Diverse Gene Expression Changes Research Articles

IGHMBP2 deletion suppresses translation and activates the integrated stress response.

IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 up-regulation. With recent studies showing the integrated stress response (ISR) can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.

Establishment of a system to analyze effects of airborne ultra-fine particulate matter from brake wear on plants under realistic exposure conditions

Research on airborne ultrafine particles (UFP) is driven by an increasing awareness of their potential effects on human health and on ecosystems. Brake wear is an important UFP source releasing largely metallic and potentially hazardous emissions. UFP uptake into plant tissues could mediate entry into food webs. Still, the effects of these particles on plants have barely been studied, especially in a realistic setting with aerial exposure. In this study, we established a system designed to mimic airborne exposure to ultrafine brake dust particles and performed experiments with the model species Arabidopsis thaliana. Using advanced analytical methods, we characterized the conditions in our exposure experiments. A comparison with data we obtained on UFP release at different outdoor stations showed that our controlled exposures are within the same order of magnitude regarding UFP deposition on plants at a traffic-heavy site. In order to assess the physiological implications of exposure to brake derived-particles we generated transcriptomic data with RNA sequencing. The UFP treatment led to diverse changes in gene expression, including the deregulation of genes involved in Fe and Cu homeostasis. This suggests a major contribution of metallic UFPs to the elicitation of physiological responses by brake wear derived emissions.

Transcriptomic analysis reveals diverse gene expression changes in airway macrophages during experimental allergic airway disease.

Background: Airway macrophages (AMs) are the most abundant leukocytes in the healthy airway lumen and have a highly specialised but plastic phenotype that is governed by signals in the local microenvironment. AMs are thought to maintain immunological homeostasis in the steady state, but have also been implicated in the pathogenesis of allergic airway disease (AAD). In this study, we aimed to better understand these potentially contrasting AM functions using transcriptomic analysis. Methods: Bulk RNA sequencing was performed on AMs (CD11c + Siglec F + CD64 + CD45 + SSC hi) flow cytometry sorted from C57BL/6 mice during experimental AAD driven by repeated house dust mite inhalation (AMs HDM), compared to control AMs from non-allergic mice. Differentially expressed genes were further analysed by hierarchical clustering and biological pathway analysis. Results: AMs HDM showed increased expression of genes associated with antigen presentation, inflammatory cell recruitment and tissue repair, including several chemokine and matrix metalloproteinase genes. This was accompanied by increased expression of mitochondrial electron transport chain subunit genes and the retinoic acid biosynthetic enzyme gene Raldh2. Conversely, AMs HDM displayed decreased expression of a number of cell cycle genes, genes related to cytoskeletal functions and a subset of genes implicated in antimicrobial innate immunity, such as Tlr5, Il18 and Tnf. Differential gene expression in AMs HDM was consistent with upstream effects of the cytokines IL-4 and IFN-γ, both of which were present at increased concentrations in lung tissue after HDM treatment. Conclusions: These data highlight diverse gene expression changes in the total AM population in a clinically relevant mouse model of AAD, collectively suggestive of contributions to inflammation and tissue repair/remodelling, but with decreases in certain steady state cellular and immunological functions.

Transcriptomic analysis reveals diverse gene expression changes in airway macrophages during experimental allergic airway disease

Background: Airway macrophages (AMs) are the most abundant leukocytes in the healthy airway lumen and have a highly specialised but plastic phenotype that is governed by signals in the local microenvironment. AMs are thought to maintain immunological homeostasis in the steady state, but have also been implicated in the pathogenesis of allergic airway disease (AAD). In this study, we aimed to better understand these potentially contrasting AM functions using transcriptomic analysis. Methods: Bulk RNA sequencing was performed on AMs (CD11c + Siglec F + CD64 + CD45 + SSC hi) flow cytometry sorted from C57BL/6 mice during experimental AAD driven by repeated house dust mite inhalation (AMs HDM), compared to control AMs from non-allergic mice. Differentially expressed genes were further analysed by hierarchical clustering and biological pathway analysis. Results: AMs HDM showed increased expression of genes associated with antigen presentation, inflammatory cell recruitment and tissue repair, including several chemokine and matrix metalloproteinase genes. This was accompanied by increased expression of mitochondrial electron transport chain subunit genes and the retinoic acid biosynthetic enzyme gene Raldh2. Conversely, AMs HDM displayed decreased expression of a number of cell cycle genes, genes related to cytoskeletal functions and a subset of genes implicated in antimicrobial innate immunity, such as Tlr5, Il18 and Tnf. Differential gene expression in AMs HDM was consistent with upstream effects of the cytokines IL-4 and IFN-γ, both of which were present at increased concentrations in lung tissue after HDM treatment. Conclusions: These data highlight diverse gene expression changes in the total AM population in a clinically relevant mouse model of AAD, collectively suggestive of contributions to inflammation and tissue repair/remodelling, but with decreases in certain steady state cellular and immunological functions.

Transcriptomic analysis reveals diverse gene expression changes in airway macrophages during experimental allergic airway disease

Background: Airway macrophages (AMs) are the most abundant leukocytes in the healthy airway lumen and have a highly specialised but plastic phenotype that is governed by signals in the local microenvironment. AMs are thought to maintain immunological homeostasis in the steady state, but have also been implicated in the pathogenesis of allergic airway disease (AAD). In this study, we aimed to better understand these potentially contrasting AM functions using transcriptomic analysis. Methods: Bulk RNA sequencing was performed on AMs flow cytometry sorted from C57BL/6 mice during experimental AAD driven by repeated house dust mite inhalation (AMs HDM), compared to control AMs from non-allergic mice. Differentially expressed genes were further analysed by hierarchical clustering and biological pathway analysis. Results: AMs HDM showed increased expression of genes associated with antigen presentation, inflammatory cell recruitment and tissue repair, including several chemokine and matrix metalloproteinase genes. This was accompanied by increased expression of mitochondrial electron transport chain subunit genes and the retinoic acid biosynthetic enzyme gene Raldh2. Conversely, AMs HDM displayed decreased expression of a number of cell cycle genes, genes related to cytoskeletal functions and a subset of genes implicated in antimicrobial innate immunity, such as Tlr5, Il18 and Tnf. Differential gene expression in AMs HDM was consistent with upstream effects of the cytokines IL-4 and IFN-γ, both of which were present at increased concentrations in lung tissue after HDM treatment. Conclusions: These data highlight diverse gene expression changes in the total AM population in a clinically relevant mouse model of AAD, collectively suggestive of contributions to inflammation and tissue repair/remodelling, but with decreases in certain steady state cellular and immunological functions.

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity.

Proteasome activity is required for diverse cellular processes, including transcriptional and epigenetic regulation. However, inhibiting proteasome activity can lead to an increase in transcriptional output that is correlated with enriched levels of trimethyl H3K4 and phosphorylated forms of RNA polymerase (Pol) II at the promoter and gene body. Here, we perform gene expression analysis and ChIP followed by sequencing (ChIP-seq) in MCF-7 breast cancer cells treated with the proteasome inhibitor MG132, and we further explore genome-wide effects of proteasome inhibition on the chromatin state and RNA Pol II transcription. Analysis of gene expression programs and chromatin architecture reveals that chemically inhibiting proteasome activity creates a distinct chromatin state, defined by spreading of the H3K4me3 mark into the gene bodies of differentially-expressed genes. The distinct H3K4me3 chromatin profile and hyperacetylated nucleosomes at transcription start sites establish a chromatin landscape that facilitates recruitment of Ser-5- and Ser-2-phosphorylated RNA Pol II. Subsequent transcriptional events result in diverse gene expression changes. Alterations of H3K36me3 levels in the gene body reflect productive RNA Pol II elongation of transcripts of genes that are induced, underscoring the requirement for proteasome activity at multiple phases of the transcriptional cycle. Finally, by integrating genomics data and pathway analysis, we find that the differential effects of proteasome inhibition on the chromatin state modulate genes that are fundamental for cancer cell survival. Together, our results uncover underappreciated downstream effects of proteasome inhibitors that may underlie targeting of distinct chromatin states and key steps of RNA Pol II-mediated transcription in cancer cells.

Regulatory discrimination of mRNAs by FMRP controls mouse adult neural stem cell differentiation

Fragile X syndrome (FXS) is caused by the loss of fragile X mental retardation protein (FMRP), an RNA binding protein whose deficiency impacts many brain functions, including differentiation of adult neural stem cells (aNSCs). However, the mechanism by which FMRP influences these processes remains unclear. Here, we performed ribosome profiling and transcriptomic analysis of aNSCs in parallel from wild-type and Fmr1 knockout mice. Our data revealed diverse gene expression changes at both mRNA and translation levels. Many mitosis and neurogenesis genes were dysregulated primarily at the mRNA level, while numerous synaptic genes were mostly dysregulated at the translation level. Translational "buffering", whereby changes in ribosome association with mRNA are compensated by alterations in RNA abundance, was also evident. Knockdown of NECDIN, an FMRP-repressed transcriptional factor, rescued neuronal differentiation. In addition, we discovered that FMRP regulates mitochondrial mRNA expression and energy homeostasis. Thus, FMRP controls diverse transcriptional and posttranscriptional gene expression programs critical for neural differentiation.

Chronic spinal cord changes in a high-fat diet-fed male rat model of thoracic spinal contusion.

Individuals that suffer injury to the spinal cord can result in long-term, debilitating sequelae. Spinal cord-injured patients have increased risk for the development of metabolic disease, which can further hinder the effectiveness of treatments to rehabilitate the cord and improve quality of life. In the present study, we sought to understand the impact of high-fat diet (HFD)-induced obesity on spinal cord injury (SCI) by examining transcriptome changes in the area of the injury and rostral and caudal to site of damage 12 wk after injury. Adult, male Long-Evans rats received either thoracic level contusion of the spinal cord or sham laminectomy and then were allowed to recover on normal rat chow for 4 wk and further on HFD for an additional 8 wk. Spinal cord tissues harvested from the rats were processed for Affymetrix microarray and further transcriptomic analysis. Diverse changes in gene expression were identified in the injured cord in genes such as MMP12, APOC4, GPNMB, and IGF1 and 2. The greatest signaling changes occurred in pathways involved in cholesterol biosynthesis and immune cell trafficking. Together, the cord changes in the chronically obese rat following thoracic SCI reveal further potential targets for therapy. These could be further explored as they overlap with genes involved in metabolic disease.

Abstract 3306: Wnt pathway dynamics in breast cancer progression

Abstract Background: The canonical Wnt/β-catenin signaling pathway directs self-renewal cues for stem cells in multiple tissues. Through alternative receptors, noncanonical Wnt/β-catenin-independent pathways exist, mediating cellular processes that include planar cell polarity, convergent extension, actin/cytoskeletal rearrangements, and in some contexts, antagonism of canonical Wnt signaling. How different Wnt pathways cooperate within heterogeneous cellular landscapes in breast cancers remains an outstanding question. Within the current study, we investigated the role of the alternative Wnt receptor, Ror2, in mediating Wnt/β-catenin-independent functions during tumor progression. Experimental design and methods: A TP53 null transplantable GEM model was used in the current study, which exhibits subtype-specific molecular signatures characteristic of human breast cancers. A lentiviral-based shRNA strategy was developed for gene silencing of noncanonical Wnt pathway components in vivo, with emphasis on the alternative Wnt receptor, Ror2. This strategy was combined with pathway-specific Wnt reporters to survey the interplay of canonical and noncanonical Wnt pathways in vivo. Results: TP53 null mammary tumors differentially expressed the noncanonical Wnt receptor Ror2, with the highest levels of expression evident in basal-like tumors relative to luminal and claudin-low subtypes analyzed. Basal-like tumors also displayed Wnt/β-catenin active populations, which were inversely correlated with Ror2 expression. Lentiviral shRNA silencing of Ror2 within 3 independent basal-like models (T1, T2, 2225L) resulted in elevated Wnt/β-catenin activity along with various phenotypic outcomes, including squamous differentiation. Ror2, therefore, functions to restrain Wnt/β-catenin-dependent signaling in vivo. Despite the similarity in Wnt/β-catenin-dependent signaling outcome upon Ror2 depletion, gene expression profiling of Ror2-intact (shLUC) vs. Ror2-depleted (shRor2) tumors unveiled diverse gene expression changes within basal-like tumor models. Intriguingly, Ror2 silencing within 2225L basal-like tumors resulted in a change in the composition of tumor cell subpopulations. Additionally, gene expression analysis of shLUC vs. shRor2 tumors revealed that genes upregulated in shRor2 tumors were associated with claudin-low features, while genes downregulated were associated with basal-like features, reflecting changes in the cellular landscape upon Ror2 loss. Gene ontology analysis also revealed alterations in actin/cytoskeletal- and cell adhesion-associated genes upon Ror2 depletion. Conclusion: The current study highlights the differential effects of Ror2 loss within TP53 null basal-like models. In particular, Ror2 can regulate the plasticity between basal-like and claudin-low states, impacting cytoskeletal and cell adhesion dynamics. Current efforts are focused on how these changes impact tumor progression and metastasis. Citation Format: Kevin Roarty, Adam D. Pfefferle, Chad J. Creighton, Charles M. Perou, Jeffrey M. Rosen. Wnt pathway dynamics in breast cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3306.

Altered expression of mir-222 and mir-25 influences diverse gene expression changes in transformed normal and anaplastic thyroidcells, and impacts on MEK and TRAIL protein expression.

Thyroid cancer is the most common endocrine malignancy and accounts for the majority of endocrine cancer-related deaths each year. Our group and others have previously demonstrated dysfunctional microRNA (miRNA or miR) expression in the context of thyroid cancer. The objective of the present study was to investigate the impact of synthetic manipulation of expression of miR-25 and miR-222 in benign and malignant thyroid cells. miR-25 and miR-222 expression was upregulated in 8505C (an anaplastic thyroid cell line) and Nthy-ori (a SV40-immortalised thyroid cell line) cells, respectively. A transcriptomics-based approach was utilised to identify targets of the two miRNAs and real-time PCR and western blotting were used to validate a subset of the targets. Almost 100 mRNAs of diverse functions were found to be either directly or indirectly targeted by both miR-222 and miR-25 [fold change ≥2, false discovery rate (FDR) ≤0.05]. Gene ontology analysis showed the miR-25 gene target list to be significantly enriched for genes involved in cell adhesion. Fluidigm real-time PCR technologies were used to validate the downregulation of 23 and 22 genes in response to miR-25 and miR-222 overexpression, respectively. The reduction of the expression of two miR-25 protein targets, TNF-related apoptosis-inducing ligand (TRAIL) and mitogen-activated protein kinase kinase 4 (MEK4), was also validated. Manipulating the expression of both miR-222 and miR-25 influenced diverse gene expression changes in thyroid cells. Increased expression of miR-25 reduced MEK4 and TRAIL protein expression, and cell adhesion and apoptosis are important aspects of miR-25 functioning in thyroid cells.

Transcriptome profiling of brown adipose tissue during cold exposure reveals extensive regulation of glucose metabolism.

We applied digital gene expression profiling to determine the transcriptome of brown and white adipose tissues (BAT and WAT, respectively) during cold exposure. Male C57BL/6J mice were exposed to cold for 2 or 4 days. A notable induction of genes related to glucose uptake, glycolysis, glycogen metabolism, and the pentose phosphate pathway was observed in BAT from cold-exposed animals. In addition, glycerol-3-phosphate dehydrogenase 1 expression was induced in BAT from cold-challenged mice, suggesting increased synthesis of glycerol from glucose. Similarly, expression of lactate dehydrogenases was induced by cold in BAT. Pyruvate dehydrogenase kinase 2 (Pdk2) and Pdk4 were expressed at significantly higher levels in BAT than in WAT, and Pdk2 was induced in BAT by cold. Of notice, only a subset of the changes detected in BAT was observed in WAT. Based on changes in gene expression during cold exposure, we propose a model for the intermediary glucose metabolism in activated BAT: 1) fluxes through glycolysis and the pentose phosphate pathway are induced, the latter providing reducing equivalents for de novo fatty acid synthesis; 2) glycerol synthesis from glucose is increased, facilitating triacylglycerol synthesis/fatty acid re-esterification; 3) glycogen turnover and lactate production are increased; and 4) entry of glucose carbon into the tricarboxylic acid cycle is restricted by PDK2 and PDK4. In summary, our results demonstrate extensive and diverse gene expression changes related to glucose handling in activated BAT.

Study on correlation of signal molecule genes and their receptor-associated genes with rat liver regeneration

To investigate the effect of signal molecules and their receptor-associated genes on rat liver regeneration (LR) at the transcriptional level, the associated genes were originally obtained by retrieving the databases and related scientific publications; their expression profiles in rat LR were then checked using the Rat Genome 230 2.0 microarray. The LR-associated genes were identified by comparing gene expression difference between partial hepatectomy groups and operation-control groups. A total of 454 genes were proved to be LR related. The genes associated with the seven kinds of signal molecules (steroid hormones, fatty acid derivatives, protein and polypeptide hormones, amino acids and their derivatives, choline, cytokines, and gas signal molecules) were detected to be enriched in a cluster characterized by upregulated expression in LR. The number of genes related to the seven kinds of signal molecules was, in sequence, 63, 27, 100, 102, 16, 166, and 18. The 1027 frequencies of upregulation and 823 frequencies of downregulation in total as well as 42 types of different expression patterns suggest the complex and diverse gene expression changes in LR. It is presumed that signal molecules played an important role in metabolism, inflammation, cell proliferation, growth and differentiation, etc., during rat LR.

Gene expression profile analyses of cortical dysplasia by cDNA arrays

Cortical dysplasia (CD) is a well-recognized cause of intractable epilepsy, especially in children and is characterized histologically by derangements in cortical development and organization. The objective of this study was to expand the current knowledge of altered gene expression in CD as a first step towards in the identification of additional genes operative in the evolution of CD. Surgical specimens were obtained from eight patients (4 males and 4 females; age range 2–38 years; mean 15 years) with a pathologic diagnosis of CD. Nondysplastic temporal neocortex was obtained from a 2-year-old boy with intractable epilepsy and medial temporal lobe ganglioglioma. After total RNA isolation from frozen brain tissues, we carried out gene expression profiling using a cDNA expression array. Differences in gene expressions between CD and the nondysplastic neocortex were confirmed by semi-quantitative conventional reverse transcription-PCR. Three genes (recombination activating gene 1 (RAG1), heat shock 60 kDa protein 1 (HSP-60), and transforming growth factor β1 (TGF β1)) were found to be up-regulated more than two-fold in CD, whereas four genes (phosphoinositide-3-kinase regulatory subunit polypeptide 1 [p85 α] (PI3K), frizzled homolog 2 [Drosophila], Bcl-2/adenovirus E1B 19 kDa interacting protein (NIP3), and glia maturation factor β (GMF β)) were down-regulated to less than 50% of their normal levels. Interestingly, the majority of genes showing altered expression were associated with apoptosis. Our study demonstrates diverse changes in gene expression in CD. However, it remains to be shown which of these are causally related to the evolution of CD.