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Histone H4K20/H3K9 demethylase PHF8 regulates zebrafish brain and craniofacial development

Abstract

X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability1. Causal mutations have been found in approximately 90 X-linked genes2; however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD (plant homeo domain) finger protein 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of 7,000 RefSeq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in upregulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signalling and developmental pathways3. Our findings indicate that an imbalance of histone methylation dynamics has a critical role in XLMR.

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Figure 1: PHF8 and KIA1718 demethylate multiple lysines of histone 3 and 4 in vitro.
Figure 2: PHF8 differently regulates H4K20me1, H3K9me1 and H3K9me2.
Figure 3: Depletion of PHF8 increases H3K9me1, H4K20me1 and L3MBTL1 expression at TSS-bound PHF8 target genes.
Figure 4: zPHF8 regulates brain and craniofacial development in part through regulation of msxb.

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Gene Expression Omnibus

Data deposits

Genome-wide mapping data of PHF8, H4K20me1, H3K9me1, 2 and gene expression microarray analysis in HeLa cells reported in this paper are available at the Gene Expression Omnibus server (http://www.ncbi.nlm.nih.gov/geo/) with the accession numbers GSE21108 and GSE21555, respectively.

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Acknowledgements

We thank Shi laboratory members for helpful discussions and N. Mosammaparast and S. Chen for providing the nucleosomes. We thank G. Rosenfeld for sharing unpublished PHF8 results, and G. Rosenfeld, A. Schier and P. Hinds for discussions. H.H.Q. is a recipient of Ruth L. Kirschstein-National Service Research Award (T32 NS007473 and T32 CA09031-32). N.K.Y. was supported by Harvard SHURP (Summer Honors Undergraduate Research Program). H.L. is a recipient of the Agency for Science, Technology and Research (A*STAR) National Science Scholarship. This work was supported by grants from the National Science Foundation (CBET-0941143) to B.A.G.; NIH (GM 071004 and NCI118487) to Y.S.; a Senior Scholar Grant from the Ellison Foundation, and an NIH PO1 grant (CA50661) to T.M.R.

Author information

Authors and Affiliations

Authors

Contributions

Y.S. and H.H.Q. conceived, designed the study, and co-wrote the manuscript. H.H.Q. performed cloning, demethylation, PHF8 knockdown, conventional ChIP, gene expression, cell cycle analysis and histone profiling experiments. T.M.R. directed the zebrafish work and M.S. performed zebrafish experiments; both T.M.R. and M.S. contributed to the writing of the manuscript. Z.W. performed the ChIP-Seq experiments; G.-Q.H. analysed the ChIP-Seq data; K.Z. directed the ChIP-Seq analysis; Z.W. and G.-Q.H. contributed equally to ChIP-Seq experiments. A.B., D.B.G. and L.B. contributed to the ChIP-chip experiments and data analysis. M.G. and B.A.G. performed mass spectrometry experiments and data analysis. F.L. identified that the PHD domain of PHF8 and KIAA1718 bind to H3K4me3. P.P.O. designed and helped in the cell cycle work. M.H. cloned the full-length KIAA1718 cDNA. N.K.Y. generated mutations in the PHD domain of PHF8 and H.L. helped in the demethylation assays.

Corresponding authors

Correspondence to Thomas M. Roberts or Yang Shi.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-15 with legends and Supplementary Tables 1, 2, 5, 7, 8 and 9 (see separate files for Supplementary Tables 3, 4 and 6). (PDF 1668 kb)

Supplementary Table 3

This table contains ChIP-seq results of PHF8 binding events around TSS. (XLS 1638 kb)

Supplementary Table 4

This table contains ChIP-chip results of PHF8 binding events around TSS. (XLS 283 kb)

Supplementary Table 6

This table contains gene expression microarray data (Phalanx) from HeLa cells and IPA Analysis. (XLS 272 kb)

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Qi, H., Sarkissian, M., Hu, GQ. et al. Histone H4K20/H3K9 demethylase PHF8 regulates zebrafish brain and craniofacial development. Nature 466, 503–507 (2010). https://doi.org/10.1038/nature09261

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