Person:

Leng, Yumei

Cullin-RING ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes, and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. We demonstrate that one substrate, NUSAP1, is an SCFCyclin F substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology and provides a set of proteins likely to be key indicators of cellular physiology.

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS) candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose) polymerase (PARP)-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, similar to 90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins.