Regulation of the p300 HAT domain via a novel activation loop

Article metrics

Abstract

The transcriptional coactivator p300 is a histone acetyltransferase (HAT) whose function is critical for regulating gene expression in mammalian cells. However, the molecular events that regulate p300 HAT activity are poorly understood. We evaluated autoacetylation of the p300 HAT protein domain to determine its function. Using expressed protein ligation, the p300 HAT protein domain was generated in hypoacetylated form and found to have reduced catalytic activity. This basal catalytic rate was stimulated by autoacetylation of several key lysine sites within an apparent activation loop motif. This post-translational modification and catalytic regulation of p300 HAT activity is conceptually analogous to the activation of most protein kinases by autophosphorylation. We therefore propose that this autoregulatory loop could influence the impact of p300 on a wide variety of signaling and transcriptional events.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Purification and partial proteolysis of p300(1195–1673) HAT domain.
Figure 2: Expression and purification of semisynthetic p300 HAT domain by expressed protein ligation.
Figure 3: Autoacetylation of p300.
Figure 4: A proteolytically sensitive loop region that regulates p300 HAT activity.
Figure 5: Role of p300 autoacetylation in vivo.

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. 1

    Ogryzko, V.V., Schiltz, R.L., Russanova, V., Howard, B.H. & Nakatani, Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953– 959 (1996).

  2. 2

    Bannister, A.J. & Kouzarides, T. The CBP co-activator is a histone acetyltransferase. Nature 384, 641– 643 (1996).

  3. 3

    Goodman, R.H. & Smolik, S. CBP/p300 in cell growth, transformation, and development. Genes Dev. 14, 1553– 1577 (2000).

  4. 4

    Gayther, S.A. et al. Mutations truncating the EP300 acetylase in human cancers. Nat. Genet. 24, 300– 303 (2000).

  5. 5

    Borrow, J. et al. The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein. Nat. Genet. 14, 33– 41 (1996).

  6. 6

    Bandyopadhyay, D. et al. Down-regulation of p300/CBP histone acetyltransferase activates a senescence checkpoint in human melanocytes. Cancer Res. 62, 6231– 6239 (2002).

  7. 7

    Muraoka, M. et al. p300 gene alterations in colorectal and gastric carcinomas. Oncogene 12, 1565– 1569 (1996).

  8. 8

    Deguchi, K. et al. MOZ-TIF2-induced acute myeloid leukemia requires the MOZ nucleosome binding motif and TIF2-mediated recruitment of CBP. Cancer Cell 3, 259– 271 (2003).

  9. 9

    Ross, C.A. Polyglutamine pathogenesis: emergence of unifying mechanisms for Huntington's disease and related disorders. Neuron 35, 819– 822 (2002).

  10. 10

    Gusterson, R.J., Jazrawi, E., Adcock, I.M. & Latchman, D.S. The transcriptional co-activators CREB-binding protein (CBP) and p300 play a critical role in cardiac hypertrophy that is dependent on their histone acetyltransferase activity. J. Biol. Chem. 278, 6838– 6847 (2003).

  11. 11

    Chan, H.M. & La Thangue, N.B. p300/CBP proteins: HATs for transcriptional bridges and scaffolds. J. Cell Sci. 114, 2363– 2373 (2001).

  12. 12

    Girdwood, D. et al. p300 Transcriptional repression is mediated by SUMO modification. Mol. Cell 11, 1043– 1054 (2003).

  13. 13

    Yadav, N. et al. Specific protein methylation defects and gene expression perturbations in coactivator-associated arginine methyltransferase 1-deficient mice. Proc. Natl. Acad. Sci. USA 100, 6464– 6468 (2003).

  14. 14

    Chevillard-Briet, M., Trouche, D. & Vandel, L. Control of CBP co-activating activity by arginine methylation. EMBO J. 21, 5457– 5466 (2002).

  15. 15

    Banerjee, A.C. et al. The adenovirus E1A 289R and 243R proteins inhibit the phosphorylation of p300. Oncogene 9, 1733– 1737 (1994).

  16. 16

    Yaciuk, P. & Moran, E. Analysis with specific polyclonal antiserum indicates that the E1A-associated 300-kDa product is a stable nuclear phosphoprotein that undergoes cell cycle phase-specific modification. Mol. Cell. Biol. 11, 5389– 5397 (1991).

  17. 17

    Grossman, S.R. et al. Polyubiquitination of p53 by a ubiquitin ligase activity of p300. Science 300, 342– 344 (2003).

  18. 18

    Hamamori, Y. et al. Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein twist and adenoviral oncoprotein E1A. Cell 96, 405– 413 (1999).

  19. 19

    Thompson, P.R., Kurooka, H., Nakatani, Y. & Cole, P.A. Transcriptional coactivator protein p300. Kinetic characterization of its histone acetyltransferase activity. J. Biol. Chem. 276, 33721– 33729 (2001).

  20. 20

    Roth, S.Y., Denu, J.M. & Allis, C.D. Histone acetyltransferases. Annu. Rev. Biochem. 70, 81– 120 (2001).

  21. 21

    Marmorstein, R. Structure of histone acetyltransferases. J. Mol. Biol. 311, 433– 444 (2001).

  22. 22

    Bordoli, L., Netsch, M., Luthi, U., Lutz, W. & Eckner, R. Plant orthologs of p300/CBP: conservation of a core domain in metazoan p300/CBP acetyltransferase-related proteins. Nucleic Acids Res. 29, 589– 597 (2001).

  23. 23

    Muir, T.W., Sondhi, D. & Cole, P.A. Expressed protein ligation: a general method for protein engineering. Proc. Natl. Acad. Sci. USA 95, 6705– 6710 (1998).

  24. 24

    Evans, T.C. Jr., Benner, J. & Xu, M.Q. Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci. 7, 2256– 2264 (1998).

  25. 25

    Biemann, K. Contributions of mass spectrometry to peptide and protein structure. Biomed. Environ. Mass Spectrom. 16, 99– 111 (1988).

  26. 26

    Costanzo, A. et al. DNA damage-dependent acetylation of p73 dictates the selective activation of apoptotic target genes. Mol. Cell 9, 175– 186 (2002).

  27. 27

    Hong, R. & Chakravarti, D. The human proliferating cell nuclear antigen regulates transcriptional coactivator p300 activity and promotes transcriptional repression. J. Biol. Chem. 278, 44505– 44513 (2003).

  28. 28

    Seo, S.B. et al. Regulation of histone acetylation and transcription by INHAT, a human cellular complex containing the set oncoprotein. Cell 104, 119– 130 (2001).

  29. 29

    Turlais, F. et al. High-throughput screening for identification of small molecule inhibitors of histone acetyltransferases using scintillating microplates (FlashPlate). Anal. Biochem. 298, 62– 68 (2001).

  30. 30

    Lau, O.D. et al. HATs off: selective synthetic inhibitors of the histone acetyltransferases p300 and PCAF. Mol. Cell 5, 589– 595 (2000).

  31. 31

    Huse, M. & Kuriyan, J. The conformational plasticity of protein kinases. Cell 109, 275– 282 (2002).

  32. 32

    Adams, J.A. Activation loop phosphorylation and catalysis in protein kinases: is there functional evidence for the autoinhibitor model? Biochemistry 42, 601– 607 (2003).

  33. 33

    Winston, F. & Allis, C.D. The bromodomain: a chromatin-targeting module? Nat. Struct. Biol. 6, 601– 604 (1999).

  34. 34

    Riddles, P.W., Blakeley, R.L. & Zerner, B. Reassessment of Ellman's reagent. Methods Enzymol. 91, 49– 60 (1983).

  35. 35

    Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673– 4680 (1994).

  36. 36

    Huang, Z.Q., Li, J., Sachs, L.M., Cole, P.A. & Wong, J. A role for cofactor-cofactor and cofactor-histone interaction in targeting of CBP/p300, SWI/SNF and mediator for transcription. EMBO J. 22, 2146– 2155 (2003).

Download references

Acknowledgements

This work was supported by grants from the US National Institutes of Health to P.A.C. and J.W. and from the Ellison Medical Foundation to P.A.C., by a Canadian Institutes for Health Research postdoctoral fellowship to P.R.T., and by grants from AIRC, MURST-Cofin and MURST-FIRB to M.L. W.A. was supported by a National Research Fellowship Award. We thank C. Wolberger, M. Ott and J. Boeke for helpful discussions and for reagents. We thank N. Rust for technical assistance. We thank D. Leahy, R. Alani and J. Liu for comments on the manuscript.

Author information

Correspondence to Philip A Cole.

Ethics declarations

Competing interests

Q.G. works for Cell Signaling Technology Inc. and could benefit by selling antibody. R.J.C. is a paid consultant for Shimadzu Co. and has licensed his invention to the company.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Thompson, P., Wang, D., Wang, L. et al. Regulation of the p300 HAT domain via a novel activation loop. Nat Struct Mol Biol 11, 308–315 (2004) doi:10.1038/nsmb740

Download citation

Further reading