Kimberly Glass

Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD

The pathogenesis of chronic obstructive pulmonary disease (COPD) remains unclear, but involves loss of alveolar surface area (emphysema) and airway inflammation (bronchitis) as the consequence of cigarette smoke (CS) exposure. Previously, we demonstrated that autophagy proteins promote lung epithelial cell death, airway dysfunction, and emphysema in response to CS; however, the underlying mechanisms have yet to be elucidated. Here, using cultured pulmonary epithelial cells and murine models, we demonstrated that CS causes mitochondrial dysfunction that is associated with a reduction of mitochondrial membrane potential. CS induced mitophagy, the autophagy-dependent elimination of mitochondria, through stabilization of the mitophagy regulator PINK1. CS caused cell death, which was reduced by administration of necrosis or necroptosis inhibitors. Genetic deficiency of PINK1 and the mitochondrial division/mitophagy inhibitor Mdivi-1 protected against CS-induced cell death and mitochondrial dysfunction in vitro and reduced the phosphorylation of MLKL, a substrate for RIP3 in the necroptosis pathway. Moreover, Pink1(-/-) mice were protected against mitochondrial dysfunction, airspace enlargement, and mucociliary clearance (MCC) disruption during CS exposure. Mdivi-1 treatment also ameliorated CS-induced MCC disruption in CS-exposed mice. In human COPD, lung epithelial cells displayed increased expression of PINK1 and RIP3. These findings implicate mitophagy-dependent necroptosis in lung emphysematous changes in response to CS exposure, suggesting that this pathway is a therapeutic target for COPD.

CpG methylation of half-CRE sequences creates C/EBPα binding sites that activate some tissue-specific genes

DNA methylation of the cytosine in the CpG dinucleotide is typically associated with gene silencing. Genomic analyses have identified low CpG promoters that are both methylated and transcriptionally active, but the mechanism underlying the activation of these methylated promoters remains unclear. Here we show that CpG methylation of the CRE sequence (TGACGTCA) enhances the DNA binding of the C/EBPα transcription factor, a protein critical for activation of differentiation in various cell types. Transfection assays also show that C/EBPα activates the CRE sequence only when it is methylated. The biological significance of this observation was seen in differentiating primary keratinocyte cultures from newborn mice where certain methylated promoters are both bound by C/EBPα and activated upon differentiation. Experimental demethylation by either 5-azacytidine treatment or DNMT1 depletion diminished both C/EBPα binding and activation of the same methylated promoters upon differentiation suggesting that CpG methylation can localize C/EBPα. Transfection studies in cell cultures using methylated tissue-specific proximal promoters identified half-CRE (CGTCA) and half-C/EBP (CGCAA) sequences that need to be methylated for C/EBPα mediated activation. In primary dermal fibroblasts, C/EBPα activates a different set of methylated tissue-specific promoters upon differentiation into adipocytes. These data identify a new function for methyl CpGs: producing DNA binding sites at half-CRE and half-C/EBP sequences for C/EBPα that are needed to activate tissue-specific genes.

Combinatorial Assembly of Developmental Stage-Specific Enhancers Controls Gene Expression Programs during Human Erythropoiesis

Gene-distal enhancers are critical for tissue-specific gene expression, but their genomic determinants within a specific lineage at different stages of development are unknown. Here we profile chromatin state maps, transcription factor occupancy, and gene expression profiles during human erythroid development at fetal and adult stages. Comparative analyses of human erythropoiesis identify developmental stage-specific enhancers as primary determinants of stage-specific gene expression programs. We find that erythroid master regulators GATA1 and TAL1 act cooperatively within active enhancers but confer little predictive value for stage specificity. Instead, a set of stage-specific coregulators collaborates with master regulators and contributes to differential gene expression. We further identify and validate IRF2, IRF6, and MYB as effectors of an adult-stage expression program. Thus, the combinatorial assembly of lineage-specific master regulators and transcriptional coregulators within developmental stage-specific enhancers determines gene expression programs and temporal regulation of transcriptional networks in a mammalian genome.

KDM4A Lysine Demethylase Induces Site-Specific Copy Gain and Rereplication of Regions Amplified in Tumors

Acquired chromosomal instability and copy number alterations are hallmarks of cancer. Enzymes capable of promoting site-specific copy number changes have yet to be identified. Here, we demonstrate that H3K9/36me3 lysine demethylase KDM4A/JMJD2A overexpression leads to localized copy gain of 1q12, 1q21, and Xq13.1 without global chromosome instability. KDM4A-amplified tumors have increased copy gains for these same regions. 1q12h copy gain occurs within a single cell cycle, requires S phase, and is not stable but is regenerated each cell division. Sites with increased copy number are rereplicated and have increased KDM4A, MCM, and DNA polymerase occupancy. Suv39h1/KMT1A or HP1γ overexpression suppresses the copy gain, whereas H3K9/K36 methylation interference promotes gain. Our results demonstrate that overexpression of a chromatin modifier results in site-specific copy gains. This begins to establish how copy number changes could originate during tumorigenesis and demonstrates that transient overexpression of specific chromatin modulators could promote these events.

ETV1 directs androgen metabolism and confers aggressive prostate cancer in targeted mice and patients.

Distinguishing aggressive from indolent disease and developing effective therapy for advanced disease are the major challenges in prostate cancer research. Chromosomal rearrangements involving ETS transcription factors, such as ERG and ETV1, occur frequently in prostate cancer. How they contribute to tumorigenesis and whether they play similar or distinct in vivo roles remain elusive. Here we show that in mice with ERG or ETV1 targeted to the endogenous Tmprss2 locus, either factor cooperated with loss of a single copy of Pten, leading to localized cancer, but only ETV1 appeared to support development of invasive adenocarcinoma under the background of full Pten loss. Mechanistic studies demonstrated that ERG and ETV1 control a common transcriptional network but largely in an opposing fashion. In particular, while ERG negatively regulates the androgen receptor (AR) transcriptional program, ETV1 cooperates with AR signaling by favoring activation of the AR transcriptional program. Furthermore, we found that ETV1 expression, but not that of ERG, promotes autonomous testosterone production. Last, we confirmed the association of an ETV1 expression signature with aggressive disease and poorer outcome in patient data. The distinct biology of ETV1-associated prostate cancer suggests that this disease class may require new therapies directed to underlying programs controlled by ETV1.

Mitochondrial iron chelation ameliorates cigarette smoke–induced bronchitis and emphysema in mice

Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic determinants. We have previously identified iron-responsive element–binding protein 2 (IRP2) as an important COPD susceptibility gene and have shown that IRP2 protein is increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from cigarette smoke (CS)-induced experimental COPD. By integrating RNA immunoprecipitation followed by sequencing (RIP-seq), RNA sequencing (RNA-seq), and gene expression and functional enrichment clustering analysis, we identified Irp2 as a regulator of mitochondrial function in the lungs of mice. Irp2 increased mitochondrial iron loading and levels of cytochrome c oxidase (COX), which led to mitochondrial dysfunction and subsequent experimental COPD. Frataxin-deficient mice, which had higher mitochondrial iron loading, showed impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, whereas mice deficient in the synthesis of cytochrome c oxidase, which have reduced COX, were protected from CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a mitochondrial iron chelator or mice fed a low-iron diet were protected from CS-induced COPD. Mitochondrial iron chelation also alleviated CS-induced impairment of MCC, CS-induced pulmonary inflammation and CS-associated lung injury in mice with established COPD, suggesting a critical functional role and potential therapeutic intervention for the mitochondrial-iron axis in COPD.

Passing Messages between Biological Networks to Refine Predicted Interactions

Regulatory network reconstruction is a fundamental problem in computational biology. There are significant limitations to such reconstruction using individual datasets, and increasingly people attempt to construct networks using multiple, independent datasets obtained from complementary sources, but methods for this integration are lacking. We developed PANDA (Passing Attributes between Networks for Data Assimilation), a message-passing model using multiple sources of information to predict regulatory relationships, and used it to integrate protein-protein interaction, gene expression, and sequence motif data to reconstruct genome-wide, condition-specific regulatory networks in yeast as a model. The resulting networks were not only more accurate than those produced using individual data sets and other existing methods, but they also captured information regarding specific biological mechanisms and pathways that were missed using other methodologies. PANDA is scalable to higher eukaryotes, applicable to specific tissue or cell type data and conceptually generalizable to include a variety of regulatory, interaction, expression, and other genome-scale data. An implementation of the PANDA algorithm is available at www.sourceforge.net/projects/panda-net.

A network model for angiogenesis in ovarian cancer

BackgroundWe recently identified two robust ovarian cancer subtypes, defined by the expression of genes involved in angiogenesis, with significant differences in clinical outcome. To identify potential regulatory mechanisms that distinguish the subtypes we applied PANDA, a method that uses an integrative approach to model information flow in gene regulatory networks.ResultsWe find distinct differences between networks that are active in the angiogenic and non-angiogenic subtypes, largely defined by a set of key transcription factors that, although previously reported to play a role in angiogenesis, are not strongly differentially-expressed between the subtypes. Our network analysis indicates that these factors are involved in the activation (or repression) of different genes in the two subtypes, resulting in differential expression of their network targets. Mechanisms mediating differences between subtypes include a previously unrecognized pro-angiogenic role for increased genome-wide DNA methylation and complex patterns of combinatorial regulation.ConclusionsThe models we develop require a shift in our interpretation of the driving factors in biological networks away from the genes themselves and toward their interactions. The observed regulatory changes between subtypes suggest therapeutic interventions that may help in the treatment of ovarian cancer.

Sexually-dimorphic targeting of functionally-related genes in COPD

BackgroundThere is growing evidence that many diseases develop, progress, and respond to therapy differently in men and women. This variability may manifest as a result of sex-specific structures in gene regulatory networks that influence how those networks operate. However, there are few methods to identify and characterize differences in network structure, slowing progress in understanding mechanisms driving sexual dimorphism.ResultsHere we apply an integrative network inference method, PANDA (Passing Attributes between Networks for Data Assimilation), to model sex-specific networks in blood and sputum samples from subjects with Chronic Obstructive Pulmonary Disease (COPD). We used a jack-knifing approach to build an ensemble of likely networks for each sex. By adapting statistical methods to compare these network ensembles, we were able to identify strong differential-targeting patterns associated with functionally-related sets of genes, including those involved in mitochondrial function and energy metabolism. Network analysis also identified several potential sex- and disease-specific transcriptional regulators of these pathways.ConclusionsNetwork analysis yielded insight into potential mechanisms driving sexual dimorphism in COPD that were not evident from gene expression analysis alone. We believe our ensemble approach to network analysis provides a principled way to capture sex-specific regulatory relationships and could be applied to identify differences in gene regulatory patterns in a wide variety of diseases and contexts.

Suppression of the C/EBP family of transcription factors in adipose tissue causes lipodystrophy

Adipose-specific inactivation of both AP-1 and CCAAT-enhancer-binding protein (C/EBP) families of B-ZIP transcription factors in transgenic mice causes severe lipoatrophy. To evaluate whether inactivation of only C/EBP members was critical for lipoatrophy, A-C/EBP, a dominant-negative protein that specifically inhibits the DNA binding of the C/EBP members, was expressed in adipose tissue. For the first 2 weeks after birth, aP2-A-C/EBP mice had no white adipose tissue (WAT), drastically reduced brown adipose tissue (BAT), and exhibited marked hepatic steatosis, hyperinsulinemia, and hyperlipidemia. However, WAT appeared during the third week, coinciding with significantly improved metabolic functioning. In adults, BAT remained reduced, causing cold intolerance. At 30 weeks, the aP2-A-C/EBP mice had only 35% reduced WAT, with clear morphological signs of lipodystrophy in subcutaneous fat. Circulating leptin and adiponectin levels were less than the wild-type levels, and these mice exhibited impaired triglyceride clearance. Insulin resistance, glucose intolerance, and reduced free fatty acid release in response to β3-adrenergic agonist suggest improper functioning of the residual WAT. Gene expression analysis of inguinal WAT identified reduced mRNA levels of several enzymes involved in fatty acid synthesis and glucose metabolism that are known C/EBPα transcriptional targets. There were increased levels for genes involved in inflammation and muscle differentiation. However, when dermal fibroblasts from aP2-A-C/EBP mice were differentiated into adipocytes in tissue culture, muscle markers were elevated more than the inflammatory markers. These results demonstrate that the C/EBP family is essential for adipose tissue development during the early postnatal period, the regulation of glucose and lipid homeostasis in adults, and the suppression of the muscle lineage.