Human HMGN1 and HMGN2 are not required for transcription-coupled DNA repair

Katja Apelt,Martijn S. Luijsterburg,Iris Zoutendijk,Diana van den Heuvel,Dennis Y. Gout,Annelotte P. Wondergem

doi:10.1038/s41598-020-61243-4

Katja Apelt, Martijn S. Luijsterburg + Show 4 more

Open Access

https://doi.org/10.1038/s41598-020-61243-4

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Journal: Scientific Reports	Publication Date: Mar 9, 2020
Citations: 7	License type: open-access

Affiliation: Leiden University Medical Center

Abstract

Transcription-coupled repair (TCR) removes DNA lesions from the transcribed strand of active genes. Stalling of RNA polymerase II (RNAPII) at DNA lesions initiates TCR through the recruitment of the CSB and CSA proteins. The full repertoire of proteins required for human TCR – particularly in a chromatin context - remains to be determined. Studies in mice have revealed that the nucleosome-binding protein HMGN1 is required to enhance the repair of UV-induced lesions in transcribed genes. However, whether HMGN1 is required for human TCR remains unaddressed. Here, we show that knockout or knockdown of HMGN1, either alone or in combination with HMGN2, does not render human cells sensitive to UV light or Illudin S-induced transcription-blocking DNA lesions. Moreover, transcription restart after UV irradiation was not impaired in HMGN-deficient cells. In contrast, TCR-deficient cells were highly sensitive to DNA damage and failed to restart transcription. Furthermore, GFP-tagged HMGN1 was not recruited to sites of UV-induced DNA damage under conditions where GFP-CSB readily accumulated. In line with this, HMGN1 did not associate with the TCR complex, nor did TCR proteins require HMGN1 to associate with DNA damage-stalled RNAPII. Together, our findings suggest that HMGN1 and HMGN2 are not required for human TCR.

Highlights

Transcription-coupled repair (TCR) removes DNA lesions from the transcribed strand of active genes
These findings reveal that loss of HMGN1 is viable in human cells and provide a new tool to study the role of HMGN1 in human cells
We immunoprecipitated endogenous RNAPIIo, which strongly interacted with CSB, CSA and the TFIIH complex after UV irradiation (Fig. 8a). While all these interactions with RNAPIIo were abolished in CSB-KO cells (Fig. 8a), in line with the essential role of this protein in transcription-coupled repair (TCR), the UV-induced association of these TCR proteins with RNAPIIo was not affected in two independent HMGN1/ HMGN2-double knock-out (dKO) clones in either osteosarcoma (Fig. 8a, Supplementary Fig. 2A,B) or retinal epithelial cells (Fig. 8b, Supplementary Fig. 3A,B). These findings show that human HMGN1 does not interact with DNA damage-stalled RNA polymerase II (RNAPII) and associated TCR proteins, and that both HMGN1 and HMGN2 are dispensable for human TCR

Summary

Introduction

Transcription-coupled repair (TCR) removes DNA lesions from the transcribed strand of active genes. Mouse embryonic fibroblasts deficient in HMGN1 show a decreased repair rate of UV-induced DNA lesions in transcribed genes[27], suggesting an involvement in modulating chromatin structure during murine TCR.

Results

Conclusion