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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 p21 promoter nucleosome 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.   In (a), DNA was isolated from mononucleosomes and amplified by qPCR using overlapping primers tiling the p21 promoter (see Table  S1(c)) and presented relative to the amount of DNA amplified at position −2249. The graph represents the mean result and standard error of three or more independent ChIP experiments.  In (b), knockdown of SRCAP or p400 protein, compared to control-transfected cells, was confirmed by Western blot.  Beta actin was used as a loading control.
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fig1: The density of nucleosomes at the p21 promoter nucleosome 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. In (a), DNA was isolated from mononucleosomes and amplified by qPCR using overlapping primers tiling the p21 promoter (see Table  S1(c)) and presented relative to the amount of DNA amplified at position −2249. The graph represents the mean result and standard error of three or more independent ChIP experiments. In (b), knockdown of SRCAP or p400 protein, compared to control-transfected cells, was confirmed by Western blot. Beta actin was used as a loading control.

Mentions: The results of this approach indicate that the regions of highest mononucleosome DNA density overlaps the same DNA sequence where the highest p400 binding was observed in U2OS cells, from approximately-2668 to-2092 bp upstream of the transcription start site (TSS) (Figure 1(a), black bars). To ask if the nucleosomes are repositioned in the absence of SRCAP or p400, the expression of each protein was reduced by siRNA treatment. Knockdown of SRCAP or p400 was confirmed by Western blot (Figure 1(b)) and did not significantly alter nucleosome density at any position (open and gray bars in Figure 1(a)).


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 p21 promoter nucleosome 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.   In (a), DNA was isolated from mononucleosomes and amplified by qPCR using overlapping primers tiling the p21 promoter (see Table  S1(c)) and presented relative to the amount of DNA amplified at position −2249. The graph represents the mean result and standard error of three or more independent ChIP experiments.  In (b), knockdown of SRCAP or p400 protein, compared to control-transfected cells, was confirmed by Western blot.  Beta actin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3140016&req=5

fig1: The density of nucleosomes at the p21 promoter nucleosome 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. In (a), DNA was isolated from mononucleosomes and amplified by qPCR using overlapping primers tiling the p21 promoter (see Table  S1(c)) and presented relative to the amount of DNA amplified at position −2249. The graph represents the mean result and standard error of three or more independent ChIP experiments. In (b), knockdown of SRCAP or p400 protein, compared to control-transfected cells, was confirmed by Western blot. Beta actin was used as a loading control.
Mentions: The results of this approach indicate that the regions of highest mononucleosome DNA density overlaps the same DNA sequence where the highest p400 binding was observed in U2OS cells, from approximately-2668 to-2092 bp upstream of the transcription start site (TSS) (Figure 1(a), black bars). To ask if the nucleosomes are repositioned in the absence of SRCAP or p400, the expression of each protein was reduced by siRNA treatment. Knockdown of SRCAP or p400 was confirmed by Western blot (Figure 1(b)) and did not significantly alter nucleosome density at any position (open and gray bars in Figure 1(a)).

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