The p400 and SRCAP (Snf2-related CBP activator protein) complexes remodel chromatin by catalyzing deposition of histone H2A.Z into nucleosomes. This remodeling activity has been proposed as a basis for regulation of transcription by these complexes. Transcript levels of p21 or Sp1 mRNAs after knockdown of p400 or SRCAP reveals that each regulates transcription of these promoters differently. In this study, we asked whether deposition of H2A.Z within specific nucleosomes by p400 or SRCAP dictates transcriptional activity. Our data indicates that nucleosome density at specific p21 or Sp1 promoter positions is not altered by the loss of either remodeling complex. However, knockdown of SRCAP or p400 reduces deposition of H2A.Z~50% into all p21 and Sp1 promoter nucleosomes. Thus, H2A.Z deposition is not targeted to specific nucleosomes. These results indicate that the deposition of H2A.Z by the p400 or SRCAP complexes is not sufficient to determine how each regulates transcription. This conclusion is further supported by studies that demonstrate a SRCAPΔATP mutant unable to deposit H2A.Z has similar transcriptional activity as wild-type SRCAP. 1. Introduction The histone variant H2A.Z has been shown to have multiple functions in mammalian cells. It is essential for embryonic development, proper segregation of chromosomes [1, 2], and a number of studies indicate it plays a role in both activation and repression of transcription [3–5]. Aberrant H2A.Z expression may also play a role in some human diseases, since it has been demonstrated to play a role in cardiac hypertrophy [6] and H2A.Z levels are elevated in breast cancer [7, 8]. Recent studies have examined the genomewide distribution of H2A.Z in human cells. These studies found that nucleosomes located in promoter regions are highly enriched in H2A.Z, indicating a positive correlation that exists between gene activity and H2A.Z deposition [9, 10]. The deposition of H2A.Z at these sites has been hypothesized to promote organization of nucleosomes at the promoter providing the optimal architecture for activation of transcription. How incorporation of H2A.Z into nucleosomes functions to increase promoter organization and contribute to regulation of transcription is not clear. Several studies indicate that nucleosomes comprised of recombinant H2A.Z or native chicken erythrocyte H2A.Z are more stable than H2A-containing nucleosomes [11, 12]. Other studies, however, have raised the possibility that in certain circumstances, when nucleosomes also contain the histone variant H3.3, H2A.Z decreases nucleosome
References
[1]
R. Faast, V. Thonglairoam, T. C. Schulz et al., “Histone variant H2A.Z is required for early mammalian development,” Current Biology, vol. 11, no. 15, pp. 1183–1187, 2001.
[2]
D. Rangasamy, I. Greaves, and D. J. Tremethick, “RNA interference demonstrates a novel role for H2A.Z in chromosome segregation,” Nature Structural and Molecular Biology, vol. 11, no. 7, pp. 650–655, 2004.
[3]
M. M. Wong, L. K. Cox, and J. C. Chrivia, “The chromatin remodeling protein, SRCAP, is critical for deposition of the histone variant H2A.Z at promoters,” Journal of Biological Chemistry, vol. 282, no. 36, pp. 26132–26139, 2007.
[4]
Z. Wang, C. Zang, J. A. Rosenfeld et al., “Combinatorial patterns of histone acetylations and methylations in the human genome,” Nature Genetics, vol. 40, no. 7, pp. 897–903, 2008.
[5]
N. Gévry, M. C. Ho, L. Laflamme, D. M. Livingston, and L. Gaudreau, “p21 transcription is regulated by differential localization of histone H2A.Z,” Genes and Development, vol. 21, no. 15, pp. 1869–1881, 2007.
[6]
I. Y. Chen, J. Lypowy, J. Pain et al., “Histone H2A.z is essential for cardiac myocyte hypertrophy but opposed by silent information regulator 2α,” Journal of Biological Chemistry, vol. 281, no. 28, pp. 19369–19377, 2006.
[7]
A. Svotelis, N. Gévry, G. Grondin, and L. Gaudreau, “H2A.Z overexpression promotes cellular proliferation of breast cancer cells,” Cell Cycle, vol. 9, no. 2, pp. 364–370, 2010.
[8]
S. Hua, C. B. Kallen, R. Dhar et al., “Genomic analysis of estrogen cascade reveals histone variant H2A.Z associated with breast cancer progression,” Molecular Systems Biology, vol. 4, article 188, 2008.
[9]
A. Barski, S. Cuddapah, K. Cui et al., “High-resolution profiling of histone methylations in the human genome,” Cell, vol. 129, no. 4, pp. 823–837, 2007.
[10]
D. E. Schones, K. Cui, S. Cuddapah et al., “Dynamic regulation of nucleosome positioning in the human genome,” Cell, vol. 132, no. 5, pp. 887–898, 2008.
[11]
Y. J. Park, P. N. Dyer, D. J. Tremethick, and K. Luger, “A new fluorescence resonance energy transfer approach demonstrates that the histone variant H2AZ stabilizes the histone octamer within the nucleosome,” Journal of Biological Chemistry, vol. 279, no. 23, pp. 24274–24282, 2004.
[12]
A. A. Thambirajah, D. Dryhurst, T. Ishibashi, A. Li, A. H. Maffey, and J. Ausió, “H2A.Z stabilizes chromatin in a way that is dependent on core histone acetylation,” Journal of Biological Chemistry, vol. 281, no. 29, pp. 20036–20044, 2006.
[13]
C. Jin and G. Felsenfeld, “Nucleosome stability mediated by histone variants H3.3 and H2A.Z,” Genes and Development, vol. 21, no. 12, pp. 1519–1529, 2007.
[14]
C. Jin, C. Zang, G. Wei et al., “H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions,” Nature Genetics, vol. 41, no. 8, pp. 941–945, 2009.
[15]
A. Thakar, P. Gupta, T. Ishibashi et al., “H2A.Z and H3.3 histone variants affect nucleosome structure: biochemical and biophysical studies,” Biochemistry, vol. 48, no. 46, pp. 10852–10857, 2009.
[16]
D. D. Ruhl, J. Jin, Y. Cai et al., “Purification of a human SRCAP complex that remodels chromatin by incorporating the histone variant H2A.Z into nucleosomes,” Biochemistry, vol. 45, no. 17, pp. 5671–5677, 2006.
[17]
Y. Cai, J. Jin, C. Tomomori-Sato et al., “Identification of new subunits of the multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase complex,” Journal of Biological Chemistry, vol. 278, no. 44, pp. 42733–42736, 2003.
[18]
Y. Cai, J. Jin, L. Florens et al., “The mammalian YL1 protein is a shared subunit of the TRRAP/TIP60 histone acetyltransferase and SRCAP complexes,” Journal of Biological Chemistry, vol. 280, no. 14, pp. 13665–13670, 2005.
[19]
M. A. Monroy, D. D. Ruhl, X. Xu, D. K. Granner, P. Yaciuk, and J. C. Chrivia, “Regulation of cAMP-responsive element-binding protein-mediated transcription by the SNF2/SWI-related protein, SRCAP,” Journal of Biological Chemistry, vol. 276, no. 44, pp. 40721–40726, 2001.
[20]
J. H. Park, X. J. Sun, and R. G. Roeder, “The SANT domain of p400 ATPase represses acetyltransferase activity and coactivator function of TIP60 in basal p21 gene expression,” Molecular and Cellular Biology, vol. 30, no. 11, pp. 2750–2761, 2010.
[21]
P. D. Hartley and H. D. Madhani, “Mechanisms that specify promoter nucleosome location and identity,” Cell, vol. 137, no. 3, pp. 445–458, 2009.
[22]
R. M. Raisner, P. D. Hartley, M. D. Meneghini et al., “Histone variant H2A.Z Marks the 5′ ends of both active and inactive genes in euchromatin,” Cell, vol. 123, no. 2, pp. 233–248, 2005.
[23]
J. C. Eissenberg, M. Wong, and J. C. Chrivia, “Human SRCAP and Drosophila melanogaster DOM are homologs that function in the Notch signaling pathway,” Molecular and Cellular Biology, vol. 25, no. 15, pp. 6559–6569, 2005.
[24]
A. Flaus, C. Rencurel, H. Ferreira, N. Wiechens, and T. Owen-Hughes, “Sin mutations alter inherent nucleosome mobility,” EMBO Journal, vol. 23, no. 2, pp. 343–353, 2004.
[25]
B. Guillemette, A. R. Bataille, N. Gévry et al., “Variant histone H2A.z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning,” PLoS Biology, vol. 3, no. 12, article 384, pp. 1–11, 2005.
[26]
J. A. Goldman, J. D. Garlick, and R. E. Kingston, “Chromatin remodeling by imitation switch (ISWI) class ATP-dependent remodelers is stimulated by histone variant H2A.Z,” Journal of Biological Chemistry, vol. 285, no. 7, pp. 4645–4651, 2010.
[27]
N. Gévry, S. Hardy, P. é. Jacques et al., “Histone H2A.Z is essential for estrogen receptor signaling,” Genes and Development, vol. 23, no. 13, pp. 1522–1533, 2009.
[28]
M. Adam, F. Robert, M. Larochelle, and L. Gaudreau, “H2A.Z is required for global chromatin integrity and for recruitment of RNA polymerase II under specific conditions,” Molecular and Cellular Biology, vol. 21, no. 18, pp. 6270–6279, 2001.
[29]
Y. Wan, R. A. Saleem, A. V. Ratushny et al., “Role of the histone variant H2A.Z/Htz1p in TBP recruitment, chromatin dynamics, and regulated expression of oleate-responsive genes,” Molecular and Cellular Biology, vol. 29, no. 9, pp. 2346–2358, 2009.
[30]
M. Larochelle and L. Gaudreau, “H2A.Z has a function reminiscent of an activator required for preferential binding to intergenic DNA,” EMBO Journal, vol. 22, no. 17, pp. 4512–4522, 2003.
[31]
M. J. Clarkson, J. R. E. Wells, F. Gibson, R. Saint, and D. J. Tremethick, “Regions of variant histone His2AvD required for Drosophila development,” Nature, vol. 399, no. 6737, pp. 694–697, 1999.
[32]
R. K. Suto, M. J. Clarkson, D. J. Tremethick, and K. Luger, “Crystal structure of a nucleosome core particle containing the variant histone H2A.Z,” Nature Structural Biology, vol. 7, no. 12, pp. 1121–1124, 2000.
