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Immunoaffinity purification allows for the purification of epitope-tagged proteins and their associated multisubunit complexes from mammalian cells. Subsequent identification of the proteins by proteomic analysis enables unbiased biochemical characterization of their associated partners, potentially revealing the physiological or functional context of any given protein. Here, we use immunoaffinity isolation of the Activating Signal Co-integrator Complex (ASCC) from human cells as an example, demonstrating the utility of the approach in revealing protein complexes involved in genotoxic stress responses.

The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance., SOX2 is required for the maintenance of glioblastoma stem cells (GSCs). Here the authors identify that the RING family E3 ubiquitin ligase TRIM26 promotes SOX2 stability in a non-canonical ligase-independent manner and thus, increases the tumorigenicity of GSCs.

Chen et al. show that XLF functions to limit fork reversal during DNA replication. H2AX prevents MRE11-dependent replication stress in XLF-deficient cells, suggesting that H2AX prevents the resection of regressed arms at reversed forks., XRCC4-like factor (XLF) is a non-homologous end joining (NHEJ) DNA double strand break repair protein. However, XLF deficiency leads to phenotypes in mice and humans that are not necessarily consistent with an isolated defect in NHEJ. Here we show that XLF functions during DNA replication. XLF undergoes cell division cycle 7–dependent phosphorylation; associates with the replication factor C complex, a critical component of the replisome; and is found at replication forks. XLF deficiency leads to defects in replication fork progression and an increase in fork reversal. The additional loss of H2AX, which protects DNA ends from resection, leads to a requirement for ATR to prevent an MRE11-dependent loss of newly synthesized DNA and activation of DNA damage response. Moreover, H2ax−/−:Xlf−/− cells exhibit a marked dependence on the ATR kinase for survival. We propose that XLF and H2AX function in series to prevent replication stress induced by the MRE11-dependent resection of regressed arms at reversed replication forks.

Background Reversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifications and whether cells distinguish aberrant from canonical methylations. Methods Here we use quantitative mass spectrometry to determine the fate of chemically induced methylbases in the mRNA of human cells. Concomitant alteration in the mRNA binding proteome was analyzed by SILAC mass spectrometry. Results MMS induced prominent direct mRNA methylations that were chemically identical to endogenous methylbases. Transient loss of 40S ribosomal proteins from isolated mRNA suggests that aberrant methylbases mediate arrested translational initiation and potentially also no-go decay of the affected mRNA. Four proteins (ASCC3, YTHDC2, TRIM25 and GEMIN5) displayed increased mRNA binding after MMS treatment. ASCC3 is a binding partner of the DNA/RNA demethylase ALKBH3 and was recently shown to promote disassembly of collided ribosomes as part of the ribosome quality control (RQC) trigger complex. We find that ASCC3-deficient cells display delayed removal of MMS-induced 1-methyladenosine (m1A) and 3-methylcytosine (m3C) from mRNA and impaired formation of MMS-induced P-bodies. Conclusions Our findings conform to a model in which ASCC3-mediated disassembly of collided ribosomes allows demethylation of aberrant m1A and m3C by ALKBH3. Our findings constitute first evidence of selective sanitation of aberrant mRNA methylbases over their endogenous counterparts and warrant further studies on RNA-mediated effects of chemical alkylators commonly used in the clinic.

Cellular RNAs are modified by both physiological factors and exogenous agents, such as methyl methanesulfonate (MMS). However, techniques for analyzing how proteins may interact with these modified RNAs are limited. Here, we provide a protocol combining RNA immunoprecipitation (RIP) with mass spectrometry (MS) to analyze the methylation state of the RNAs bound by Flag-tagged proteins in mammalian cells. The approach is highly quantitative and can simultaneously detect several methylated nucleosides in a single experiment. For complete details on the use and execution of this protocol, please refer to Tsao et al. (2021).

Adding to the cross-presentation family Immune responses to viral or tumor antigens are typically initiated by the process of cross-presentation. Cross-presentation is believed to be the major way that innate immune cells, such as the classical dendritic cell 1 (cDC1) subset, activate and prime immunological T cells. Theisen et al. used CRISPR-based screening to identify regulators of cross-presentation by cDC1s (see the Perspective by Barbet and Blander). One such regulator that was identified, WDFY4 (WD repeat- and FYVE domain–containing protein 4), was required for cross-presentation of cell- and bacterial-associated antigens. WDFY4 played a critical role in cDC1-mediated viral and tumor immunity yet did not seem necessary for major histocompatibility complex class II presentation or for cross-presentation by monocyte-derived DCs. Science , this issue p. 694 ; see also p. 641

SUMMARY Early B cell development is regulated by stage-specific transcription factors. PU.1, an ETS-family transcription factor, is essential for coordination of early B cell maturation and immunoglobulin gene (Ig) rearrangement. Here we show that RAG DNA double-strand breaks (DSBs) generated during Ig light chain gene (Igl) rearrangement in pre-B cells induce global changes in PU.1 chromatin binding. RAG DSBs activate a SPIC/BCLAF1 transcription factor complex that displaces PU.1 throughout the genome and regulates broad transcriptional changes. SPIC recruits BCLAF1 to gene-regulatory elements that control expression of key B cell developmental genes. The SPIC/BCLAF1 complex suppresses expression of the SYK tyrosine kinase and enforces the transition from large to small pre-B cells. These studies reveal that RAG DSBs direct genome-wide changes in ETS transcription factor activity to promote early B cell development., In Brief ETS-family transcription factors are key regulators of early B cell development. Soodgupta et al. show that RAG-induced DNA breaks generated during antigen receptor gene recombination activate a SPIC/BCLAF1 transcription factor complex that counters PU.1 activity and regulates gene expression changes to promote transition from large to small pre-B cells., Graphical Abstract

The TPX2/Aurora A heterodimeric kinase canonically orchestrates mitotic events. Byrum et al. identify two new roles for this complex in regulating DNA double-stranded break repair and the protection of DNA forks during replication stress., 53BP1 is a chromatin-associated protein that regulates the DNA damage response. In this study, we identify the TPX2/Aurora A heterodimer, nominally considered a mitotic kinase complex, as a novel binding partner of 53BP1. We find that TPX2/Aurora A plays a previously unrecognized role in DNA damage repair and replication fork stability by counteracting 53BP1 function. Loss of TPX2 or Aurora A compromises DNA end resection, BRCA1 and Rad51 recruitment, and homologous recombination. Furthermore, loss of TPX2 or Aurora A causes deprotection of stalled replication forks upon replication stress induction. This fork protection pathway counteracts MRE11 nuclease activity but functions in parallel to BRCA1. Strikingly, concurrent loss of 53BP1 rescues not only BRCA1/Rad51 recruitment but also the fork instability induced upon TPX2 loss. Our work suggests the presence of a feedback mechanism by which 53BP1 is regulated by a novel binding partner and uncovers a unique role for 53BP1 in replication fork stability.

Ubiquitination is a major mechanism that regulates numerous cellular processes, including autophagy, DNA damage signaling, and inflammation. While hundreds of ubiquitin ligases exist to conjugate ubiquitin onto substrates, approximately one hundred deubiquitinases are encoded by the human genome. Thus, deubiquitinases are likely regulated by unidentified mechanisms to target distinct substrates and cellular functions. Here, we demonstrate that the deubiquitinase OTUD4, which nominally encodes a K48-specific deubiquitinase, is phosphorylated near its catalytic domain, activating a latent K63-specific deubiquitinase. Besides phosphorylation, this latter activity requires an adjacent ubiquitin-interacting motif, which increases the affinity of OTUD4 for K63-linked chains. We reveal the Toll-like receptor (TLR) associated factor MyD88 as a target of this K63 deubiquitinase activity. Consequently, TLR-mediated activation of NF-κB is negatively regulated by OTUD4, and macrophages from Otud4(−/−) mice exhibit increased inflammatory signaling upon TLR stimulation. Our results reveal insights into how a deubiquitinase may modulate diverse processes through post-translational modification.

Summary 53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5′-3′ nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 “end-blocking” at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.

The response to DNA damage intersects with many other physiological processes in the cell, such as DNA replication, chromatin remodeling, and the cell cycle. Certain damaging lesions, such as UV-induced pyrimidine dimers, also strongly block RNA polymerases, necessitating the coordination of the repair mechanism with remodeling of the elongating transcriptional machinery, in a process called transcription-coupled nucleotide excision repair (TC-NER). This pathway is typically not thought to be engaged with smaller lesions such as base alkylation. However, recent work has uncovered the potential for shared molecular components between the cellular response to alkylation and UV damage. Here, we review our current understanding of the alkylation damage response and its impacts on RNA biogenesis. We give particular attention to the Activating Signal Cointegrator Complex (ASCC), which plays important roles in the transcriptional response during UV damage as well as alkylation damage reversal, and intersects with trichothiodystrophy, an inherited disease associated with TC-NER.

The modulator of retrovirus infection (MRI or CYREN) is a 30-kDa protein with a conserved N-terminal Ku-binding motif (KBM) and a C-terminal XLF-like motif (XLM). We show that MRI is intrinsically disordered and interacts with many DNA damage response (DDR) proteins, including the kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs and the classical non-homologous end joining (cNHEJ) factors Ku70, Ku80, XRCC4, XLF, PAXX, and XRCC4. MRI forms large multimeric complexes that depend on its N and C termini and localizes to DNA double-strand breaks (DSBs), where it promotes the retention of DDR factors. Mice deficient in MRI and XLF exhibit embryonic lethality at a stage similar to those deficient in the core cNHEJ factors XRCC4 or DNA ligase IV. Moreover, MRI is required for cNHEJ-mediated DSB repair in XLF-deficient lymphocytes. We propose that MRI is an adaptor that, through multivalent interactions, increases the avidity of DDR factors to DSB-associated chromatin to promote cNHEJ.

The resection of broken DNA ends is required for DNA double-strand break (DSB) repair by homologous recombination (HR) but can inhibit normal repair by nonhomologous end joining (NHEJ), the main DSB repair pathway in G1-phase cells. Antigen receptor gene assembly proceeds through DNA DSB intermediates generated in G1-phase lymphocytes by the RAG endonuclease. These DSBs activate ATM, which phosphorylates H2AX, forming γ-H2AX in flanking chromatin. γ-H2AX prevents CtIP from initiating resection of RAG DSBs. Whether there are additional proteins required to promote resection of these DNA ends is not known. KRAB-associated protein 1 (KAP-1) (TRIM28) is a transcriptional repressor that modulates chromatin structure and has been implicated in the repair of DNA DSBs in heterochromatin. Here, we show that in murine G1-phase lymphocytes, KAP-1 promotes resection of DSBs that are not protected by H2AX and its downstream effector 53BP1. In these murine cells, KAP-1 activity in DNA end resection is attenuated by a single-amino-acid change that reflects a KAP-1 polymorphism between primates and other mammalian species. These findings establish KAP-1 as a component of the machinery that can resect DNA ends in G1-phase cells and suggest that there may be species-specific features to this activity.

The cellular response to DNA double-stranded breaks (DSBs) involves a conserved mechanism of recruitment and activation of numerous proteins involved in this pathway. The events that trigger this response in mammalian cells involve several post-translational modifications, but the role of non-proteasomal ubiquitin signaling is particularly central to this pathway. Recent work has demonstrated that ubiquitination does not act alone, but in concert with other post-translational modifications, including phosphorylation, methylation, acetylation, ADP-ribosylation, and other ubiquitin-like modifiers, particularly SUMOylation. We review novel and exciting crosstalk mechanisms between ubiquitination and other post-translational modifications, many of which work synergistically with each other to activate signaling events and help recruit important DNA damage effector proteins, particularly BRCA1 (breast cancer 1, early onset) and 53BP1 (tumor protein p53 binding protein 1), to sites of DNA damage.

The E3 ubiquitin ligase RNF168 recruits LSD1 to DNA damage sites, where it reduces histone methylation upstream of 53BP1 recruitment during the DNA damage response., Histone demethylation is known to regulate transcription, but its role in other processes is largely unknown. We report a role for the histone demethylase LSD1/KDM1A in the DNA damage response (DDR). We show that LSD1 is recruited directly to sites of DNA damage. H3K4 dimethylation, a major substrate for LSD1, is reduced at sites of DNA damage in an LSD1-dependent manner. The E3 ubiquitin ligase RNF168 physically interacts with LSD1 and we find this interaction to be important for LSD1 recruitment to DNA damage sites. Although loss of LSD1 did not affect the initial formation of pH2A.X foci, 53BP1 and BRCA1 complex recruitment were reduced upon LSD1 knockdown. Mechanistically, this was likely a result of compromised histone ubiquitylation preferentially in late S/G2. Consistent with a role in the DDR, knockdown of LSD1 resulted in moderate hypersensitivity to γ-irradiation and increased homologous recombination. Our findings uncover a direct role for LSD1 in the DDR and place LSD1 downstream of RNF168 in the DDR pathway.

The importance of histone methylation in gene regulation was suggested over 40 years ago. Yet, the dynamic nature of this histone modification was recognized only recently, with the discovery of the first histone demethylase nearly five years ago. Since then, our insight into the mechanisms, structures, and macromolecular complexes of these enzymes has grown exponentially. Overall, the evidence strongly supports a key role for histone demethylases in eukaryotic transcription and other chromatin-dependent processes. Here, we examine these and related facets of histone demethylases discovered to date, focusing on their biochemistry, structure, and enzymology.

The nuclear import of histones is a prerequisite for the downstream deposition of histones to form chromatin. However, the coordinate regulation of these processes remains poorly understood. Here we demonstrate that Kap114p, the primary karyopherin/importin responsible for the nuclear import of histones H2A and H2B, modulates the deposition of histones H2A and H2B by the histone chaperone Nap1p. We show that a complex comprising Kap114p, histones H2A and H2B, and Nap1p is present in the nucleus and that the presence of this complex is specifically promoted by Nap1p. This places Kap114p in a position to modulate Nap1p function, and we demonstrate by the use of two different assay systems that Kap114p inhibits Nap1p-mediated chromatin assembly. The inhibition of H2A and H2B deposition by Kap114p results in the concomitant inhibition of RCC1 loading onto chromatin. Biochemical evidence suggests that the mechanism by which Kap114p modulates histone deposition primarily involves direct histone binding, while the interaction between Kap114p and Nap1p plays a secondary role. Furthermore, we found that the inhibition of histone deposition by Kap114p is partially reversed by RanGTP. Our results indicate a novel mechanism by which cells can regulate histone deposition and establish a coordinate link between histone nuclear import and chromatin assembly.

The karyopherin beta (or importin beta) family comprises soluble transport factors that mediate the movement of proteins and RNAs between the nucleus and cytoplasm. Recent studies have extended the role of karyopherins to regulating assembly of the nuclear pore complex (NPC), assembly of the nuclear envelope, mitosis and replication. New data also address how karyopherins specifically recognize and transport many distinct cargoes and traverse the NPC. These data raise the possibility that, although there might be a universal mechanism for nuclear transport, specific interactions between karyopherins and components of the NPC might function to regulate differentially the ability of the different karyopherins to cross the NPC.