RPA Interacts with HIRA and Regulates H3.3 Deposition at Gene Regulatory Elements in Mammalian Cells

Abstract

SUMMARY The histone chaperone HIRA is involved in depositing histone variant H3.3 into distinct genic regions including promoters, enhancers and gene bodies. However, how HIRA deposits H3.3 to these regions remains elusive. Through an shRNA screening, we identified single-stranded DNA binding protein RPA as a regulator of the deposition of newly synthesized H3.3 into chromatin. We show that RPA physically interacts with HIRA to form RPA-HIRA-H3.3 complexes, and co-localizes with HIRA and H3.3 at gene promoters and enhancers. Depletion of RPA1, the largest subunit of the RPA complex, dramatically reduces both HIRA association with chromatin and the deposition of newly synthesized H3.3 at promoters and enhancers and leads to altered transcription at gene promoters. These results support a model whereby RPA, best known for its role in DNA replication and repair, recruits HIRA to promoters and enhancers and regulates deposition of newly synthesized H3.3 to these regulatory elements for gene regulation.

The replisome guides nucleosome assembly during DNA replication

“…Therefore, we proposed that RPA could function as a platform for histone chaperones to connect to the replication fork, thereby promoting the coupling of nucleosome assembly with ongoing DNA replication [138]. Consistent with this idea, it has been reported that human RPA can bind the histone chaperone HIRA and histone H3.3-H4 during gene transcription [139], supporting a role of RPA in DNA replication-independent nucleosome assembly via collaboration with histone chaperones. It would be interesting to determine the regulation of the RPA-ssDNA-binding platform and its potential impact on RC nucleosome assembly.…”

Replication Protein A Availability during DNA Replication Stress Is a Major Determinant of Cisplatin Resistance in Ovarian Cancer Cells

“…Collectively, our data suggest that yet-to-be-identified cancer-associated genetic alterations resulting in failure to prevent unscheduled origin activity might enhance chemosensitivity in HGSOC cells by causing RPA exhaustion and SPR defects. We note that although RPA is well characterized for its essential roles in DNA replication and repair, this multifunctional complex has also recently been implicated in transcription (39)(40)(41). Thus, it is interesting to speculate that generation of ssDNA at sites of RNA polymerase stalling at CDDP-damaged sites in DNA might (i) sequester RPA and (ii) promote replication stress via collisions between the blocked transcriptional machineries and DNA polymerases (42).…”

Rtt105 functions as a chaperone for replication protein A to preserve genome stability

“…RPA is an evolutionarily conserved protein complex present in all eukaryotes and regulates both DNA replication initiation and elongation (Wobbe et al , ; Fairman & Stillman, ; Wold & Kelly, ; Brill & Stillman, ; Wold, ). RPA is also important during DNA damage repair and recombination, and RPA‐coated ssDNA plays a role in the activation of the DNA replication checkpoint pathway and the nucleosome assembly pathway (Zou & Elledge, ; Maréchal & Zou, ; Liu et al , ; Zhang et al , ). RPA consists of the three related subunits Rfa1, Rfa2, and Rfa3 in Saccharomyces cerevisiae , with apparent masses of approximately 70, 30, and 14 kDa, respectively (Brill & Stillman, ).…”