Transcription activity is rapidly and transiently paused in response to DNA double‐strand breaks (DSBs) to eliminate production of abnormal transcripts and to avoid deleterious collisions between transcription and repair machineries. Little is known about the mechanisms that ensure transcription repression following DNA damage. Here we describe two novel DNA‐damage responsive proteins, regulated by PARP1 activity, that foster DSB‐induced transcription silencing and promote DSB repair. First, using I‐SceI and CRISPR‐Cas9 endonucleases, we showed that the negative elongation factor member E (NELF‐E), subunit of the NELF complex, is preferentially recruited to DSBs induced upstream transcriptionally active rather than inactive genes. The preferential recruitment of NELF‐E to DNA breakage sites is dependent on the presence of RNA polymerase II [1]. Second, we showed that the chromodomain Y‐like (CDYL1) protein is rapidly recruited to damaged euchromatic regions. In addition, CDYL1 promotes the recruitment of EZH2 methyltransferase, stimulates local increase of the repressive methyl mark H3K27me3 and promotes transcription silencing at DSB sites. Furthermore, the “traffic‐light reporter” system revealed that CDYL1 mainly promotes homology‐directed repair of DSBs. Consequently, CDYL1 knockout cells display synthetic lethality with the chemotherapeutic agent, cisplatin [2]. In the meeting, I will also discuss unpublished data implicating CDYL1 in regulating histone crotonylation at DNA damage sites. Altogether, our findings identify NELF‐E and CDYL1 as novel components that play an essential role in the cellular response to DNA damage.Support or Funding InformationThis work was supported by grants from the Israel Science Foundation (ISF) (grant no. 2021242), the Israel Cancer association (grant no. 2019404), the Binational Science Foundation (grant no. 2023065), the Israel Cancer Research Fund (ICRF) (grant no. 2021762), Volkswagen Foundation (grant no. 2020594). Enas R. Abu‐Zhaiya and Samah W. Awwad are supported by the Council for Higher Education 19 fellowship for outstanding minority MSc and PhD students, respectively. N. Ayoub is supported by the Neubauer Family Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.