Using an RNA-seq analysis the transcriptional change mediated by PSIP1 in PC-3 and LNCaP prostate cancer cells as compared to normal cells was studied to understand the role of PSIP1 in mediating the cancer specific transcriptional alteration. CUT&Tag-seq for H3K27ac, H3K4me1, H4K16Ac, γH2AX and androgen receptor in control and PSIP1 KD prostate normal and cancer cells was also performed, to know the aberrant enhancer activation. Since this analysis indicated a role for PSIP1 in DNA repair, a proteomics study from immunoprecipitation of endogenous PSIP1 was performed. It identified several proteins involved in transcription, RNA processing, DNA repair and R-loop homeostasis to be interacting with PSIP1. Therefore, the role of PSIP1 in R-loop homeostasis was investigated. Immunoprecipitation and proximity ligation assay (PLA) confirmed the interaction of PSIP1 with the R-loop complex. In normal prostate epithelial cells (RWPE-1) depleted with PSIP1 (PSIP1-KD), resulted in significant increase in R-loop levels as compared to the control as seen by slot blot, immunofluorescence and CUT&Tag-seq. This PSIP1 depletion mediated accumulation of R-loops lead to accumulation of DNA damage in those sites as seen by γ-H2AX western blotting and CUT&Tag. Notably, PSIP1 is enriched at γ-H2AX peaks specific to PSIP1-KD, confirming that γ-H2AX accumulation is due to the direct consequence of PSIP1 depletion. Furthermore, PSIP1 KD cells showed increased number of PLA foci between γ-H2AX and R-loops implying the co-localisation of γ-H2AX and R-loop in PSIP1-KD. This data further confirmed that R-loops that accumulate upon PSIP1 depletion are responsible for increased DNA damage in PSIP1 KD. RNase H1 overexpression and inhibition of RNAPII transcription by triptolide rescued the elevated level of γ-H2AX demonstrating that the elevated DNA damage in the absence of PSIP1 is due to R-loop accumulation and is RNAPII transcription-dependent. Further analysis on genomic context revealed that PSIP1 binding is enriched around gene promoters, and PSIP1 depletion led to an increase in R-loop levels and DNA damage at those promoter sites and gene bodies. Transient transcriptome sequencing (TT-seq) revealed a stark reduction in the nascent transcript levels at R-loop peaks detected explicitly in the PSIP1-KD. Further results indicated that this accumulation of R-loops lead to the collision between transcription and replication machinery leading to the DNA damage. Further, PSIP1 was involved in repair of these DNA damage by facilitating homologous repair pathway. Hence, the sensitivity of cancer cells with low PSIP1 to PARP1 inhibitors and clastogens that cause transcription-coupled DNA damage. Results showed that the prostate cancer cells with lower levels of PSIP1, to more sensitive to PARP1 inhibitor – Olaparib compared to prostate normal cells. Depletion of PSIP1 led to hypersensitivity of prostate cancer cells to illudin-S –a drug that cause transcription coupled DNA damage.
The results of these projects were regularly discussed and disseminated in Lab meetings, joint Lab meetings, QMUL Epigenetics-Hub meetings, Blizard Institute centre seminars and at the Gordon International Research conference on Chromatin Structure and Function.
The results of these projects have been published two open access publications.
