementary Table 1 from Identification of SULF2 as a Novel Transcriptional Target of p53 by Use of Integrated Genomic Analyses

Supplementary Figure and Table Legend from Identification of SULF2 as a Novel Transcriptional Target of p53 by Use of Integrated Genomic Analyses

Supplementary Table D from Coordinated Regulation of Cell Cycle Transcripts by p53-Inducible microRNAs, miR-192 and miR-215

Supplementary Table A from Coordinated Regulation of Cell Cycle Transcripts by p53-Inducible microRNAs, miR-192 and miR-215

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the loss of repression at the D4Z4 locus leading to aberrant double homeobox 4 (DUX4) expression in skeletal muscle. Activation of this early embryonic transcription factor results in the expression of its target genes causing muscle fiber death. Although progress toward understanding the signals driving DUX4 expression has been made, the factors and pathways involved in the transcriptional activation of this gene remain largely unknown. Here, we describe the identification and characterization of p38α as a novel regulator of DUX4 expression in FSHD myotubes. By using multiple highly characterized, potent, and specific inhibitors of p38α/β, we show a robust reduction of DUX4 expression, activity, and cell death across patient-derived FSHD1 and FSHD2 lines. RNA-seq profiling reveals that a small number of genes are differentially expressed upon p38α/β inhibition, the vast majority of which are DUX4 target genes. Our results reveal a novel and apparently critical role for p38α in the aberrant activation of DUX4 in FSHD and support the potential of p38α/β inhibitors as effective therapeutics to treat FSHD at its root cause. SIGNIFICANCE STATEMENT: Using patient-derived facioscapulohumeral muscular dystrophy (FSHD) myotubes, we characterize the pharmacological relationships between p38α/β inhibition, double homeobox 4 (DUX4) expression, its downstream transcriptional program, and muscle cell death. p38α/β inhibition results in potent and specific DUX4 downregulation across multiple genotypes without significant effects in the process of myogenesis in vitro. These findings highlight the potential of p38α/β inhibitors for the treatment of FSHD, a condition that today has no approved therapies.

Fragile X syndrome (FXS) is characterized by physical abnormalities, anxiety, intellectual disability, hyperactivity, autistic behaviors, and seizures. Abnormal neuronal development in FXS is poorly understood. Data on patients with FXS remain scarce, and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons.To mimic human neuron development in vivo, we coinjected neural precursor cells derived from FXS patient-derived induced pluripotent stem cells and neural precursor cells derived from corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice.The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Immunofluorescence and single and bulk RNA sequencing analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, we found increased percentages of Arc- and Egr-1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons.This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3-dimensional context.

Abnormal neuronal development in Fragile X syndrome (FXS) is poorly understood. Data on FXS patients remain scarce and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. Here, we co-injected neural precursor cells (NPCs) from FXS patient-derived and corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Single-cell RNA sequencing of transplanted cells revealed upregulated excitatory synaptic transmission and neuronal differentiation pathways in FXS neurons. Immunofluorescence analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, increased percentages of Arc- and Egr1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons pointed to an increase in synaptic activity and synaptic strength as compared to control. This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3D context, and could be used to test new therapeutic compounds correcting neuronal development defects in FXS.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the loss of repression at the

We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual-guide RNAs in three cell lines, altogether comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified and these patterns replicated with combinatorial drugs at 75% precision. Based on these results we anticipate cellular context will be critical to synthetic-lethal therapies.

Previous studies have demonstrated that E proteins induce activation-induced deaminase (AID) expression in activated B cells. Here, we examined the role of Id3 in germinal center (GC) cells. We found that Id3 expression is high in follicular B lineage cells but declines in GC cells. Immunized mice with Id3 expression depleted displayed a block in germinal center B cell maturation, showed reduced numbers of marginal zone B cells and class-switched cells, and were associated with decreased antibody titers and lower numbers of plasma cells. In vitro, Id3-depleted B cells displayed a defect in class switch recombination. Whereas AID levels were not altered in Id3-depleted activated B cells, the expression of a subset of genes encoding signaling components of antigen receptor-, cytokine receptor-, and chemokine receptor-mediated signaling was significantly impaired. We propose that during the GC reaction, Id3 levels decline to activate the expression of genes encoding signaling components that mediate B cell receptor- and or cytokine receptor-mediated signaling to promote the differentiation of GC B cells.

FSHD is caused by the loss of repression at the D4Z4 locus leading to DUX4 expression in skeletal muscle, activation of its early embryonic transcriptional program and muscle fiber death. While progress toward understanding the signals driving DUX4 expression has been made, the factors and pathways involved in the transcriptional activation of this gene remain largely unknown. Here, we describe the identification and characterization of p38α as a novel regulator of DUX4 expression in FSHD myotubes. By using multiple highly characterized, potent and specific inhibitors of p38α/β, we show a robust reduction of DUX4 expression, activity and cell death across FSHD1 and FSHD2 patient-derived lines. RNA-seq profiling reveals that a small number of genes are differentially expressed upon p38α/β inhibition, the vast majority of which are DUX4 target genes. Our results reveal a novel and apparently critical role for p38α in the aberrant activation of DUX4 in FSHD and support the potential of p38α/β inhibitors as effective therapeutics to treat FSHD at its root cause.VISUAL ABSTRACT

Summary: Tumors that overexpress the MYC oncogene are frequently aneuploid, a state associated with highly aggressive cancers and tumor evolution. However, how MYC causes aneuploidy is not well understood. Here, we show that MYC overexpression induces mitotic spindle assembly defects and chromosomal instability (CIN) through effects on microtubule nucleation and organization. Attenuating MYC expression reverses mitotic defects, even in established tumor cell lines, indicating an ongoing role for MYC in CIN. MYC reprograms mitotic gene expression, and we identify TPX2 to be permissive for spindle assembly in MYC-high cells. TPX2 depletion blocks mitotic progression, induces cell death, and prevents tumor growth. Further elevating TPX2 expression reduces mitotic defects in MYC-high cells. MYC and TPX2 expression may be useful biomarkers to stratify patients for anti-mitotic therapies. Our studies implicate MYC as a regulator of mitosis and suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution. : Rohrberg et al. identify a reversible role of the MYC oncogene for inducing chromosomal instability by inducing error-prone mitosis. MYC-high tumor cells rely on the mitotic regulator TPX2 to survive the altered mitotic program, revealing a synthetic-lethal interaction between MYC overexpression and TPX2 loss as a potential therapeutic strategy. Keywords: MYC, TPX2, mitotic spindle assembly, chromosomal instability, CIN, mitosis, microtubules, synthetic-lethality, receptor triple-negative breast cancer, TNBC

The MYC oncogene promotes tumorigenesis in part by facilitating cell cycle entry thus driving cellular proliferation. Tumors that overexpress MYC frequently demonstrate aneuploidy, numerical chromosome alterations associated with highly aggressive cancers, rapid tumor evolution, and poor patient outcome. While the role of MYC in overcoming the G1/S checkpoint is well established, it remains poorly understood whether MYC induces chromosomal instability (CIN). Here, we identify a direct influence of MYC on mitotic progression. MYC overexpression induces defects in microtubule nucleation and spindle assembly promoting chromosome segregation defects, micronuclei and CIN. We examined which mitotic regulators are required for the survival of MYC-overexpressing cells and found a reliance on high TPX2 expression. TPX2, a master microtubule regulator, is overexpressed together with MYC in multiple cell lines, in mouse tumor models and in aggressive human breast cancers. High TPX2 expression is permissive for mitotic spindle assembly and chromosome segregation in cells with deregulated MYC, whereas TPX2 depletion blocks mitotic progression, induces cell death and prevents tumor growth. Importantly, attenuation of MYC expression reverses the mitotic defects observed, even in established tumor cell lines, implicating an ongoing role for high MYC in the persistence of a CIN phenotype in tumors. Here, we implicate the MYC oncogene as a regulator of spindle assembly and dynamics and identify a new MYC-TPX2 synthetic-lethal interaction that could represent a future therapeutic strategy in MYC-overexpressing cancers. Our studies suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution.

Tumors that overexpress the MYC oncogene frequently demonstrate aneuploidy, numerical chromosome alterations associated with highly aggressive cancers, rapid tumor evolution, and poor patient outcome. Here, we identify that MYC overexpression induces reversible defects in microtubule nucleation and mitotic spindle assembly, promoting chromosome segregation defects, micronuclei and chromosomal instability (CIN). High TPX2 expression is permissive for mitotic spindle assembly and chromosome segregation in cells with MYC overexpression; whereas TPX2 depletion blocks mitotic progression, induces cell death and prevents tumor growth. Attenuating MYC expression reverses mitotic defects, even in established tumor cell lines, implicating an ongoing role for high MYC in the persistence of CIN in tumors. Our studies implicate the MYC oncogene as a regulator of spindle assembly and identify a new MYC-TPX2 synthetic-lethal interaction that could represent a future therapeutic strategy in MYC-overexpressing cancers. Moreover, our studies suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution.

During an immune response, CD8+ T lymphocytes can undergo asymmetric division, giving rise to daughter cells that exhibit distinct tendencies to adopt terminal effector and memory cell fates. Here we show that "pre-effector" and "pre-memory" cells resulting from the first CD8+ T cell division in vivo exhibited low and high rates of endogenous proteasome activity, respectively. Pharmacologic reduction of proteasome activity in CD8+ T cells early during differentiation resulted in acquisition of terminal effector cell characteristics, whereas enhancement of proteasome activity conferred attributes of memory lymphocytes. Transcriptomic and proteomic analyses revealed that modulating proteasome activity in CD8+ T cells affected cellular metabolism. These metabolic changes were mediated, in part, through differential expression of Myc, a transcription factor that controls glycolysis and metabolic reprogramming. Taken together, these results demonstrate that proteasome activity is an important regulator of CD8+ T cell fate and raise the possibility that increasing proteasome activity may be a useful therapeutic strategy to enhance the generation of memory lymphocytes.

It is now well established that the E and Id protein axis regulates multiple steps in lymphocyte development. However, it remains unknown how E and Id proteins mechanistically enforce and maintain the naïve T-cell fate. Here we show that Id2 and Id3 suppressed the development and expansion of innate variant follicular helper T (TFH) cells. Innate variant TFH cells required major histocompatibility complex (MHC) class I-like signaling and were associated with germinal center B cells. We found that Id2 and Id3 induced Foxo1 and Foxp1 expression to antagonize the activation of a TFH transcription signature. We show that Id2 and Id3 acted upstream of the Hif1a/Foxo/AKT/mTORC1 pathway as well as the c-myc/p19Arf module to control cellular expansion. We found that mice depleted for Id2 and Id3 expression developed colitis and αβ T-cell lymphomas. Lymphomas depleted for Id2 and Id3 expression displayed elevated levels of c-myc, whereas p19Arf abundance declined. Transcription signatures of Id2- and Id3-depleted lymphomas revealed similarities to genetic deficiencies associated with Burkitt lymphoma. We propose that, in response to antigen receptor and/or cytokine signaling, the E–Id protein axis modulates the activities of the PI3K–AKT–mTORC1–Hif1a and c-myc/p19Arf pathways to control cellular expansion and homeostatic proliferation.

Early B cell development is orchestrated by the combined activities of the transcriptional regulators E2A, EBF1, Foxo1 and Ikaros. However, how the genome-wide binding patterns of these regulators are modulated during B lineage development remains to be determined. Here we found that in lymphoid progenitor cells, the chromatin remodeler Brg1 specified the B cell fate. In committed pro-B cells, Brg1 regulated contraction of the locus encoding the immunoglobulin heavy chain (Igh) and controlled expression of the gene encoding the transcription factor c-Myc (Myc) to modulate the expression of genes encoding products that regulate ribosome biogenesis. In committed pro-B cells, Brg1 suppressed a pre-B lineage-specific pattern of gene expression. Finally, we found that Brg1 acted mechanistically to establish B cell fate and modulate cell growth by facilitating access of lineage-specific transcription factors to enhancer repertoires.

Hepatocellular carcinoma (HCC) is associated with poor survival for patients and few effective treatment options, raising the need for novel therapeutic strategies. MicroRNAs (miRNAs) play important roles in tumor development and show deregulated patterns of expression in HCC. Because of the liver’s unique affinity for small nucleic acids, miRNA-based therapy has been proposed in the treatment of liver disease. Thus, there is an urgent need to identify and characterize aberrantly expressed miRNAs in HCC. In our study, we profiled miRNA expression changes in de novo liver tumors driven by MYC and/or RAS, two canonical oncogenes activated in a majority of human HCCs. We identified an up-regulated miRNA megacluster comprised of 53 miRNAs on mouse chromosome 12qF1 (human homolog 14q32). This miRNA megacluster is up-regulated in all three transgenic liver models and in a subset of human HCCs. An unbiased functional analysis of all miRNAs within this cluster was performed. We found that miR-494 is overexpressed in human HCC and aids in transformation by regulating the G1/S cell cycle transition through targeting of the Mutated in Colorectal Cancer tumor suppressor. miR-494 inhibition in human HCC cell lines decreases cellular transformation, and anti-miR-494 treatment of primary MYC-driven liver tumor formation significantly diminishes tumor size. Conclusion: Our findings identify a new therapeutic target (miR-494) for the treatment of HCC. (Hepatology 2014;58:202–215)

The epithelial-mesenchymal transition (EMT) endows carcinoma cells with phenotypic plasticity that can facilitate the formation of cancer stem cells (CSCs) and contribute to the metastatic cascade. While there is substantial support for the role of EMT in driving cancer cell dissemination, less is known about the intracellular molecular mechanisms that govern formation of CSCs via EMT. Here we show that β2 and β5 proteasome subunit activity is downregulated during EMT in immortalized human mammary epithelial cells. Moreover, selective proteasome inhibition enabled mammary epithelial cells to acquire certain morphologic and functional characteristics reminiscent of cancer stem cells, including CD44 expression, self-renewal, and tumor formation. Transcriptomic analyses suggested that proteasome-inhibited cells share gene expression signatures with cells that have undergone EMT, in part, through modulation of the TGF-β signaling pathway. These findings suggest that selective downregulation of proteasome activity in mammary epithelial cells can initiate the EMT program and acquisition of a cancer stem cell-like phenotype. As proteasome inhibitors become increasingly used in cancer treatment, our findings highlight a potential risk of these therapeutic strategies and suggest a possible mechanism by which carcinoma cells may escape from proteasome inhibitor-based therapy.

The hypoxia-inducible factor (HIF) pathway is essential for cell survival under low oxygen and plays an important role in tumor cell homeostasis. We investigated the function of miR-210, the most prominent microRNA upregulated by hypoxia and a direct transcriptional target of HIFs. miR-210 expression was elevated in multiple cancer types and correlated with metastasis of breast and melanoma tumors. miR-210 overexpression in cancer cell lines bypassed hypoxia-induced cell cycle arrest and partially reversed the hypoxic gene expression signature. We identified MNT, a known MYC antagonist, as a miR-210 target. MNT mRNA contains multiple miR-210 binding sites in the 3' UTR and its knockdown phenocopied miR-210 overexpression. Furthermore, loss of MYC abolished miR-210-mediated override of hypoxia-induced cell cycle arrest. Comparison of miR-210 and MYC overexpression with MNT knockdown signatures also indicated that miR-210 triggered a "MYC-like" transcriptional response. Thus, miR-210 influences the hypoxia response in tumor cells through targeting a key transcriptional repressor of the MYC-MAX network.

Cell cycle arrest in response to DNA damage is an important antitumorigenic mechanism. MicroRNAs (miRNAs) were recently shown to play key regulatory roles in cell cycle progression. For example, miR-34a is induced in response to p53 activation and mediates G1 arrest by down-regulating multiple cell cycle–related transcripts. Here we show that genotoxic stress promotes the p53-dependent up-regulation of the homologous miRNAs miR-192 and miR-215. Like miR-34a, activation of miR-192/215 induces cell cycle arrest, suggesting that multiple miRNA families operate in the p53 network. Furthermore, we define a downstream gene expression signature for miR-192/215 expression, which includes a number of transcripts that regulate G1 and G2 checkpoints. Of these transcripts, 18 transcripts are direct targets of miR-192/215, and the observed cell cycle arrest likely results from a cooperative effect among the modulations of these genes by the miRNAs. Our results showing a role for miR-192/215 in cell proliferation combined with recent observations that these miRNAs are underexpressed in primary cancers support the idea that miR-192 and miR-215 function as tumor suppressors. [Cancer Res 2008;68(24):10105–12]

Transcription factors GATA-1 and GATA-2 are required for normal hematopoiesis. The loss of either leads to embryonic lethality in knockout mice because of the failure of erythroid maturation and the expansion of progenitors, respectively. As the expression of GATA-1 and GATA-2 overlaps within hematopoietic progenitors, the extent to which these factors functionally compensate for each other during embryogenesis is unknown. As shown here, we have analyzed double-knockout embryos at the yolk sac stage of development and have shown that the combined absence of these GATA factors virtually ablates primitive erythroid cell formation. Thus, the function of GATA-1 and GATA-2 overlaps at the yolk sac stage. Moreover, a GATA factor, either GATA-1 or GATA-2, is required to initiate blood formation in the embryo.

Scarring of the kidney is a major public health concern, directly promoting loss of kidney function. In order to understand the role of microRNA (miRNA) in the progression of kidney scarring in response to injury, we investigated changes in miRNA expression in two kidney fibrosis models, and identified 24 commonly upregulated miRNAs. Among them, miR-21 was highly elevated in both animal models and human transplant kidney nephropathy. Deletion of miR-21 in mice resulted in no overt abnormality. However, miR-21-/- mice suffered far less interstitial fibrosis in response to kidney injury, which was pheno-copied in wild-type mice treated with anti-miR-21 oligonucleotides. Surprisingly, global de-repression of miR-21 target messenger RNAs was only readily detectable in miR-21-/- kidneys after injury. Analysis of gene expression profiles identified groups of genes involved in metabolic pathways that were up-regulated in the absence of miR-21, including the lipid metabolism pathway regulated by Peroxisome proliferator activated receptor-α (Pparα), a direct miR-21 target. Over-expression of Pparα prevented UUO-induced injury and fibrosis. Pparα deficiency abrogated the anti-fibrotic effect of anti-miR21 oligonucleotides. miR-21 also regulates the redox metabolic pathway. The mitochondrial inhibitor of reactive oxygen species generation, Mpv17l, was repressed by miR-21, correlating closely with enhanced oxidative kidney damage. These studies demonstrate that miR-21 contributes to fibrogenesis and epithelial injury in the kidney in two mouse models and is a candidate target for anti-fibrotic therapies.

Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL levels have remained elusive. Recent advances in the understanding of lipid metabolism have revealed that miR-33, an intronic microRNA located within the SREBF2 gene, suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and circulating HDL levels, suggesting that antagonism of miR-33 may be atheroprotective. As the regression of atherosclerosis is clinically desirable, we assessed the impact of miR-33 inhibition in mice deficient for the LDL receptor (Ldlr–/– mice), with established atherosclerotic plaques. Mice treated with anti-miR33 for 4 weeks showed an increase in circulating HDL levels and enhanced reverse cholesterol transport to the plasma, liver, and feces. Consistent with this, anti-miR33–treated mice showed reductions in plaque size and lipid content, increased markers of plaque stability, and decreased inflammatory gene expression. Notably, in addition to raising ABCA1 levels in the liver, anti-miR33 oligonucleotides directly targeted the plaque macrophages, in which they enhanced ABCA1 expression and cholesterol removal. These studies establish that raising HDL levels by anti-miR33 oligonucleotide treatment promotes reverse cholesterol transport and atherosclerosis regression and suggest that it may be a promising strategy to treat atherosclerotic vascular disease.

Prioritization, or ranking, of gene lists is becoming increasingly important for analyzing data generated from high-throughput assays like expression profiling and RNAi-based screening. This is especially the case when specific genes in a list need to be further validated using low-throughput experiments. In addition to gene set overlap enrichment methods, a complementary approach is to examine molecular interaction networks. These can provide putative functional insights based on gene connectivity, especially when many genes contain little or no annotation. For bench and computational biologists alike, using networks requires an informed selection of interaction data for network construction and strategies for managing network complexity. Moreover, graph theory and social network analysis methods can be used to isolate critical subnetworks and quantify network properties. Here, I discuss the basic components of networks, implications of their structure for functional interpretation, and common metrics used for prioritization. Although this is still an ongoing area of research, networks are providing new ways for gauging pathway impact in large-scale data sets.

Microarray analysis has been useful for identifying the targets of many transcription factors. However, gene expression changes in response to transcription factor perturbation reveal both direct transcriptional targets and secondary gene regulation. By integrating RNA interference, gene expression profiling, and chromatin immunoprecipitation technologies, we identified a set of 32 direct transcriptional targets of the tumor suppressor p53. Of these 32 genes, 11 are not currently associated with the core p53 pathway. From among these novel pathway members, we focused on understanding the connection between p53 and SULF2, which encodes an extracellular heparan sulfate 6-O-endosulfatase that modulates the binding of growth factors to their cognate receptors and that has been shown to function as a tumor suppressor. Genetic and pharmacologic perturbation of p53 directly influences SULF2 expression, and similar to silencing of TP53, RNA interference–mediated suppression of SULF2 results in an impaired senescence response of cells to genotoxic stress. Thus, our integrated genomic approach has led to the identification of a novel mediator of p53 network biology. [Cancer Res 2009;69(4):1368–74]

Prioritization, or ranking, of gene lists is becoming increasingly important for analyzing data generated from high-throughput assays like expression profiling and RNAi-based screening. This is especially the case when specific genes in a list need to be further validated using low-throughput experiments. In addition to gene set overlap enrichment methods, a complementary approach is to examine molecular interaction networks. These can provide putative functional insights based on gene connectivity, especially when many genes contain little or no annotation. For bench and computational biologists alike, using networks requires an informed selection of interaction data for network construction and strategies for managing network complexity. Moreover, graph theory and social network analysis methods can be used to isolate critical subnetworks and quantify network properties. Here, I discuss the basic components of networks, implications of their structure for functional interpretation, and common metrics used for prioritization. Although this is still an ongoing area of research, networks are providing new ways for gauging pathway impact in large-scale data sets.

Background The rapid growth of protein interactome data has elevated the necessity and importance of network analysis tools. However, unlike pure text data, network search spaces are of exponential complexity. This poses special challenges for storing, searching, and navigating this data efficiently. Moreover, development of effective web interfaces has been difficult. Results We present Integrator, a web-integrated graphical search tool for protein-protein interaction networks across 50+ genomes. Conclusion Integrator provides single and multiple protein searches of the Bioverse database containing experimentally-derived and predicted protein-protein interactions. The interface provides animated local network views, rapid subgraph manipulation, and cross-referencing of functional annotations. Integrator is available at http://bioverse.compbio.washington.edu/integrator.

Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤ 0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins.

Summary: We have developed several new navigation features for a Java graph applet previously released for visualizing protein--protein interactions. This graph viewer can be used to navigate any molecular interactome dataset. We have successfully implemented this tool for exploring protein networks stored in the Bioverse interaction database. Availability: http://bioverse.compbio.washington.edu/viewer Contact: ram@compbio.washington.edu

The function of GATA transcription factors in diverse developmental contexts depends in part on physical interaction with cofactors of the Friend of GATA (FOG) family. However, previous studies indicate that FOG-1 may play a GATA-1-independent role in early megakaryopoiesis, suggesting that FOG proteins might act in a GATA factor-independent manner. Here, we have generated mouse knock-in (KI) mutants harboring a critical valine-to-glycine substitution in the amino-terminal zinc fingers of GATA-1 and GATA-2 to ablate FOG interaction. In contrast to male GATA-1 KI (GATA-1 is located on the X-chromosome) or GATA-2 KI/KI mice, compound GATA-1 KI GATA-2 KI/KI mutant mice display complete megakaryopoietic failure, a phenocopy of FOG-1 −/− mice. We conclude that FOG-1 requires an interaction with either GATA-1 or -2 as part of its essential role in early megakaryopoiesis. On the basis of these and previous reports, we infer that GATA factor dependence is a critical aspect of FOG protein function.

LBA204 Background: Noninvasive biomarkers are urgently needed in HCC. MicroRNA (miRNA) are small, noncoding RNA that regulate mRNA expression and are detectable in tumor tissue and extracellular compartments. miRNA signatures in blood specimens show association with HCC diagnosis but have not been explored as pharmacodynamic or predictive biomarkers. We present a pilot study to identify differentially expressed miRNA using NGS on serum from HCC patients at baseline and on targeted therapy, compared to controls. Methods: Serum samples were obtained from HCC patients enrolled on a clinical trial of sorafenib plus temsirolimus. Control sera were obtained from patients with non-malignant liver diseases (NMLD) and healthy volunteers (HV). Total RNA was extracted using RNAzol followed by library construction for each sample. Small RNA were separated by gel purification and sequenced by Illumina HiSeq 2500 at 5M+ reads per sample. Raw sequencing reads were mapped using Novoalign and quantified. Counts were normalized using an exogenous spike-in miRNA. Comparisons between HCC vs. control and baseline vs. on treatment cohorts were performed by linear regression (one-way ANOVA) and multiple-test corrected using Benjamini-Hochberg statistics. Candidate signature miRNA were derived using fold change, p-value, and abundance cutoffs. Results: Cohorts: HCC baseline (n=23) and paired on treatment (n=20), NMLD (n=12), and HV (n=10). HCC cohort: HBV+/dual 40%, HCV+ 32%. NMLD cohort: HCV+ 83%. Median RNA yield/200 µL was 410 ng (range: 69-1066) for HCC, 406 ng (range: 224-1100) for NMLD. The mapping rate ranged from 10-70%. Elevated AFP was associated with up-regulated miRNA identified in TCGA HCC cases, including miR-10a/b. No significant differences were observed for HBV+ vs. HCV+. Multiple oncomiRs appeared downregulated on treatment including Let-7 and miR-17/92 family members. Some miRNA isoforms were differentially regulated in each cohort. Conclusions: Serum miRNA in HCC patients demonstrate changes on treatment and can be characterized by NGS. Certain miRNA implicated in HCC appeared related to clinical covariates and warrant further study as novel biomarkers.

We have made a number of enhancements to the previously described Bioverse web server and computational biology framework (http://bioverse.compbio.washington.edu). In this update, we provide an overview of the new features available that include: (i) expansion of the number of organisms represented in the Bioverse and addition of new data sources and novel prediction techniques not available elsewhere, including network-based annotation; (ii) reengineering the database backend and supporting code resulting in significant speed, search and ease-of use improvements; and (iii) creation of a stateful and dynamic web application frontend to improve interface speed and usability. Integrated Java-based applications also allow dynamic visualization of real and predicted protein interaction networks.

micro-RNA-21 is frequently over-expressed and has been shown to correlate with poor outcome in multiple cancer types. Its genomic locus is amplified in some cancer types and miR-21 is also transactivated by several known cancer drivers (e.g. STAT3 and Ras). We have used a publically available data set from 86 patients to show that miR-21 is over-expressed in hepatocellular carcinoma (HCC). The treatment options for HCC are limited, thus making it the third leading cause of cancer related deaths worldwide. Additional treatments for HCC are greatly needed. We investigated the therapeutic potential of inhibiting miR-21 with chemically modified oligonucleotides known as anti-miRs using models of HCC, including a genetically engineered mouse model of HCC driven by an inducible, activated form of Ras. Short term treatment with the anti-miR led to a reduction in tumor formation. Furthermore, inhibition of miR-21 was clearly demonstrated by analysis of genome wide mRNA expression data from treated versus untreated tumors. This data showed that genes containing the mir-21 octamer sequence were the most significantly over-enriched among up-regulated genes compared to all of the other 65,535 possible octamers. We also determined the effect of long-term anti-miR-21 treatment on the survival in this mouse model of HCC. anti-miR-21 treated mice demonstrated a highly significant survival advantage (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4708. doi:10.1158/1538-7445.AM2011-4708

Genetic interactions occur when a combination of mutations results in a surprising phenotype. These interactions capture functional redundancy, and thus are important for predicting function, dissecting protein complexes into functional pathways, and exploring the mechanistic underpinnings of common human diseases. Synthetic sickness and lethality are the most studied types of genetic interactions in yeast. However, even in yeast, only a small proportion of gene pairs have been tested for genetic interactions due to the large number of possible combinations of gene pairs. To expand the set of known synthetic lethal (SL) interactions, we have devised an integrative, multi-network approach for predicting these interactions that significantly improves upon the existing approaches. First, we defined a large number of features for characterizing the relationships between pairs of genes from various data sources. In particular, these features are independent of the known SL interactions, in contrast to some previous approaches. Using these features, we developed a non-parametric multi-classifier system for predicting SL interactions that enabled the simultaneous use of multiple classification procedures. Several comprehensive experiments demonstrated that the SL-independent features in conjunction with the advanced classification scheme led to an improved performance when compared to the current state of the art method. Using this approach, we derived the first yeast transcription factor genetic interaction network, part of which was well supported by literature. We also used this approach to predict SL interactions between all non-essential gene pairs in yeast (http://sage.fhcrc.org/downloads/downloads/predicted_yeast_genetic_interactions.zip). This integrative approach is expected to be more effective and robust in uncovering new genetic interactions from the tens of millions of unknown gene pairs in yeast and from the hundreds of millions of gene pairs in higher organisms like mouse and human, in which very few genetic interactions have been identified to date., Author Summary Genetic interactions occur when a combination of mutations results in a surprising phenotype. These interactions capture functional redundancy, and thus are important for predicting function, dissecting protein complexes into functional pathways, and exploring the mechanistic underpinnings of common human diseases. Due to the large number of possible combinations of genes, only a small portion of gene pairs in yeast and only a few pairs in higher organisms like mouse and human have been tested. Therefore, predicting genetic interactions has received significant attention in the past several years. The existing methods primarily rely on the known genetic interactions, and thus are far less effective in classifying most gene pairs not well connected with known genetic interactions. Here we developed a non-parametric multi-classifier system for predicting genetic interactions based on a large number of novel features independent of the known genetic interactions. This approach led to an improved performance when compared to the current state-of-the-art method. Using this approach, we derived the first yeast transcription factor genetic interaction network, part of which was well supported by literature. This integrative approach is expected to be more effective and robust in uncovering new genetic interactions in yeast and other species.