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Chd1 co-localizes with early transcription elongation factors independently of H3K36 methylation and releases stalled RNA polymerase II at introns.

Park D, Shivram H, Iyer VR - Epigenetics Chromatin (2014)

Bottom Line: Using genome-wide approaches, we found that the loss of Chd1 significantly disrupted nucleosome arrays within the gene bodies of highly transcribed genes.We also found that Chd1 is physically recruited to gene bodies, and that its occupancy specifically corresponds to that of the early elongating form of RNA polymerase, RNAPII Ser 5-P.We also found that deletion of the histone methyltransferase for H3K36 (SET2) did not affect either Chd1 occupancy or nucleosome organization genome-wide.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, TX 78712 USA.

ABSTRACT

Background: Chromatin consists of ordered nucleosomal arrays that are controlled by highly conserved adenosine triphosphate (ATP)-dependent chromatin remodeling complexes. One such remodeler, chromodomain helicase DNA binding protein 1 (Chd1), is believed to play an integral role in nucleosomal organization, as the loss of Chd1 is known to disrupt chromatin. However, the specificity and basis for the functional and physical localization of Chd1 on chromatin remains largely unknown.

Results: Using genome-wide approaches, we found that the loss of Chd1 significantly disrupted nucleosome arrays within the gene bodies of highly transcribed genes. We also found that Chd1 is physically recruited to gene bodies, and that its occupancy specifically corresponds to that of the early elongating form of RNA polymerase, RNAPII Ser 5-P. Conversely, RNAPII Ser 5-P occupancy was affected by the loss of Chd1, suggesting that Chd1 is associated with early transcription elongation. Surprisingly, the occupancy of RNAPII Ser 5-P was affected by the loss of Chd1 specifically at intron-containing genes. Nucleosome turnover was also affected at these sites in the absence of Chd1. We also found that deletion of the histone methyltransferase for H3K36 (SET2) did not affect either Chd1 occupancy or nucleosome organization genome-wide.

Conclusions: Chd1 is specifically recruited onto the gene bodies of highly transcribed genes in an elongation-dependent but H3K36me3-independent manner. Chd1 co-localizes with the early elongating form of RNA polymerase, and affects the occupancy of RNAPII only at genes containing introns, suggesting a role in relieving splicing-related pausing of RNAPII.

No MeSH data available.


Related in: MedlinePlus

Chd1 localization on chromatin is Set2-independent. (A, B) The binding profiles of Chd1 are indistinguishable between WT and set2Δ. (C) shapeDiff analysis confirms that set2Δ has little effect on Chd1 occupancy on a genome wide scale.
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Fig6: Chd1 localization on chromatin is Set2-independent. (A, B) The binding profiles of Chd1 are indistinguishable between WT and set2Δ. (C) shapeDiff analysis confirms that set2Δ has little effect on Chd1 occupancy on a genome wide scale.

Mentions: Based on these lines of evidence, we hypothesized that the critical function of Chd1 at gene bodies is mediated by its recruitment in an H3K36me3-dependent manner. Although Chd1 occupancy has been reported to be independent of H3K36 methylation, no published data is available, and it is unclear how the genome-wide occupancy of Chd1 is affected by levels of H3K36 methylation[30]. To test our hypothesis, we measured the genome-wide occupancy of our epitope-tagged Chd1 after we deleted the H3K36 methyltransferase gene SET2. Examination of the ChIP-seq data confirmed the successful deletion of SET2 in that only a few reads were mapped to the SET2 gene body (see Additional file1: Figure S5A). Immunoblotting revealed that the levels of H3K36me3 were greatly reduced by loss of Set2, while Chd1 expression levels were unaltered (see Additional file1: Figure S5B). However, global Chd1 occupancy in set2Δ appeared identical to that in WT (Figure 6A). No significant changes were detectable in the pattern of Chd1 occupancy at several individual genes (Figure 6B). Additionally, shapeDiff analysis also revealed a high similarity between Chd1 occupancy in WT and set2Δ (median correlation coefficient =0.71) (Figure 6C), which was comparable to the similarity of two independent ChIP-seq datasets that also measured Chd1 occupancy in a WT strain (median correlation coefficient =0.65). Thus, loss of Set2 had no effect on Chd1 occupancy, suggesting that Chd1 occupancy within gene bodies is Set2-independent. This observation is also supported by the fact that set2Δ shows normal nucleosome organization and does not recapitulate the loss of Chd1 [see Additional file1: Figure S6A and S6B]. Therefore, we conclude that Chd1 is recruited onto chromatin in a H3K36 methylation-independent manner, and that methylation at H3K36 has no effect on well-organized nucleosome arrays. Based on the known interacting partners of Chd1 such as the PAF and SAGA complexes and Spt5[14, 15, 17], it is instead possible that early transcription elongation factors are likely to recruit Chd1 to gene bodies.Figure 6


Chd1 co-localizes with early transcription elongation factors independently of H3K36 methylation and releases stalled RNA polymerase II at introns.

Park D, Shivram H, Iyer VR - Epigenetics Chromatin (2014)

Chd1 localization on chromatin is Set2-independent. (A, B) The binding profiles of Chd1 are indistinguishable between WT and set2Δ. (C) shapeDiff analysis confirms that set2Δ has little effect on Chd1 occupancy on a genome wide scale.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230344&req=5

Fig6: Chd1 localization on chromatin is Set2-independent. (A, B) The binding profiles of Chd1 are indistinguishable between WT and set2Δ. (C) shapeDiff analysis confirms that set2Δ has little effect on Chd1 occupancy on a genome wide scale.
Mentions: Based on these lines of evidence, we hypothesized that the critical function of Chd1 at gene bodies is mediated by its recruitment in an H3K36me3-dependent manner. Although Chd1 occupancy has been reported to be independent of H3K36 methylation, no published data is available, and it is unclear how the genome-wide occupancy of Chd1 is affected by levels of H3K36 methylation[30]. To test our hypothesis, we measured the genome-wide occupancy of our epitope-tagged Chd1 after we deleted the H3K36 methyltransferase gene SET2. Examination of the ChIP-seq data confirmed the successful deletion of SET2 in that only a few reads were mapped to the SET2 gene body (see Additional file1: Figure S5A). Immunoblotting revealed that the levels of H3K36me3 were greatly reduced by loss of Set2, while Chd1 expression levels were unaltered (see Additional file1: Figure S5B). However, global Chd1 occupancy in set2Δ appeared identical to that in WT (Figure 6A). No significant changes were detectable in the pattern of Chd1 occupancy at several individual genes (Figure 6B). Additionally, shapeDiff analysis also revealed a high similarity between Chd1 occupancy in WT and set2Δ (median correlation coefficient =0.71) (Figure 6C), which was comparable to the similarity of two independent ChIP-seq datasets that also measured Chd1 occupancy in a WT strain (median correlation coefficient =0.65). Thus, loss of Set2 had no effect on Chd1 occupancy, suggesting that Chd1 occupancy within gene bodies is Set2-independent. This observation is also supported by the fact that set2Δ shows normal nucleosome organization and does not recapitulate the loss of Chd1 [see Additional file1: Figure S6A and S6B]. Therefore, we conclude that Chd1 is recruited onto chromatin in a H3K36 methylation-independent manner, and that methylation at H3K36 has no effect on well-organized nucleosome arrays. Based on the known interacting partners of Chd1 such as the PAF and SAGA complexes and Spt5[14, 15, 17], it is instead possible that early transcription elongation factors are likely to recruit Chd1 to gene bodies.Figure 6

Bottom Line: Using genome-wide approaches, we found that the loss of Chd1 significantly disrupted nucleosome arrays within the gene bodies of highly transcribed genes.We also found that Chd1 is physically recruited to gene bodies, and that its occupancy specifically corresponds to that of the early elongating form of RNA polymerase, RNAPII Ser 5-P.We also found that deletion of the histone methyltransferase for H3K36 (SET2) did not affect either Chd1 occupancy or nucleosome organization genome-wide.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas, 2500 Speedway, Austin, TX 78712 USA.

ABSTRACT

Background: Chromatin consists of ordered nucleosomal arrays that are controlled by highly conserved adenosine triphosphate (ATP)-dependent chromatin remodeling complexes. One such remodeler, chromodomain helicase DNA binding protein 1 (Chd1), is believed to play an integral role in nucleosomal organization, as the loss of Chd1 is known to disrupt chromatin. However, the specificity and basis for the functional and physical localization of Chd1 on chromatin remains largely unknown.

Results: Using genome-wide approaches, we found that the loss of Chd1 significantly disrupted nucleosome arrays within the gene bodies of highly transcribed genes. We also found that Chd1 is physically recruited to gene bodies, and that its occupancy specifically corresponds to that of the early elongating form of RNA polymerase, RNAPII Ser 5-P. Conversely, RNAPII Ser 5-P occupancy was affected by the loss of Chd1, suggesting that Chd1 is associated with early transcription elongation. Surprisingly, the occupancy of RNAPII Ser 5-P was affected by the loss of Chd1 specifically at intron-containing genes. Nucleosome turnover was also affected at these sites in the absence of Chd1. We also found that deletion of the histone methyltransferase for H3K36 (SET2) did not affect either Chd1 occupancy or nucleosome organization genome-wide.

Conclusions: Chd1 is specifically recruited onto the gene bodies of highly transcribed genes in an elongation-dependent but H3K36me3-independent manner. Chd1 co-localizes with the early elongating form of RNA polymerase, and affects the occupancy of RNAPII only at genes containing introns, suggesting a role in relieving splicing-related pausing of RNAPII.

No MeSH data available.


Related in: MedlinePlus