Epigenetic regulation of cell fate determination and functional specification during lung development

• Applicant: Professor Guillermo Barreto, Ph.D.
• Subject Area: Pneumology, Thoracic Surgery ; Biochemistry ; Developmental Biology ; Cell Biology
• Term: from 2010 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 164686135

• Final Report Year: 2017

Final Report Abstract

During the first period, in which the project was funded by the DFG, we performed a siRNA-mediated loss-of-function (LOF) screen and identified 32 general cis-acting factors that direct lung specific gene expression and are required for respiratory cell fate determination. Some of these factors, as GATA6 and NKX2-1, were already known as key regulators of lung development, thereby supporting the quality of our screen. Interestingly, even though they were already known, further characterization of GATA6 and NKX2-1 during the second period of the project led us to the development of a new non-invasive method for early lung cancer (LC) diagnosis using exhaled breath condensates, which will dramatically improve the prognosis of LC patients undergoing classical anti-cancer therapies. About our work was reported in the media, such as newspapers, radio and television. We also found in our screen new regulators of lung development. Hmga2 regulates canonical WNT signaling during lung development at different points of the pathway. Furthermore we deciphered the molecular mechanism of transcription regulation mediated by HMGA2 . We showed that HMGA2-induced transcription requires phosphorylation of the histone variant H2AX at S139 (H2AXS139ph; γ-H2AX), which in turn is mediated by the protein kinase ataxia telangiectasia mutated (ATM). In addition, we demonstrated the biological relevance of this novel mechanism of transcription initiation within the context of TGFβ1 signaling. Our results link H2AXS139ph to transcription, assigning a new function for this classical DNA damage marker. Controlled chromatin opening during transcription may involve intermediates with DNA breaks that may require mechanisms that ensure the integrity of the genome. Due to the abundance of HMG proteins in the cell nucleus, this might be a general mechanism of transcription regulation. In addition, we were able to identify 16 proteins that are enriched in the membrane fraction of alveolar type II (ATII) cells. ATII cells have been related to diverse lung diseases including pulmonary fibrosis, chronic obstructive pulmonary disease, emphysema, cystic fibrosis as well as lung cancer. Characterization of the mechanisms regulating the expansion and differentiation of ATII cells will have a profound impact on the understanding and treatment of lung diseases. The 16 membrane proteins that we have identified will be a powerful tool for the isolation and further characterization of ATII cells. Our work was featured by Global Medical Discovery in 2014 as one of the key scientific articles in medical science in 2013 confirming the translational potential of our findings.

Publications

• Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS One; 2012;7(2):e32366
  Saffarzadeh M, Juenemann C, Queisser MA, Lochnit G, Barreto G, Galuska SP, Lohmeyer J and Preissner KT
  
• Quantitative Proteome Analysis of Alveolar Type-II Cells Reveals a Connection of Integrin Receptor Subunits Beta 2/6 and WNT Signaling. J Proteome Res; 2013 Dec 6;12(12):5598-608
  Mukhametshina RT, Ruhs A, Singh I, Hasan D, Contreras A, Mehta A, Nikam VS, Ahlbrecht K, Carraro G, Cabrera-Fuentes HA, Jiang D, Voswinckel R, Seeger W, Bellusci S, Scharffetter-Kochanek K, Bagaeva TV, Preissner KT, Boettger T, Braun T, Krüger M and Barreto G
  
• Transient Inhibition of FGFR2b-Ligands Signaling Leads to Irreversible Loss of Cellular β-Catenin Organization and Signaling in AER during Mouse Limb Development. PLoS One; 2013 Oct 22;8(10):e7624
  Danopoulos S, Parsa S, Al Alam D, Tabatabai R, Baptista S, Tiozzo C, Carraro G, Wheeler M, Barreto G, Braun T, Li X, Hajihosseini MK and Bellusci S
  
• Hmga2 is required for canonical WNT signaling during lung development. BMC Biol; 2014 Mar 24;12:21
  Singh I, Mehta A, Contreras A, Boettger T, Carraro G, Wheeler M, Cabrera-Fuentes HA, Bellusci S, Seeger W, Braun T and Barreto G
  
• miR-142-3p balances proliferation and differentiation of mesenchymal cells during lung development. Development; 2014 Mar;141(6):1272-81
  Carraro G, Shrestha A, Rostkovius J, Contreras A, Chao CM, El Agha E, Mackenzie B, Dilai S, Guidolin D, Taketo MM, Günther A, Kumar ME, Seeger W, De Langhe S, Barreto G and Bellusci S
  
• Epigenetics in lung cancer diagnosis and therapy. Cancer Metast Rev; 2015 Jun;34(2):229-41
  Mehta A, Dobersch S, Romero-Olmedo AJ and Barreto G
  
• High Mobility Group protein mediated transcription requires DNA damage marker γ-H2AX. Cell Research; 2015 Jul;25(7):837-50
  Singh I, Ozturk N, Mehta A, Cordero J, Hasan D, Cosentino C, Sebastian C, Krüger M, Looso M, Carraro G, Bellusci S, Seeger W, Braun T, Mostoslavsky R and Barreto G
  
• Direct inhibition of oncogenic KRAS by Bacillus pumilus ribonuclease (binase). BBA-Mol Cell Res; 2016 Jul;1863(7 Pt A):1559-67
  Ilinskaya ON, Singh I, Dudkina E, Ulyanova V, Kayumov A and Barreto G
  
• Non-invasive lung cancer diagnosis by detection of GATA6 and NKX2-1 isoforms in exhaled breath condensate. EMBO Mol Med; 2016; 8, 1380-1389
  Mehta A, Cordero J, Dobersch S, Romero-Olmedo AJ, Savai R, Bodner J, Chao CM, Fink L, Guzmán-Díaz E, Singh I, Dobreva G, Rapp UR, Günther S, Ilinskaya ON, Bellusci S, Dammann RH, Braun T, Seeger W, Gattenlöhner S, Tresch A, Günther A and Barreto G