Cellular signaling pathway analysis of transcriptomics profiling as a key tool to improve marbling in beef cattle.

A possible approach for artificial meat quality improvement

Susan Steinbusch-Coort, PhD

Dept. Bioinformatics-BiGCaT, NUTRIM, Maastricht University susan.coort@maastrichtuniversity.nl

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Marbling

• Intramuscular fat – the fat present within a cut of meat and between the muscle fibers.

• Breed, genotype, age, diet, husbandry and growth stages.

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Aim

To identify genes and biological pathways regulating marbling of muscle tissue in beef cattle.

Gene 1

Gene 2

Gene 3

Gene 4

Gene 5

Gene 6

Gene 7

Gene 8

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WikiPathways (www.wikipathways.org)

Community curated pathway database

o everybody can contribute and share pathways o everybody can edit and curate pathways o everybody can use the pathway collection o not just diagrams but fully annotated pathways o new findings can be added immediately

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Bos taurus collection at WikiPathways

282 Bt pathways

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Analysis workflow

Adapted from: Kutmon M, Evelo CT, Coort SL. (2014) A network biology workflow to study transcriptomics data of the diabetic liver. BMC Genomics. 15:971. doi: 10.1186/1471-2164-15-971.

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Transcriptomics data

Limousin - Low marbling

Hereford - High marbling

N=4 N=

N=4

Agilent Two-Color Mi Bovine (V2) 4 x 44K Gene Expression Microarray

T. Sadkowski et al “Transcriptional background of beef marbling – Novel genes implicated in intramuscular fat deposition,” Meat Sci., vol. 97, no. 1, pp. 32–41, 2014.

Semitendinosus muscle of 15-month-old bulls

Isolation RNA

Gene expression

Publicly available at ArrayExpress (accession number E-GEOD-46411).

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Differential expression

Differentially expressed genes comparing Hereford with Limousin

- Absolute log10 fold change > 0.11

- Absolute fold change of 1.3

- P-value < 0.05

28,753 genes measured and annotated

2,873 differentially expressed genes

1421 genes

1452 genes

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Over-representation analysis

PathVisio: a tool to edit and analyze biological pathways

Transcriptomics data

Pathway collection

Analysis collection from WikiPathways (www.wikipathways.org)

Differentially expressed

genes

Z-score per pathway

Over-representation analysis

Ranking of pathways based on z-score

Kutmon, M et al. PathVisio 3: An Extendable Pathway Analysis Toolbox. (Accepted for publication in PLOS Computational Biology)

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Pathway Positive(r) Measured(n) Total Z Score P-value Marbling

Hypertrophy Model 8 15 19 3.94 0.000 *

MAPK signaling pathway 34 124 167 3.51 0.002 *

Histone Modifications 13 35 43 3.41 0.000 -

IL-1 signaling pathway 15 41 54 3.17 0.003 *

P38 MAPK signaling pathway 10 27 36 2.98 0.006 *

Cardiac progenitor differentiation 11 33 54 2.72 0.006 -

TCR signaling pathway 19 68 89 2.70 0.011 *

MicroRNAs in cardiomyocyte hypertrophy

20 73 102 2.67 0.006 *

Mitochondrial gene expression 5 11 23 2.66 0.006 *

Physiological and pathological hypertrophy of the heart

7 19 26 2.47 0.012 *

Parkin-Ubiquitin proteasomal system pathway

13 44 64 2.45 0.022 -

Insulin Signaling 27 111 157 2.41 0.012 -

Transcriptional activation by NRF2

5 13 19 2.2 0.020 -

The highest ranked pathways based on Z score in Hereford vs Limousin

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Gene expression in Hereford vs Limousin visualized on the Hypertrophy model.

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Conclusions

• MAPK and P38 MAPK signaling, two pathways well known to affect lipid metabolism, were enriched in the high-marbling breed.

• The signaling pathways “Hypertrophy Model”, “MicroRNAs in cardiomyocyte hypertrophy” and “Physiological and pathological hypertrophy of the heart” that play a role in tissue development were affected.

• Immune response pathways - IL signaling, TCR signaling and Toll-like receptor signaling pathways – were changed.

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Future directions

• Integration of different types of omics data in pathway analysis, like proteomics and metabolomics.

• Effect of environmental factors on marbling and the underlying biological processes.

• Extending the analysis with network analysis to add a regulatory level.

• Analyzing cultured meat quality at a molecular level.

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Acknowledgements

Chris Evelo

Maastricht University (Maastricht – NL)

- Lars Eijssen - Egon Willighagen - Anwesha Bohler-Dutta - Elisa Cirillo - Nuno Nunes - Jonathan Mellius - Linda Rieswijk - Frederieke Ehrhart

Dept. Animal Science, University of Ferdowsi Mashhad (Mashhad – Iran)

Zahra Roudbari

Gladstone Institute (San Francisco - USA) - Alexander Pico - Samad Lotia - Kristina Hanspers

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Questions