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. 2016 Mar 18;44(5):2125-35.
doi: 10.1093/nar/gkv1219. Epub 2015 Nov 17.

The CSB chromatin remodeler and CTCF architectural protein cooperate in response to oxidative stress

Robert J Lake  1 , Erica L Boetefuer  2 , Kyoung-Jae Won  3 , Hua-Ying Fan  4
Affiliations

The CSB chromatin remodeler and CTCF architectural protein cooperate in response to oxidative stress

Robert J Lake et al. Nucleic Acids Res. .

Abstract

Cockayne syndrome is a premature aging disease associated with numerous developmental and neurological abnormalities, and elevated levels of reactive oxygen species have been found in cells derived from Cockayne syndrome patients. The majority of Cockayne syndrome cases contain mutations in the ATP-dependent chromatin remodeler CSB; however, how CSB protects cells from oxidative stress remains largely unclear. Here, we demonstrate that oxidative stress alters the genomic occupancy of the CSB protein and increases CSB occupancy at promoters. Additionally, we found that the long-range chromatin-structure regulator CTCF plays a pivotal role in regulating sites of genomic CSB occupancy upon oxidative stress. We show that CSB directly interacts with CTCF in vitro and that oxidative stress enhances the CSB-CTCF interaction in cells. Reciprocally, we demonstrate that CSB facilitates CTCF-DNA interactions in vitro and regulates CTCF-chromatin interactions in oxidatively stressed cells. Together, our results indicate that CSB and CTCF can regulate each other's chromatin association, thereby modulating chromatin structure and coordinating gene expression in response to oxidative stress.

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Figures

Figure 1.
Figure 1.
Menadione sensitivity assays. (A) CS1AN-sv cells were reconstituted with CSBWT or an empty vector. Stable cell lines expressing transgenes were assayed for viability 24 h after a 1-h menadione treatment with the indicated menadione concentrations. Shown are means ± standard errors of the mean (SEM) (n = 5). A paired t-test was used to determine if the difference in menadione sensitivity of CS1AN cells before and after CSB add-back was significant. Triple asterisks indicate P values < 0.001, and double asterisks indicate P values < 0.01. (B) Analysis of CSB partitioning in cells after a 1-h menadione treatment, with menadione concentrations as indicated. Western blots were probed with antibodies as noted. BRG1 was used as a loading control. GAPDH and acetylated histone H3 were used as markers for soluble and chromatin-enriched fractions, respectively. Total core histones were visualized by Ponceau S staining. (C) Quantification of CSB levels in the soluble versus chromatin-enriched fraction. Shown are means ± SEM (n = 4).
Figure 2.
Figure 2.
Comparison of CSB occupancy in cells with or without menadione treatment. (A) A volcano plot showing the correlation between CSB ChIP-seq results from cells with or without a 1-h menadione treatment (100 μM). (B) Screen shots of CSB ChIP-seq results from seven genomic regions, displayed using the UCSC genome browser. The y-axis is number of normalized sequencing reads. The x-axis represents the genomic coordinates; chrX-1, chrX:73766518–73766600; chr17-1, chr17:49770395–49770537; chr19-2, chr19:45793789–45793877; chrX-2, chrX:48568220–48568299, chr12-7, chr12:13679173–13679256; chr2-2, chr2:180325437–180325517; chr7-1, chr7:2001695–2001793. The directions of nearby transcription (arrows) are noted at the bottom. (C) Validation of CSB ChIP-seq results by ChIP-qPCR. Bar graphs showing CSB ChIP-qPCR results with matched beads-only controls. Shown are means ± SEM (n = 3). A paired t-test was used to determine if the difference in CSB enrichment before and after menadione treatment was significant. Single asterisks indicate P values < 0.05, and double asterisks indicate P values < 0.01. (D–F) Genomic distribution of CSB occupancy sites. The genome was divided into seven categories, as defined by the UCSC RefSeq gene annotation. (D) Menadione-induced CSB occupancy. (E) Common CSB occupancy. (F) Menadione-repressed CSB occupancy.
Figure 3.
Figure 3.
CTCF collaborates with CSB in response to oxidative stress. (A) Motif analysis of CSB ChIP-seq data. (B) Menadione sensitivity assays on CSB expressing and non-expressing (Vector) cells with decreased CTCF levels. Shown are means ± SEM (n = 4). (C) Western blot showing a reduction in the CTCF protein level in cells expressing CTCF shRNA. Relative CTCF levels are shown below the CTCF blot. (D) CSB ChIP-qPCR assays in cells infected with lentivirus expressing control or CTCF shRNA, with or without with a 1-h menadione treatment (100 μM). Shown are means ± SEM (n = 3). A paired t-test was used to determine if the difference in CSB enrichment with and without CTCF shRNA treatment was significant. Asterisks indicate P-values < 0.05.
Figure 4.
Figure 4.
CSB interacts with CTCF in cells and in vitro. (A) Co-immunoprecipitation of CSB and CTCF in 293T transiently transfected with Flag-tagged CTCF, with or without a 1-h treatment of 100 μM menadione. 3.3% of the lysates used for IP were loaded as input. (B) Schematics of recombinant proteins used in (C–E). All CSB derivatives were N-terminally tagged with the Flag epitope. (C) Coomassie-stained gel showing that CSB directly interacts with CTCF. CSB-C, but not CSB-N, is sufficient for the CTCF association. MBP was used as a negative control. (D and E) EMSA assays showing that CSB enhances CTCF association with DNA. (D) Varying amounts of purified MBP-CTCF (lane 2 in C) or MBP (lane 1 in C) were incubated with a 32P-labeled, 200 bp DNA fragment containing a CTCF-binding motif (Supplementary Figure S4B). Protein–DNA complexes were resolved in a native 5% polyacrylamide gel. (E) Varying amounts of purified CSB were incubated with the radiolabeled DNA fragment in the presence or absence of MBP-CTCF. Reactions were subsequently resolved in a 5% native polyacrylamide gel. Protein–DNA complexes marked by ‘•’ and ‘••’ contain the MBP-CTCF protein, as they interacted with an anti-MBP antibody (Supplementary Figure S4A).
Figure 5.
Figure 5.
CSB regulates a subset of CTCF occupancy sites upon oxidative stress. (A) CTCF ChIP-qPCR assays in CSB expressing (WT) and non-expressing (CS1AN) cells, with or without a 1-h menadione treatment (100 μM). Shown are means ± SEM (n = 3). A paired t-test was used to determine if the difference in CTCF enrichment before and after menadione treatment was significant. Single asterisks indicate P values < 0.05, and double asterisks indicate P values < 0.01, as determined by a paired t-test. (B) Model depicting possible modes of CSB–CTCF chromatin association in response to oxidative stress. CTCF can recruit CSB to CTCF-binding sites or enhance the association of CSB with distal sites. See text for details.
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