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Coordination of the recruitment of the FANCD2 and PALB2 Fanconi anemia proteins by an ubiquitin signaling network

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Abstract

Fanconi anemia (FA) is a chromosome instability syndrome and the 20 identified FA proteins are organized into two main arms which are thought to function at distinct steps in the repair of DNA interstrand crosslinks (ICLs). These two arms include the upstream FA pathway, which culminates in the monoubiquitination of FANCD2 and FANCI, and downstream breast cancer (BRCA)-associated proteins that interact in protein complexes. How, and whether, these two groups of FA proteins are integrated is unclear. Here, we show that FANCD2 and PALB2, as indicators of the upstream and downstream arms, respectively, colocalize independently of each other in response to DNA damage induced by mitomycin C (MMC). We also show that ubiquitin chains are induced by MMC and colocalize with both FANCD2 and PALB2. Our finding that the RNF8 E3 ligase has a role in recruiting FANCD2 and PALB2 also provides support for the hypothesis that the two branches of the FA-BRCA pathway are coordinated by ubiquitin signaling. Interestingly, we find that the RNF8 partner, MDC1, as well as the ubiquitin-binding protein, RAP80, specifically recruit PALB2, while a different ubiquitin-binding protein, FAAP20, functions only in the recruitment of FANCD2. Thus, FANCD2 and PALB2 are not recruited in a single linear pathway, rather we define how their localization is coordinated and integrated by a network of ubiquitin-related proteins. We propose that such regulation may enable upstream and downstream FA proteins to act at distinct steps in the repair of ICLs.

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Acknowledgments

The authors thank the DNA Sequencing Core and Comprehensive Mouse and Cancer Core of CCRF for their services and Dr. Alan D’Andrea (Dana-Farber Cancer Institute, Boston) for PD20 fibroblasts reconstituted with HA-tagged FANCD2. This work was funded by the following grants: NIH R01 HL084082 (A.R.M.) and NIH R01 HL085587 (P.R.A).

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Authors and Affiliations

  1. Division of Experimental Hematology and Cancer Biology, Cancer and Blood Diseases Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229, USA

    Gregory Bick, Fan Zhang, A. Ruhikanta Meetei & Paul R. Andreassen

  2. Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, 45229, USA

    A. Ruhikanta Meetei & Paul R. Andreassen

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  1. Gregory Bick
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  2. Fan Zhang
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Fig. S1

The roles of RNF8 and MDC1 in recruiting PALB2 and FANCD2 to foci, in response to MMC, in a second cell type. a Immunoblot showing depletion of RNF8 and MDC1 in U2OS cells. b-c Quantification of MMC-induced FANCD2 foci (b) or PALB2 foci (c) in U2OS cells transfected with a control siRNA (siLacZ) or with siRNAs directed against RNF8 or MDC1 and treated with 0.5 μM MMC for 16 h. Values in each bar graph represent the mean of three independent counts of at least 150 cells each ±standard deviation. *indicates p < 0.005, **indicates p < 0.05. (GIF 25 kb)

High resolution image (TIFF 219 kb)

Fig. S2

FANCD2 and PALB2 foci display colocalization with γH2AX foci following treatment with MMC. a Quantification of the percentage of corrected FANCD2-deficient PD20 fibroblasts with FANCD2 nuclear foci which display five or more colocalized γH2AX foci at 4 or 16 h of treatment with 0.5 μM MMC. b The percentage of FANCD2-deficient cells, PD20 fibroblasts, corrected with HA-FANCD2, that display three or more FANCD2 (HA), PALB2, or γH2AX foci at different time points of treatment with 0.5 μM MMC. Results for γH2AX foci are added here to those already shown in Fig. 1b for FANCD2 and PALB2. Values in a-b represent the mean + standard deviation of three counts of 150 cells each. c-d Representative images of FANCD2 (c) and PALB2 (d) foci in which colocalization with γH2AX foci is detected. Images of DAPI-stained cells are shown to indicate the position of nuclei. Single channel images are shown in black-white, while merged images with FANCD2 or PALB2 foci in red and γH2AX foci in green are also included to display colocalization. Colocalization is indicated by a yellow signal in the merged images. (GIF 105 kb)

High resolution image (TIFF 1.23 mb)

Fig. S3

Knockdown of RNF8 and MDC1 does not alter cell cycle status. a Representative dot plots of the levels of BrdU versus DNA content measured with propidium iodide that were obtained using flow cytometry. Overlayed separation bins are shown for quantification of different cell cycle phases. The upper bin in each dot plot represents S phase. Bins at the lower left and lower right represent G1 and G2-M, respectively. b A bar graph representing quantification of G1, S, and G2-M in HeLa cells treated with each of the indicated siRNAs. (GIF 47 kb)

High resolution image (TIFF 369 kb)

Fig. S4

RNF8 and MDC1 have distinct roles in the recruitment of FANCD2 and PALB2 in response to ionizing radiation, similar to the response to ICLs. a Quantification of IR-induced FANCD2 or PALB2 foci in HeLa cells transfected with siRNAs targeting LacZ or RNF8 at 16 h following exposure to 10 Gy IR. b Quantification of IR-induced FANCD2 or PALB2 foci in cells treated with siRNAs targeting LacZ or MDC1. Values represent the mean of three independent counts of at least 150 cells each ±standard deviation *indicates p < 0.005. (GIF 18 kb)

High resolution image (TIFF 124 kb)

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Bick, G., Zhang, F., Meetei, A.R. et al. Coordination of the recruitment of the FANCD2 and PALB2 Fanconi anemia proteins by an ubiquitin signaling network. Chromosoma 126, 417–430 (2017). https://doi.org/10.1007/s00412-016-0602-9

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