Tony T. Huang

FANCI is a second monoubiquitinated member of the Fanconi anemia pathway

Activation of the Fanconi anemia (FA) DNA damage–response pathway results in the monoubiquitination of FANCD2, which is regulated by the nuclear FA core ubiquitin ligase complex. A FANCD2 protein sequence–based homology search facilitated the discovery of FANCI, a second monoubiquitinated component of the FA pathway. Biallelic mutations in the gene coding for this protein were found in cells from four FA patients, including an FA-I reference cell line.

The Zinc Finger Domain of NEMO Is Selectively Required for NF-κB Activation by UV Radiation and Topoisomerase Inhibitors

ABSTRACT Exposure of mammalian cells to UV radiation was proposed to stimulate the transcription factor NF-κB by a unique mechanism. Typically, rapid and strong inducers of NF-κB, such as tumor necrosis factor alpha (TNF-α) and bacterial lipopolysaccharide (LPS), lead to rapid phosphorylation and proteasomal degradation of its inhibitory protein, IκBα. In contrast, UV, a relatively slower and weaker inducer of NF-κB, was suggested not to require phosphorylation of IκBα for its targeted degradation by the proteasome. We now provide evidence to account for this peculiar degradation process of IκBα. The phospho-IκBα generated by UV is only detectable by expressing a ΔF-box mutant of the ubiquitin ligase β-TrCP, which serves as a specific substrate trap for serine 32 and 36 phosphorylated IκBα. In agreement with this finding, we also find that the IκB kinase (IKK) phospho-acceptor sites on IκBα, core components of the IKK signalsome, and IKK catalytic activity are all required for UV signaling. Furthermore, deletion and point mutation analyses reveal that both the amino-terminal IKK-binding and the carboxy-terminal putative zinc finger domains of NEMO (IKKγ) are critical for UV-induced NF-κB activation. Interestingly, the zinc finger domain is also required for NF-κB activation by two other slow and weak inducers, camptothecin and etoposide. In contrast, the zinc finger module is largely dispensable for NF-κB activation by the rapid and strong inducers LPS and TNF-α. Thus, we suggest that the zinc finger domain of NEMO likely represents a point of convergence for signaling pathways initiated by slow and weak NF-κB-activating conditions.

Postrepression Activation of NF-κB Requires the Amino-Terminal Nuclear Export Signal Specific to IκBα

ABSTRACT One of the most prominent NF-κB target genes in mammalian cells is the gene encoding one of its inhibitor proteins, IκBα. The increased synthesis of IκBα leads to postinduction repression of nuclear NF-κB activity. However, it is unknown why IκBα, among multiple IκB family members, is involved in this process and what significance this feedback regulation has beyond terminating NF-κB activity. Herein, we report an important IκBα-specific function dictated by its amino-terminal nuclear export sequence (N-NES). The IκBα N-NES is necessary for the postinduction export of nuclear NF-κB, which is a critical event in reestablishing a permissive condition for NF-κB to be rapidly reactivated. We show that although IκBα and another IκB member, IκBβ, can enter the nucleus and repress NF-κB DNA-binding activity during the postinduction phase, only IκBα allows the efficient export of nuclear NF-κB. Moreover, swapping the N-terminal region of IκBβ for the corresponding IκBα sequence is sufficient for the IκB chimera protein to export NF-κB similarly to IκBα during the postinduction state. Our findings provide a mechanistic explanation of why IκBα but not other IκB members is crucial for postrepression activation of NF-κB. We propose that this IκBα-specific function is important for certain physiological and pathological conditions where NF-κB needs to be rapidly reactivated.

Dysregulation of DNA polymerase κ recruitment to replication forks results in genomic instability

Translesion synthesis polymerases (TLS Pols) are required to tolerate DNA lesions that would otherwise cause replication arrest and cell death. Aberrant expression of these specialized Pols may be responsible for increased mutagenesis and loss of genome integrity in human cancers. The molecular events that control the usage of TLS Pols in non‐pathological conditions remain largely unknown. Here, we show that aberrant recruitment of TLS Polκ to replication forks results in genomic instability and can be mediated through the loss of the deubiquitinase USP1. Moreover, artificial tethering of Polκ to proliferating cell nuclear antigen (PCNA) circumvents the need for its ubiquitin‐binding domain in the promotion of genomic instability. Finally, we show that the loss of USP1 leads to a dramatic reduction of replication fork speed in a Polκ‐dependent manner. We propose a mechanism whereby reversible ubiquitination of PCNA can prevent spurious TLS Pol recruitment and regulate replication fork speed to ensure the maintenance of genome integrity.

Role of Deubiquitinating Enzymes in DNA Repair.

ABSTRACT Both proteolytic and nonproteolytic functions of ubiquitination are essential regulatory mechanisms for promoting DNA repair and the DNA damage response in mammalian cells. Deubiquitinating enzymes (DUBs) have emerged as key players in the maintenance of genome stability. In this minireview, we discuss the recent findings on human DUBs that participate in genome maintenance, with a focus on the role of DUBs in the modulation of DNA repair and DNA damage signaling.

Co-opting the Fanconi anemia genomic stability pathway enables herpesvirus DNA synthesis and productive growth.

DNA damage associated with viral DNA synthesis can result in double-strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi anemia (FA) genomic stability pathway is exploited by herpes simplex virus 1 (HSV-1) to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV-1-infected cells resulted in monoubiquitination of FA effector proteins FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments, and FANCI-D2 interacted with a multisubunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, whereas HSV-1 productive growth was impaired in monoubiquitination-defective FA cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for nonhomologous end-joining (NHEJ). This identifies the FA-pathway as a cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral life cycle.

SENP8 limits aberrant neddylation of NEDD8 pathway components to promote cullin-RING ubiquitin ligase function

NEDD8 is a ubiquitin-like modifier most well-studied for its role in activating the largest family of ubiquitin E3 ligases, the cullin-RING ligases (CRLs). While many non-cullin neddylation substrates have been proposed over the years, validation of true NEDD8 targets has been challenging, as overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery. Here, we developed a deconjugation-resistant form of NEDD8 to stabilize the neddylated form of cullins and other non-cullin substrates. Using this strategy, we identified Ubc12, a NEDD8-specific E2 conjugating enzyme, as a substrate for auto-neddylation. Furthermore, we characterized SENP8/DEN1 as the protease that counteracts Ubc12 auto-neddylation, and observed aberrant neddylation of Ubc12 and other NEDD8 conjugation pathway components in SENP8-deficient cells. Importantly, loss of SENP8 function contributes to accumulation of CRL substrates and defective cell cycle progression. Thus, our study highlights the importance of SENP8 in maintaining proper neddylation levels for CRL-dependent proteostasis. DOI: http://dx.doi.org/10.7554/eLife.24325.001

How SUMOylation Fine-Tunes the Fanconi Anemia DNA Repair Pathway.

Fanconi anemia (FA) is a rare human genetic disorder characterized by developmental defects, bone marrow failure and cancer predisposition, primarily due to a deficiency in the repair of DNA interstrand crosslinks (ICLs). ICL repair through the FA DNA repair pathway is a complicated multi-step process, involving at least 19 FANC proteins and coordination of multiple DNA repair activities, including homologous recombination, nucleotide excision repair and translesion synthesis (TLS). SUMOylation is a critical regulator of several DNA repair pathways, however, the role of this modification in controlling the FA pathway is poorly understood. Here, we summarize recent advances in the fine-tuning of the FA pathway by small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligases (STUbLs) and other SUMO-related interactions, and discuss the implications of these findings in the design of novel therapeutics for alleviating FA-associated condition, including cancer.

Transcription drives DNA replication initiation and termination in human cells

The locations of active DNA replication origins in the human genome, and the determinants of origin activation, remain controversial. Additionally, neither the predominant sites of replication termination nor the impact of transcription on replication-fork mobility have been defined. We demonstrate that replication initiation occurs preferentially in the immediate vicinity of the transcription start site of genes occupied by high levels of RNA polymerase II, ensuring co-directional replication of the most highly transcribed genes. Further, we demonstrate that dormant replication origin firing represents the global activation of pre-existing origins. We also show that DNA replication naturally terminates at the polyadenylation site of transcribed genes. During replication stress, termination is redistributed to gene bodies, generating a global reorientation of replication relative to transcription. Our analysis provides a unified model for the coupling of transcription with replication initiation and termination in human cells.

HUWE1 comes to the rescue at stalled replication forks

HUWE1 is a multi‐faceted E3 ubiquitin ligase of the HECT family with many confirmed substrates, but mechanistic understanding of its functional roles in signaling pathways remains limited. In this issue of EMBO Reports, Choe et al demonstrate a novel function for HUWE1 in promoting DNA damage tolerance mechanisms to bypass DNA lesions during replication stress, thereby preserving genome stability. The authors connect this role for HUWE1 with its function in maintaining H2AX monoubiquitination levels for efficient signaling at stalled replication forks . Thus, this work highlights HUWE1 as a novel player in the replication stress response and prompts further investigation of its regulation during replication and other cellular processes.