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Loss of H2A.Z Is Not Sufficient to Determine Transcriptional Activity of Snf2-Related CBP Activator Protein or p400 Complexes.

Bowman TA, Wong MM, Cox LK, Baldassare JJ, Chrivia JC - Int J Cell Biol (2011)

Bottom Line: However, knockdown of SRCAP or p400 reduces deposition of H2A.Z∼50% into all p21 and Sp1 promoter 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 SRCAP(ΔATP ) mutant unable to deposit H2A.Z has similar transcriptional activity as wild-type SRCAP.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 South Grand Boulevard, Saint Louis, MO 63104, USA.

ABSTRACT
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 SRCAP(ΔATP ) mutant unable to deposit H2A.Z has similar transcriptional activity as wild-type SRCAP.

No MeSH data available.


Related in: MedlinePlus

The density of nucleosomes at the Sp1 promoter is not altered in the absence of SRCAP or p400.  A549 cells were transfected with control, SRCAP or p400 siRNA and harvested 72 hours later.  DNA was isolated from mononucleosomes and amplified by qPCR using overlapping primer sets tiling the Sp1 promoter (see Table  S2) and presented relative to the amount of DNA amplified at position −1241. The graph represents the mean result and standard error of three or more independent ChIP experiments.
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fig4: The density of nucleosomes at the Sp1 promoter is not altered in the absence of SRCAP or p400. A549 cells were transfected with control, SRCAP or p400 siRNA and harvested 72 hours later. DNA was isolated from mononucleosomes and amplified by qPCR using overlapping primer sets tiling the Sp1 promoter (see Table  S2) and presented relative to the amount of DNA amplified at position −1241. The graph represents the mean result and standard error of three or more independent ChIP experiments.

Mentions: To determine whether H2A.Z plays a similar role at other promoters, we also examined H2A.Z deposition into nucleosomes at the Sp1 promoter. This promoter was chosen because knockdown of SRCAP and p400 affects transcription of Sp1 differently than p21. At the Sp1 promoter, knockdown of SRCAP decreases transcription [3], whereas loss of p400 has no effect on transcription (Figure 3). To measure nucleosome density, sixteen primer sets were designed, flanking the SRCAP-binding site in the Sp1 promoter that was previously characterized [3]. Examination of the Sp1 promoter indicates that it contains nucleosomes at several positions including directly downstream of the TSS (Figure 4, black bars). This result is consistent with the findings of a recent genomewide survey of human promoters that indicate that several strongly phased nucleosomes flank the TSS of most expressed genes [10]. The Sp1 promoter, however, also contains a large nucleosome free region (NFR) at −788 to −60, where SRCAP binds the promoter. Knockdown of p400 or SRCAP expression did not alter formation of the NFR nor did it alter the density of nucleosomes at any position (gray and open bars in Figure 4).


Loss of H2A.Z Is Not Sufficient to Determine Transcriptional Activity of Snf2-Related CBP Activator Protein or p400 Complexes.

Bowman TA, Wong MM, Cox LK, Baldassare JJ, Chrivia JC - Int J Cell Biol (2011)

The density of nucleosomes at the Sp1 promoter is not altered in the absence of SRCAP or p400.  A549 cells were transfected with control, SRCAP or p400 siRNA and harvested 72 hours later.  DNA was isolated from mononucleosomes and amplified by qPCR using overlapping primer sets tiling the Sp1 promoter (see Table  S2) and presented relative to the amount of DNA amplified at position −1241. The graph represents the mean result and standard error of three or more independent ChIP experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140016&req=5

fig4: The density of nucleosomes at the Sp1 promoter is not altered in the absence of SRCAP or p400. A549 cells were transfected with control, SRCAP or p400 siRNA and harvested 72 hours later. DNA was isolated from mononucleosomes and amplified by qPCR using overlapping primer sets tiling the Sp1 promoter (see Table  S2) and presented relative to the amount of DNA amplified at position −1241. The graph represents the mean result and standard error of three or more independent ChIP experiments.
Mentions: To determine whether H2A.Z plays a similar role at other promoters, we also examined H2A.Z deposition into nucleosomes at the Sp1 promoter. This promoter was chosen because knockdown of SRCAP and p400 affects transcription of Sp1 differently than p21. At the Sp1 promoter, knockdown of SRCAP decreases transcription [3], whereas loss of p400 has no effect on transcription (Figure 3). To measure nucleosome density, sixteen primer sets were designed, flanking the SRCAP-binding site in the Sp1 promoter that was previously characterized [3]. Examination of the Sp1 promoter indicates that it contains nucleosomes at several positions including directly downstream of the TSS (Figure 4, black bars). This result is consistent with the findings of a recent genomewide survey of human promoters that indicate that several strongly phased nucleosomes flank the TSS of most expressed genes [10]. The Sp1 promoter, however, also contains a large nucleosome free region (NFR) at −788 to −60, where SRCAP binds the promoter. Knockdown of p400 or SRCAP expression did not alter formation of the NFR nor did it alter the density of nucleosomes at any position (gray and open bars in Figure 4).

Bottom Line: However, knockdown of SRCAP or p400 reduces deposition of H2A.Z∼50% into all p21 and Sp1 promoter 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 SRCAP(ΔATP ) mutant unable to deposit H2A.Z has similar transcriptional activity as wild-type SRCAP.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 1402 South Grand Boulevard, Saint Louis, MO 63104, USA.

ABSTRACT
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 SRCAP(ΔATP ) mutant unable to deposit H2A.Z has similar transcriptional activity as wild-type SRCAP.

No MeSH data available.


Related in: MedlinePlus