The nucleosomal barrier to promoter escape by RNA polymerase II is overcome by the chromatin remodeler Chd1
- Fred Hutchinson Cancer Research Center, United States
- Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, United States
- University of Washington School of Medicine, United States
Figures
Figure 1 with 2 supplements
Chd1 is recruited to promoters with high PolII occupancy and requires ATPase activity to extend into the gene body.
(A) Schematic of the domain structure of the full-length mouse Chd1 (1-1711) used to generate the N-terminal FLAG-tagged construct. A region corresponding to the Chd1 ATP-binding pocket is shown …
Figure 1—figure supplement 1
A novel crosslinking ChIP strategy allows near-complete extraction of chromatin-associated proteins and high-resolution mapping of binding sites.
(A) Extraction efficiency of mammalian chromatin associated proteins is low using native conditions. Nuclei from cells expressing FLAG-tagged wildtype Chd1 were prepared by hypotonic lysis. A …
Figure 1—figure supplement 2
Analysis of short fragments provides high-resolution mapping of Chd1 and PolII binding sites at the promoter.
(A) Size class comparison to show crosslinking of Chd1 to the promoter proximal nucleosomes. Mapped paired-end reads recovered from the ChIP-seq experiment were separated based on size and occupancy …
Figure 2 with 1 supplement
Chd1 activity is required to maintain nucleosome occupancy in the promoter region and the gene body.
Cross-linked chromatin was digested with MNase and the DNA fragments were subjected to paired-end sequencing. Mono-nucleosomal fragments (111–140 bp) were aligned relative to the TSS or TES. (A) …
Figure 2—figure supplement 1
Chd1 activity is required to maintain nucleosome occupancy in the promoter region and the gene body.
(A) Cross-linked chromatin was digested with MNase and the DNA fragments were subjected to paired-end sequencing. Mono-nucleosomal fragments (111–140 bp) were aligned relative to the TES. Nucleosome …
Figure 3 with 1 supplement
Chd1 activity has opposing effects on nucleosome turnover at the promoter and the gene body.
Nucleosome turnover is decreased over the promoter but increased over the gene body. The genome-wide average nucleosome turnover was discerned using CATCH-IT at both the (A) TSS and (B) TES. (C) …
Figure 3—figure supplement 1
Chd1 activity suppresses nucleosome turnover in the gene body, and the role of Chd1 is independent of gene length.
(A) Chd1 activity suppresses nucleosome turnover in the gene body. Nucleosome turnover is increased in the gene body in cells lacking Chd1 activity. Average CATCH-IT signal is plotted for the …
Figure 4 with 1 supplement
Chd1 activity is responsible for PolII-directed nucleosome turnover at the promoter.
(A) CATCH-IT data represented as a heatmap for the ±1.5 kb surrounding the TSS. Genes were ranked by the level of PolII occupancy at the promoter (all fragment sizes; density within −100 to +300 …
Figure 4—figure supplement 1
Knockdown of endogenous Chd1 shows a partial reduction in nucleosome turnover at the promoter.
MEFs were transduced with either a lentivirus harboring a short hairpin RNA to Chd1 or an empty vector in order to knockdown expression of Chd1. CATCH-IT was performed to measure nucleosome …
Figure 5
Chd1 activity is required to enable PolII to efficiently escape the promoter proximal region.
Genome-wide distribution of PolII was determined using ChIP-seq with antibodies against (A) the N-terminus of Rpb1 to map total PolII and (B) PolII Ser2phos to map elongating PolII. The recovered …
Figure 6 with 2 supplements
Chd1 functions to alleviate the nucleosomal barrier to PolII transit at highly processive genes.
(A) Schematic describing the calculation of the stalling index. The dark gray shading indicates the total PolII density at the promoter (−100 to +300 bp) as calculated from the ChIP-seq data using …
Figure 6—figure supplement 1
Loss of Chd1 results in an increase in the stalling index, predominantly at processive genes.
(A) Stalling index and traveling ratio are closely correlated. Genes were ranked by increasing stalling index in cells expressing wildtype-Chd1 and then the average stalling index was calculated …
Figure 6—figure supplement 2
The promoters of processive genes have a highly dynamic chromatin barrier to transcription.
(A) Distinct nucleosome organization at processive and stalled genes. Cross-linked chromatin was digested with MNase and the DNA fragments were subjected to paired-end sequencing. Mono-nucleosomal …
Figure 7
Model depicting the role of Chd1 in defining nucleosome dynamics during transcription.
At the promoter of processive genes, Chd1 is recruited directly to the promoter proximal nucleosomes, where it is required for both eviction of existing histone octamers (green) and deposition of …
