Note for: Systematic E2 screening reveals a UBE2D-RNF138-CtIP axis promoting DNA repair

Note for: Systematic E2 screening reveals a UBE2D-RNF138-CtIP axis promoting DNA repair

(doi: 10.1038/ncb3260)

Very nice piece of work, I would say. It reflects the complexity of living thing is, especially, post-translational modification processes which control the cellular function through adding the small molecule namely ubiquitin to certain protein.

Unrepaired DSB -- genome instability, tumorigenesis, neurodegeneration or premature ageing.

The team used UBE2Ds to screen for the binding partner during the DSB responses (using IR in this case). This study found RNF138 was the binding partner for UBE2Ds during the DSB responses. RNF138 promotes CtIF ubiquitination.

Key to initiating HR is DNA-end resection promoted by CtIP (RBBP8) recruitment to DSB sites, yielding single-stranded DNA (ssDNA) that is rapidly bound by RPA and subsequently replaced by RAD51, leading to strand invasion and ensuing HR processes.

Gap -- how CtIP and early HR events are regulated, however, is not known.

ubiquitination is known to play role in DDR regulator.

2 E1-activating, ~40 E2-conjugating and >600 E3-ligating enzymes, post-translational modification by ubiquitin modulates the stability, localization, activity or interaction properties of proteins.

Gap;

few systematic analyses of human E2s or E3s have been conducted, although recent proteomic approaches have identified hundreds of DDR-regulated ubiquitylation substrates, suggesting that E2s and many E3s with DDR roles await discovery.

Tool to use;

short interfering RNA (siRNA) - screening for E3 partner during the DSB - they use siRNA to downregulate E2 (37 E2s) and observe the phenotype during the DSB.

Cell line that they use as a platform: U2O2

Targeted for siRNA;

1. all known ubiquitin E2s

2. two NEDD8 E2

3. UBE2M

4. UBE2F

Module 1 evaluated impacts of E2 depletions on IRIF kinetics for DDR factors/markers by semi-automated, quantitative high- content, high-throughput (HC/HT) microscopy --> using foci formation as the readout (gH2Ax, 53BP1).

Validating the screening pipeline, depleting the ubiquitin E3s RNF8 and RNF168, or depleting UBE2N (Ubc13) that has known DDR connections, strongly impaired FK2 and 53BP1 but not H2AX IRIF.

Module 2 evaluated HR and mutagenic end-joining (mutEJ) by the traffic-light-reporter (TLR) system in U2OS cells.

proteasome inhibitor -- MG132

we found that depleting many E2s markedly inhibited HR.

Module 3 measured DDR signalling triggered by ionizing radiation treatment that generates DSBs throughout the cell cycle, or camptothecin (CPT), a topoisomerase I poison that yields S-phase DSBs.

Three different readout markers;

1. ATR -- CHK1-P (Ser345)

2. Resection markers -- RPA2-P (Ser4+Ser8)

3. ATM -- KAP1-P

Markers

1. H2Ax -- DNA damage induction

2. CyclinA -- S/G2

UBE2N, -O, -V2, -D, -R, -L and -J family members -- possess DDR functions.

UBE2T and -W linked to the Fanconi anaemia DNA-repair pathway.

it is clear that many E2-siRNA treatments affected HR. with various E2 siRNAs also impairing FK2 IRIF. In contrast, mutEJ effects were less pronounced, possibly reflecting the lower complexity of end-joining than HR.

Owing to their strong phenotypes in multiple screen read-outs.

UBE2Ds function upstream of ssDNA formation to promote ATR signalling and DSB repair by HR.

to identify the UBE2D partner E3(s). Thus, we selected UBE2D-interacting E3s retrieved from several databases (UniProtKB, MINT, STRING and I2D).

Strikingly, when we investigated the resulting six E3s in a semi-automated screen, only RNF138 (RING finger protein 138) depletion substantially impaired ssDNA formation.

RNF138 is a ubiquitin E3 ligase, highly conserved in higher eukaryotes, containing a RING domain, three zinc fingers (ZNFs) and a ubiquitin-interacting motif.

Furthermore, RNF138 depletion mimicked UBE2D1-4 depletion, reducing FK2 and BRCA1 IRIF without markedly influencing 53BP1 or H2AX IRIF.

Like UBE2Ds, RNF138 was required for cellular resistance to ionizing radiation in clonogenic survival and cell proliferation/growth rate assays.

Conversely, RNF8, RNF168 and BRCA1 clearly promote FK2 IRIF formation and HR through BRCA1 recruitment and therefore, operate through mechanisms distinct from the UBE2D-RNF138-CtIP-resection axis.

UBE2Ds and RNF138 can selectively act as a functional pair and play important roles early on in theDDR to promote ionizing-radiation-induced CtIP ubiquitylation.

to gain deeper mechanistic insights into the importance of UBE2Ds-/RNF138-dependent CtIP ubiquitylation for DNA-end resection and CtIP recruitment, we identified 13 ubiquitylated lysines on CtIP by mass spectrometry, immunoprecipitated from irradiated cells.

we have established UBE2D family proteins as important DDR enzymes, and identified RNF138 as a resection/HR-promoting factor that functionally interacts with UBE2Ds and CtIP.

we speculate that CtIP ubiquitylation in this

region may promote these functions by affecting its multimeric state and/or its interactions with other factors.

Our data thereby suggest further mechanistic studies to determine precisely how ubiquitylation of these N-terminal CtIP lysines promotes resection and HR.

Many additional avenues await exploration through exploiting our E2 screening data (yes I agree!).

these studies have highlighted links between various understudied E2 proteins and diverse aspects of the DDR, including DSB repair by HR and mutEJ, IRIF formation and ATM/ATR-mediated signaling.

FK2 antibodiesmost likely recognizemany conjugated ubiquitin species, including dierent chain linkages and/or lengths,we

speculate that these ubiquitylation events requiremultiple E2s, serving as initiators or elongators, and/or ubiquitylating the same protein at multiple sites.

To be short, one protein can be targeted by different E2s.

Collectively, our results suggest that various ubiquitylation events are required for the accumulation of various DDR proteins at DSBs.

Thus, it will be interesting to explore additional roles of UBE2Ds and RNF138 distinct from the CtIP ubiquitylation-resection axis defined here.

We speculate that follow-on studies emanating from our E2 screen will be greatly facilitated by the use of orthogonal data sets, generated from other functional screens together with proteomic, mutational and gene-expression resources.

because small-molecule targeting of DDR

enzymes and ubiquitin system components is providing opportunities for cancer therapies.

Highlight - how certain ubiquitin E2 enzymes with DDR functions may represent attractive therapeutic targets.

PS. Kind of wonder whether we can do this kind of experiments in Thailand...