A genome-wide regulatory network of INTS12 associated with pulmonary function

Genome-wide association studies of human lung function and Chronic Obstructive Pulmonary Disease have identified a highly significant and reproducible signal on 4q24. It remains unclear which of the two candidate genes within this locus may regulate lung function: GSTCD, a gene with unknown function, and/or INTS12, a member of the Integrator Complex which is currently thought to mediate 3’end processing of small nuclear RNAs. An interrogation of bioinformatic datasets showed that in lung tissue, 4q24 polymorphisms associated with lung function correlate with INTS12 but not neighboring GSTCD expression. In contrast to the previous reports in other species, a minor alteration of small nuclear RNA processing was observed following INTS12 depletion. RNA sequencing analysis of knockdown cells instead revealed dysregulation of a core subset of genes relevant to airway biology and a robust downregulation of protein synthesis pathways. Consistent with this, protein translation was decreased in INTS12 knockdown cells. In addition, chromatin immunoprecipitation and sequencing experiments demonstrated INTS12 binding throughout the genome, which was enriched in transcriptionally active regions. Finally, INTS12 regulome was defined which includes genes belonging to the protein synthesis pathways. INTS12 has functions beyond the canonical snRNA processing and evidence is presented showing that it regulates translation by directly controlling the expression of genes belonging to protein synthesis pathways. This thesis provides a detailed analysis of INTS12 activities on a genome-wide scale and contributes to the understanding of biology behind the genetic association for lung function at the 4q24

Chloride intracellular channel 1 (CLIC1) contributes to modulation of cyclic AMP-activated whole cell chloride currents in human bronchial epithelial cells

Chloride channels are known to play critical physiological roles in many cell types. Here we describe the expression of anion channels using RNA Seq in primary cultures of human Bronchial Epithelial Cells (hBECs). Chloride intracellular channel (CLIC) family members were the most abundant chloride channel transcripts, and CLIC1 showed the highest level of expression. In addition, we characterise the chloride currents in hBECs and determine how inhibition of CLIC1 via pharmacological and molecular approaches impacts these. We demonstrate that CLIC1 is able to modulate cyclic AMP-induced chloride currents and suggest that CLIC1 modulation could be important for chloride homeostasis in this cell type

Lung function associated gene Integrator Complex subunit 12 regulates protein synthesis pathways

Background: Genetic studies of human lung function and Chronic Obstructive Pulmonary Disease have identified a highly significant and reproducible signal on 4q24. It remains unclear which of the two candidate genes within this locus may regulate lung function: GSTCD, a gene with unknown function, and/or INTS12, a member of the Integrator Complex which is currently thought to mediate 3'end processing of small nuclear RNAs.Results: We found that, in lung tissue, 4q24 polymorphisms associated with lung function correlate with INTS12 but not neighbouring GSTCD expression. In contrast to the previous reports in other species, we only observed a minor alteration of snRNA processing following INTS12 depletion. RNAseq analysis of knockdown cells instead revealed dysregulation of a core subset of genes relevant to airway biology and a robust downregulation of protein synthesis pathways. Consistent with this, protein translation was decreased in INTS12 knockdown cells. In addition, ChIPseq experiments demonstrated INTS12 binding throughout the genome, which was enriched in transcriptionally active regions. Finally, we defined the INTS12 regulome which includes genes belonging to the protein synthesis pathways.Conclusion: INTS12 has functions beyond the canonical snRNA processing. We show that it regulates translation by regulating the expression of genes belonging to protein synthesis pathways. This study provides a detailed analysis of INTS12 activities on a genome-wide scale and contributes to the biology behind the genetic association for lung function at 4q24.</p

The Ser82 RAGE variant affects lung function and serum RAGE in smokers and sRAGE production in vitro

Introduction: Genome-Wide Association Studies have identified associations between lung function measures and Chronic Obstructive Pulmonary Disease (COPD) and chromosome region 6p21 containing the gene for the Advanced Glycation End Product Receptor (AGER, encoding RAGE). We aimed to (i) characterise RAGE expression in the lung, (ii) identify AGER transcripts, (iii) ascertain if SNP rs2070600 (Gly82Ser C/T) is associated with lung function and serum sRAGE levels and (iv) identify whether the Gly82Ser variant is functionally important in altering sRAGE levels in an airway epithelial cell model. Methods: Immunohistochemistry was used to identify RAGE protein expression in 26 human tissues and qPCR was used to quantify AGER mRNA in lung cells. Gene expression array data was used to identify AGER expression during lung development in 38 fetal lung samples. RNA-Seq was used to identify AGER transcripts in lung cells. sRAGE levels were assessed in cells and patient serum by ELISA. BEAS2B-R1 cells were transfected to overexpress RAGE protein with either the Gly82 or Ser82 variant and sRAGE levels identified. Results: Immunohistochemical assessment of 6 adult lung samples identified high RAGE expression in the alveoli of healthy adults and individuals with COPD. AGER/RAGE expression increased across developmental stages in human fetal lung at both the mRNA (38 samples) and protein levels (20 samples). Extensive AGER splicing was identified. The rs2070600T (Ser82) allele is associated with higher FEV1, FEV1/FVC and lower serum sRAGE levels in UK smokers. Using an airway epithelium model overexpressing the Gly82 or Ser82 variants we found that HMGB1 activation of the RAGE-Ser82 receptor results in lower sRAGE production. Conclusions: This study provides new information regarding the expression profile and potential role of RAGE in the human lung and shows a functional role of the Gly82Ser variant. These findings advance our understanding of the potential mechanisms underlying COPD particularly for carriers of this AGER polymorphism

Sixteen new lung function signals identified through 1000 Genomes Project reference panel imputation

Lung function measures are used in the diagnosis of chronic obstructive pulmonary disease. In 38,199 European ancestry individuals, we studied genome-wide association of forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC) and FEV1/FVC with 1000 Genomes Project (phase 1)-imputed genotypes and followed up top associations in 54,550 Europeans. We identify 14 novel loci (P <5 x 10(-8)) in or near ENSA, RNU5F-1, KCNS3, AK097794, ASTN2, LHX3, CCDC91, TBX3, TRIP11, RIN3, TEKT5, LTBP4, MN1 and AP1S2, and two novel signals at known loci NPNT and GPR126, providing a basis for new understanding of the genetic determinants of these traits and pulmonary diseases in which they are altered.Peer reviewe

A genome-wide regulatory network of INTS12 associated with pulmonary function

Genome-wide association studies of human lung function and Chronic Obstructive Pulmonary Disease have identified a highly significant and reproducible signal on 4q24. It remains unclear which of the two candidate genes within this locus may regulate lung function: GSTCD, a gene with unknown function, and/or INTS12, a member of the Integrator Complex which is currently thought to mediate 3’end processing of small nuclear RNAs. An interrogation of bioinformatic datasets showed that in lung tissue, 4q24 polymorphisms associated with lung function correlate with INTS12 but not neighboring GSTCD expression. In contrast to the previous reports in other species, a minor alteration of small nuclear RNA processing was observed following INTS12 depletion. RNA sequencing analysis of knockdown cells instead revealed dysregulation of a core subset of genes relevant to airway biology and a robust downregulation of protein synthesis pathways. Consistent with this, protein translation was decreased in INTS12 knockdown cells. In addition, chromatin immunoprecipitation and sequencing experiments demonstrated INTS12 binding throughout the genome, which was enriched in transcriptionally active regions. Finally, INTS12 regulome was defined which includes genes belonging to the protein synthesis pathways. INTS12 has functions beyond the canonical snRNA processing and evidence is presented showing that it regulates translation by directly controlling the expression of genes belonging to protein synthesis pathways. This thesis provides a detailed analysis of INTS12 activities on a genome-wide scale and contributes to the understanding of biology behind the genetic association for lung function at the 4q24

Dataset for: CHLORIDE INTRACELLULAR CHANNEL 1 (CLIC1) CONTRIBUTES TO MODULATION OF CYCLIC AMP-ACTIVATED WHOLE CELL CHLORIDE CURRENTS IN HUMAN BRONCHIAL EPITHELIAL CELLS

Chloride channels are known to play critical physiological roles in many cell types. Here we describe the expression of anion channels using RNA Seq in primary cultures of human Bronchial Epithelial Cells (hBECs). Chloride intracellular channel (CLIC) family members were the most abundant chloride channel transcripts, and CLIC1 showed the highest level of expression. In addition, we characterise the chloride currents in hBECs and determine how inhibition of CLIC1 via pharmacological and molecular approaches impacts these. We demonstrate that CLIC1 is able to modulate cyclic AMP-induced chloride currents and suggest that CLIC1 modulation could be important for chloride homeostasis in this cell type

<i>AGER</i> mRNA expression levels in total lung and airway cells.

<p>Q-PCR analysis identified <i>AGER</i> mRNA was highly expressed in total lung and at lower levels in human airway smooth muscle cells (HASM) cells, human bronchial epithelial cells (HBEC) and the BEAS-2BR1 bronchial epithelial cell line, (n = 3).</p

<i>AGER</i> isoform expression in three HBEC donors using RNA Seq.

<p>Structure and abundance of known <i>AGER</i> isoforms in three human bronchial epithelial cell donors illustrating heterogeneity in expression levels. Percentage abundances (% FPKM) were calculated for each donor. Transcripts for full length and soluble <i>AGER</i> were identified at similar low abundancies. FPKM; fragments per kilobase of transcript per million mapped reads.</p

Gene expression array data of 38 fetal lungs for the <i>AGER</i> probes.

<p>Expression intensities for the <i>AGER</i> probes 210081_at and 217046_s_at were plotted against the gestational age of each fetal lung sample and showed an increase in expression with fetal lung age. RMA; Robust Multi-array Average. Affymetrix U133 Plus 2 expression array probe 210081_at probes for exon 8 of AGER mRNA whilst 217046_s_at probes AGER exons 7–11’.</p