ChIP bias as a function of cross-linking time

Abstract

The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins in vivo. Formaldehyde cross-linking of DNA and proteins is a critical step required to trap their interactions inside the cells before immunoprecipitation and analysis. Yet insufficient attention has been given to variables that might give rise to artifacts in this procedure, such as the duration of cross-linking. We analyzed the dependence of the ChIP signal on the duration of formaldehyde cross-linking time for two proteins: DNA topoisomerase 1 (Top1) that is functionally associated with the double helix in vivo, especially with active chromatin, and green fluorescent protein (GFP) that has no known bona fide interactions with DNA. With short time of formaldehyde fixation, only Top1 immunoprecipation efficiently recovered DNA from active promoters, whereas prolonged fixation augmented non-specific recovery of GFP dramatizing the need to optimize ChIP protocols to minimize the time of cross-linking, especially for abundant nuclear proteins. Thus, ChIP is a powerful approach to study the localization of protein on the genome when care is taken to manage potential artifacts.

Keywords: Chromatin immunoprecipitation; DNA topoisomerase 1; Formaldehyde cross-linking; Green fluorescent protein.

Figure 1

A) RNAPII occupancy at promoters and gene bodies of selected genes. Data are visualized as custom tracks in Genome Browser. B) RNAPII ChIP was performed and qPCR quantification at the same promoters as in A) confirmed the accordance between the two experiments. Data are shown as percentage of input DNA (n=2, error bars= s.d.). Mean recovery for non-immune IgG is 0.001.

Figure 2

A) HCT116 cells were transfected with vectors expressing GFP-NLS or GFP-Top1 or mock. 24 hr following the transfection, cultures were analyzed by flow cytometry to verify that GFP-NLS and GFP-Top1 were expressed at the same level. Formaldehyde crosslinking was performed for 4, 10 and 60 minutes. Sonicated chromatin was immunoprecipitated with anti-GFP antibody or non-immune IgG. Recovered DNA was analyzed by qPCR on selected promoters (as in Figure 1). Data are shown as percentage of input DNA (n=3, error bars= s.d.). NT: non transfected control. * Low expressed gene. Mean recovery for non-immune IgG is 0.001. B) Comparison between different times of crosslinking. DNA recovery in the GFP-Top1 transfected cells was normalized for the DNA recovery in the GFP-NLS transfected cells (fold change) to better appreciate the specific promoter enrichment due to fusion with Top1.

Figure 3

A) As in Figure 2A but formaldehyde crosslinking occurred at 25°C for 60 minutes. Data are presented as percentage of input DNA (n=3, error bars= s.d.). Mean recovery for non-immune IgG is 0.0008. B) ChIP enrichment of Top1 at selected promoter regions. Data are represented as percentage of DNA input (n=2, error bars=s.d.). C) Cells were transfected with GFP-NLS or GFP-Top1 (1X) and with 4 times increased amount of these vectors (4X). Cross-linking occurred for 10 minutes. qPCR at expressed and not expressed promoters shows the ratio of non-specific over specific enrichment.

Figure 4

Non-specific chromatin binding of soluble proteins increases with long cross-linking time.