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Deep sequencing reveals novel Set7 networks

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Abstract

Background

Methyl-dependent regulation of transcription has expanded from a traditional focus on histones to encompass transcription factor modulation. While the Set7 lysine methyltransferase is associated with pro-inflammatory gene expression in vascular endothelial cells, genome-wide regulatory roles remain to be investigated. From initial characterization of Set7 as specific for methyl-lysine 4 of H3 histones (H3K4m1), biochemical activity toward non-histone substrates has revealed additional mechanisms of gene regulation.

Results

mRNA-Seq revealed transcriptional deregulation of over 8,000 genes in an endothelial model of Set7 knockdown. Gene ontology identified up-regulated pathways involved in developmental processes and extracellular matrix remodeling, whereas pathways regulating the inflammatory response as well as nitric oxide signaling were down-regulated. Chromatin maps derived from ChIP-Seq profiling of H3K4m1 identified several hundred loci with loss of H3K4m1 at gene regulatory elements associated with an unexpectedly subtle effect on gene expression. Transcription factor network analysis implicated six previously described Set7 substrates in mRNA-Seq changes, and we predict that Set7 post-translationally regulates other transcription factors associated with vascular endothelial gene expression through the presence of Set7 amino acid methylation motifs.

Conclusion

We describe a role for Set7 in regulating developmental pathways and response to stimuli (inflammation/immune response) in human endothelial cells of vascular origin. Set7-dependent gene expression changes that occurred independent of H3K4m1 may involve transcription factor lysine methylation events. The method of mapping measured transcriptional changes to transcription factors to identify putative substrates with strong associations to functional changes is applicable to substrate prediction for other broad-substrate histone modifiers.

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Acknowledgments

The authors are thankful for expert bioinformatic support by Antony Kaspi and Dr. Ross Lazarus. The authors acknowledge grant and fellowship support from the Juvenile Diabetes Research Foundation International, the Diabetes Australia Research Trust, the National Health and Medical Research Council (NHMRC) and the National Heart Foundation of Australia. STK is supported by an Australian Postgraduate Award. AE-O is a senior research fellow supported by the NHMRC. Supported in part by the Victorian Government’s Operational Infrastructure Support program.

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Authors and Affiliations

  1. Epigenetics in Human Health and Disease Laboratory, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, Melbourne, VIC, 3004, Australia

    Samuel T. Keating, Mark Ziemann, Jun Okabe, Abdul Waheed Khan, Aneta Balcerczyk & Assam El-Osta

  2. Epigenomics Profiling Facility, Baker IDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, Melbourne, VIC, 3004, Australia

    Mark Ziemann & Assam El-Osta

  3. Department of Pathology, The University of Melbourne, Melbourne, VIC, 3010, Australia

    Assam El-Osta

  4. Department of Medicine, Central Clinical School, Monash University, Melbourne, VIC, 3800, Australia

    Jun Okabe & Assam El-Osta

Authors
  1. Samuel T. Keating
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  2. Mark Ziemann
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  3. Jun Okabe
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  4. Abdul Waheed Khan
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  5. Aneta Balcerczyk
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  6. Assam El-Osta
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Corresponding author

Correspondence to Assam El-Osta.

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Supplementary material 1 (DOCX 26 kb)

18_2014_1651_MOESM2_ESM.png

Supplemental Fig.S1 shRNA knockdown of Set7 confers reproducible and widespread changes in gene expression measured by mRNA-Seq. (A) Shows the extent to which the shRNA reduced the expression of SETD7 measured across three independent biological replicates in mRNA-Seq expressed as read counts per million (CPM) **p < 0.01. (B) Multi-dimensional scaling plot shows separation of control and knockdown samples. (C) Smear plot showing widespread changes in gene expression conferred by specific knockdown of Set7 expression. False discovery rate (FDR). (PNG 298 kb)

18_2014_1651_MOESM3_ESM.jpg

Supplemental Fig.S2 Genome-wide changes to mRNA expression associated with Set7 are regulated by H3K4m1 and transcription factor-dependent mechanisms. The outer ring depicts the positions of genes down-regulated by Set7KD along individual chromosomes. Red bars depicting transcriptional repression events identified by mRNA-Seq show widespread changes in Set7KD cells. The association of gene expression with changes to H3K4m1 patterns determined by ChIP-Seq is shown in green. The abundance of genes associated with transactivity of characterized Set7 substrate NFκB-p65 represented by blue bars partly overlaps the connectivity of putative novel Set7 substrate STAT1 (orange) to transcriptional deregulation. (JPEG 3738 kb)

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Keating, S.T., Ziemann, M., Okabe, J. et al. Deep sequencing reveals novel Set7 networks. Cell. Mol. Life Sci. 71, 4471–4486 (2014). https://doi.org/10.1007/s00018-014-1651-y

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  • DOI: https://doi.org/10.1007/s00018-014-1651-y

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