PITAR, a DNA damage-inducible cancer/testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA.

In tumors with WT p53, alternate mechanisms of p53 inactivation are reported. Here, we have identified a long noncoding RNA, PITAR (p53 Inactivating TRIM28 Associated RNA), as an inhibitor of p53. PITAR is an oncogenic Cancer/testis lncRNA and is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSC). We establish that TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase, is a direct target of PITAR. PITAR interaction with TRIM28 RNA stabilized TRIM28 mRNA, which resulted in increased TRIM28 protein levels and reduced p53 steady-state levels due to enhanced p53 ubiquitination. DNA damage activated PITAR, in addition to p53, in a p53-independent manner, thus creating an incoherent feedforward loop to inhibit the DNA damage response by p53. While PITAR silencing inhibited the growth of WT p53 containing GSCs in vitro and reduced glioma tumor growth in vivo, its overexpression enhanced the tumor growth in a TRIM28-dependent manner and promoted resistance to Temozolomide. Thus, we establish an alternate way of p53 inactivation by PITAR, which maintains low p53 levels in normal cells and attenuates the DNA damage response by p53. Finally, we propose PITAR as a potential GBM therapeutic target.

ZMAT2 condensates regulate the alternative splicing of TRIM28 to reduce cellular ROS accumulation, thereby promoting the proliferation of HCC cells.

Dysregulation of splicing factor expression plays a crucial role in the progression of hepatocellular carcinoma (HCC). Our research found that the expression level of splicing factor ZMAT2 was increased in HCC, promoting the proliferation of HCC cells. RNAseq data indicated that the absence of ZMAT2 induced skipping exon of mRNA, while RIPseq data further revealed the mRNA binding motifs of ZMAT2. A comprehensive analysis of RNAseq and RIPseq data indicateed that ZMAT2 played a crucial role in the maturation process of TRIM28 mRNA. Knocking down of ZMAT2 led to the deletion of 25 bases in exon 11 of TRIM28, ultimately resulting in nonsense-mediated decay (NMD). Our data revealed that ZMAT2 could regulate TRIM28 to reduce the accumulation of ROS in HCC cells, thereby promoting their proliferation. Our research also discovered that ZMAT2 was capable of undergoing phase separation, resulting in the formation of liquid droplet condensates within HCC cells. Additionally, it was found that ZMAT2 was able to form protein-nucleic acid condensates with TRIM28 mRNA. In summary, this study is the first to reveal that ZMAT2 and TRIM28 mRNA form protein-nucleic acid condensates, thereby regulating the splicing of TRIM28 mRNA. The increased expression of ZMAT2 in HCC leads to upregulated TRIM28 expression and reduced ROS accumulation, ultimately accelerating the proliferation of HCC cells.

Satellite cell-derived TRIM28 is pivotal for mechanical load- and injury-induced myogenesis.

Satellite cells are skeletal muscle stem cells that contribute to postnatal muscle growth, and they endow skeletal muscle with the ability to regenerate after a severe injury. Here we discover that this myogenic potential of satellite cells requires a protein called tripartite motif-containing 28 (TRIM28). Interestingly, different from the role reported in a previous study based on C2C12 myoblasts, multiple lines of both in vitro and in vivo evidence reveal that the myogenic function of TRIM28 is not dependent on changes in the phosphorylation of its serine 473 residue. Moreover, the functions of TRIM28 are not mediated through the regulation of satellite cell proliferation or differentiation. Instead, our findings indicate that TRIM28 regulates the ability of satellite cells to progress through the process of fusion. Specifically, we discover that TRIM28 controls the expression of a fusogenic protein called myomixer and concomitant fusion pore formation. Collectively, the outcomes of this study expose the framework of a novel regulatory pathway that is essential for myogenesis.

The correlation between TRIM28 expression and immune checkpoints in CRPC.

This study delves into the unexplored realm of castration-resistant prostate cancer (CRPC) by investigating the role of TRIM28 and its intricate molecular mechanisms using high-throughput single-cell transcriptome sequencing and advanced bioinformatics analysis. Our comprehensive examination unveiled dynamic TRIM28 expression changes, particularly in immune cells such as macrophages and CD8+ T cells within CRPC. Correlation analyses with TCGA data highlighted the connection between TRIM28 and immune checkpoint expression and emphasized its pivotal influence on the quantity and functionality of immune cells. Using TRIM28 knockout mouse models, we identified differentially expressed genes and enriched pathways, unraveling the potential regulatory involvement of TRIM28 in the cGAS-STING pathway. In vitro, experiments further illuminated that TRIM28 knockout in prostate cancer cells induced a notable anti-tumor immune effect by inhibiting M2 macrophage polarization and enhancing CD8+ T cell activity. This impactful discovery was validated in an in situ transplant tumor model, where TRIM28 knockout exhibited a deceleration in tumor growth, reduced proportions of M2 macrophages, and enhanced infiltration of CD8+ T cells. In summary, this study elucidates the hitherto unknown anti-tumor immune role of TRIM28 in CRPC and unravels its potential regulatory mechanism via the cGAS-STING signaling pathway. These findings provide novel insights into the immune landscape of CRPC, offering promising directions for developing innovative therapeutic strategies.

KAP1 negatively regulates RNA polymerase II elongation kinetics to activate signal-induced transcription.

Signal-induced transcriptional programs regulate critical biological processes through the precise spatiotemporal activation of Immediate Early Genes (IEGs); however, the mechanisms of transcription induction remain poorly understood. By combining an acute depletion system with several genomics approaches to interrogate synchronized, temporal transcription, we reveal that KAP1/TRIM28 is a first responder that fulfills the temporal and heightened transcriptional demand of IEGs. Acute KAP1 loss triggers an increase in RNA polymerase II elongation kinetics during early stimulation time points. This elongation defect derails the normal progression through the transcriptional cycle during late stimulation time points, ultimately leading to decreased recruitment of the transcription apparatus for re-initiation thereby dampening IEGs transcriptional output. Collectively, KAP1 plays a counterintuitive role by negatively regulating transcription elongation to support full activation across multiple transcription cycles of genes critical for cell physiology and organismal functions.

The TRIM28/miR133a/CD47 axis acts as a potential therapeutic target in pancreatic necrosis by impairing efferocytosis.

Efferocytosis, the clearance of apoptotic cells by macrophages, plays a crucial role in inflammatory responses and effectively prevents secondary necrosis. However, the mechanisms underlying efferocytosis in acute pancreatitis (AP) remain unclear. In this study, we demonstrated the presence of efferocytosis in injured human and mouse pancreatic tissues. We also observed significant upregulation of CD47, an efferocytosis-related the "do not eat me" molecule in injured acinar cells. Subsequently, we used CRISPR-Cas9 gene editing, anti-adeno-associated virus (AAV) gene modification, and anti-CD47 antibody to investigate the potential therapeutic role of AP. CD47 expression was negatively regulated by upstream miR133a, which is controlled by the transcription factor TRIM28. To further investigate the regulation of efferocytosis and reduction of pancreatic necrosis in AP, we used miR-133a-agomir and pancreas-specific AAV-shTRIM28 to modulate CD47 expression. Our findings confirmed that CD47-mediated efferocytosis is critical for preventing pancreatic necrosis and suggest that targeting the TRIM28-miR133a-CD47 axis is clinically relevant for the treatment of AP.

UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168.

Maintenance of genome integrity requires tight control of DNA damage response (DDR) signalling and repair, with phosphorylation and ubiquitination representing key elements. How these events are coordinated to achieve productive DNA repair remains elusive. Here we identify the ubiquitin-conjugating enzyme UBE2D3 as a regulator of ATM kinase-induced DDR that promotes non-homologous end-joining (NHEJ) at telomeres. UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both mediated by RNF168 upon ATM activation. Additionally, UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. Mechanistically, defective KAP1-S824 phosphorylation and telomeric NHEJ upon UBE2D3-deficiency are linked to RNF168 hyperaccumulation and aberrant PP2A phosphatase activity. Together, our results identify UBE2D3 as a multi-level regulator of NHEJ that orchestrates ATM and RNF168 activities. Moreover, they reveal a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and phosphatase-mediated restriction of KAP1 phosphorylation.

Loss of RBM45 inhibits breast cancer progression by reducing the SUMOylation of IRF7 to promote IFNB1 transcription.

Type I interferons exhibit anti-proliferative and anti-cancer activities, but their detailed regulatory mechanisms in cancer have not been fully elucidated yet. RNA binding proteins are master orchestrators of gene regulation, which are closely related to tumor progression. Here we show that the upregulated RNA binding protein RBM45 correlates with poor prognosis in breast cancer. Depletion of RBM45 suppresses breast cancer progression both in cultured cells and xenograft mouse models. Mechanistically, RBM45 ablation inhibits breast cancer progression through regulating type I interferon signaling, particularly by elevating IFN-ß production. Importantly, RBM45 recruits TRIM28 to IRF7 and stimulates its SUMOylation, thereby repressing IFNB1 transcription. Loss of RBM45 reduced the SUMOylation of IRF7 by reducing the interaction between TRIM28 and IRF7 to promote IFNB1 transcription, leading to the inhibition of breast cancer progression. Taken together, our finding uncovers a vital role of RBM45 in modulating type I interferon signaling and cancer aggressive progression, implicating RBM45 as a potential therapeutic target in breast cancer.

TIF1ß activates leukemic transcriptional program in HSCs and promotes BCR::ABL1-induced myeloid leukemia.

TIF1ß/KAP1/TRIM28, a chromatin modulator, both represses and activates the transcription of genes in normal and malignant cells. Analyses of datasets on leukemia patients revealed that the expression level of TIF1ß was increased in patients with chronic myeloid leukemia at the blast crisis and acute myeloid leukemia. We generated a BCR::ABL1 conditional knock-in (KI) mouse model, which developed aggressive myeloid leukemia, and demonstrated that the deletion of the Tif1ß gene inhibited the progression of myeloid leukemia and showed longer survival than that in BCR::ABL1 KI mice, suggesting that Tif1ß drove the progression of BCR::ABL1-induced leukemia. In addition, the deletion of Tif1ß sensitized BCR::ABL1 KI leukemic cells to dasatinib. The deletion of Tif1ß decreased the expression levels of TIF1ß-target genes and chromatin accessibility peaks enriched with the Fosl1-binding motif in BCR::ABL1 KI stem cells. TIF1ß directly bound to the promoters of proliferation genes, such as FOSL1, in human BCR::ABL1 cells, in which TIF1ß and FOSL1 bound to adjacent regions of chromatin. Since the expression of Fosl1 was critical for the enhanced growth of BCR::ABL1 KI cells, Tif1ß and Fosl1 interacted to activate the leukemic transcriptional program in and cellular function of BCR::ABL1 KI stem cells and drove the progression of myeloid leukemia.

KAP1 stabilizes MYCN mRNA and promotes neuroblastoma tumorigenicity by protecting the RNA m6A reader YTHDC1 protein degradation.

BACKGROUND: Neuroblastoma (NB) patients with amplified MYCN often face a grim prognosis and are resistant to existing therapies, yet MYCN protein is considered undruggable. KAP1 (also named TRIM28) plays a crucial role in multiple biological activities. This study aimed to investigate the relationship between KAP1 and MYCN in NB. METHODS: Transcriptome analyses and luciferase reporter assay identified that KAP1 was a downstream target of MYCN. The effects of KAP1 on cancer cell proliferation and colony formation were explored using the loss-of-function assays in vitro and in vivo. RNA stability detection was used to examine the influence of KAP1 on MYCN expression. The mechanisms of KAP1 to maintain MYCN mRNA stabilization were mainly investigated by mass spectrum, immunoprecipitation, RIP-qPCR, and western blotting. In addition, a xenograft mouse model was used to reveal the antitumor effect of STM2457 on NB. RESULTS: Here we identified KAP1 as a critical regulator of MYCN mRNA stability by protecting the RNA N6-methyladenosine (m6A) reader YTHDC1 protein degradation. KAP1 was highly expressed in clinical MYCN-amplified NB and was upregulated by MYCN. Reciprocally, KAP1 knockdown reduced MYCN mRNA stability and inhibited MYCN-amplified NB progression. Mechanistically, KAP1 regulated the stability of MYCN mRNA in an m6A-dependent manner. KAP1 formed a complex with YTHDC1 and RNA m6A writer METTL3 to regulate m6A-modified MYCN mRNA stability. KAP1 depletion decreased YTHDC1 protein stability and promoted MYCN mRNA degradation. Inhibiting MYCN mRNA m6A modification synergized with chemotherapy to restrain tumor progression in MYCN-amplified NB. CONCLUSIONS: Our research demonstrates that KAP1, transcriptionally activated by MYCN, forms a complex with YTHDC1 and METTL3, which in turn maintain the stabilization of MYCN mRNA in an m6A-dependent manner. Targeting m6A modification by STM2457, a small-molecule inhibitor of METTL3, could downregulate MYCN expression and attenuate tumor proliferation. This finding provides a new alternative putative therapeutic strategy for MYCN-amplified NB.

[Expression and prognostic significance of KAP1 gene in malignant pleural mesothelioma].

Objective: To explore the expression of KAP1 (KRAB-associated protein 1, KAP1) in Malignant pleural mesothelioma (MPM) based on the cancer genome atlas (TCGA) and clinical trials. And elucidate the correlation between the expression of KAP1 and the clinical pathological parameters of patients with MPM and its prognosis. Methods: In April 2022, Based on the second generation KAP1mRNA sequencing data and clinicopathological data of MPM patients downloaded from TCGA database, the correlation between KAP1mRNA expression and clinical parameters was analyzed, and the correlation between KAP1 protein expression and clinicopathological parameters and its prognostic value were analyzed based on Chuxiong data set cohort clinical samples. The expression of KAP1 mRNA in MPM samples and matched normal tumor adjacent tissues was detected by qRT-PCR, and the expression of KAP1 protein in MPM and normal pleural tissues was detected by immunohistochemistry and Westernblotting. To construct a Kaplan-Meier model to explore the effect of KAP1 expression on the prognosis of MPM patients, and to analyze the prognostic factors of MPM patients by Cox regression. Results: qRT-PCR and Western blotting detection showed that the expression levels of KAP1 gene in four different MPM cells (NCI-H28, NCI-H2052, NCI-H2452, and MTSO-211H) were significantly higher than those in normal pleural mesothelial cells Met-5A. qRT-PCR, Western blotting and IHC results demonstrated that the mRNA and protein expression levels of KAP1 in MPM tissues was significantly higher than that in matching normal mesothelial tissues, and the expression level of KAP1 protein was correlated with TP 53 protein expression levels and serum CEA levels (P<0.05) . The mRNA expression level was significantly correlated with the prognosis, The overall survival time of mesothelioma patients with high KAP1mRNA expression was significantly shorter (HR=3.7, Logrank P<0.001) . Tumor type, age and the mRNA expression were related to the prognosis of MPM patients (P<0.05) . Multivariate analysis showed that tumor type and KAP1 mRNA expression level were independent prognostic factors of MPM patients (P<0.05) . Conclusion: In this study, TCGA database and Chuxiong cohort experiment samples were used to collect the relevant information of KAP1 expression in malignant melanoma tissues. It was confirmed that KAP1 is highly expressed in MPM tissues. The mRNA expression level and pathological type are correlated with the prognosis of patients.

Functional Analysis of KAP1/TRIM28 Requirements for HIV-1 Transcription Activation.

HIV-1 latency maintenance and reactivation are regulated by several viral and host factors. One such factor is Krüppel-associated box (KRAB)-associated protein 1 (KAP1: also named TRIM28 or TIF1ß). While initial studies have revealed KAP1 to be a positive regulator of latency reversal in transformed and primary CD4+ T cells, subsequent studies have proposed KAP1 to be a repressor required for latency maintenance. Given this discrepancy, in this study, we re-examine KAP1 transcription regulatory functions using a chemical genetics strategy to acutely deplete KAP1 expression to avoid the accumulation of indirect effects. Notably, KAP1 acute loss partially decreased HIV-1 promoter activity in response to activating signals, a function that can be restored upon complementation with exogenous KAP1, thus revealing that KAP1-mediated activation is on target. By combining comprehensive KAP1 domain deletion and mutagenesis in a cell-based reporter assay, we genetically defined the RING finger domain and an Intrinsically Disordered Region as key activating features. Together, our study solidifies the notion that KAP1 activates HIV-1 transcription by exploiting its multi-domain protein arrangement via previously unknown domains and functions.

Wilms tumour resulting from paternal transmission of a TRIM28 pathogenic variant-A first report.

Wilms tumour (nephroblastoma) is a renal embryonal tumour that is frequently caused by constitutional variants in a small range of cancer predisposition genes. TRIM28 has recently been identified as one such gene. Previously, observational data strongly suggested a parent of origin effect, whereby Wilms tumour only occurred following maternal inheritance of a pathogenic genetic variant. However, here we report a child with bilateral Wilms tumour who had inherited a pathogenic TRIM28 variant from their father. This finding suggests that genetic counselling for paternally inherited pathogenic variants in TRIM28 should include discussion of a potential risk of Wilms tumour.

TRIM28 inactivation in epithelial nephroblastoma is frequent and often associated with predisposing TRIM28 germline variants.

Wilms tumors (WTs) are histologically diverse childhood cancers with variable contributions of blastema, stroma, and epithelia. A variety of cancer genes operate in WTs, including the tripartite-motif-containing-28 gene (TRIM28). Case reports and small case series suggest that TRIM28 mutations are associated with epithelial morphology and WT predisposition. Here, we systematically investigated the prevalence of TRIM28 inactivation and predisposing mutations in a cohort of 126 WTs with >2/3 epithelial cells, spanning 20 years of biobanking in the German SIOP93-01/GPOH and SIOP2001/GPOH studies. Overall, 44.4% (56/126) cases exhibited loss of TRIM28 by immunohistochemical staining. Of these, 48 could be further analyzed molecularly, revealing TRIM28 sequence variants in each case - either homozygous (~2/3) or heterozygous with epigenetic silencing of the second allele (~1/3). The majority (80%) of the mutations resulted in premature stops and frameshifts. In addition, we detected missense mutations and small deletions predicted to destabilize the protein through interference with folding of key structural elements such as the zinc-binding clusters of the RING, B-box-2, and PHD domains or the central coiled-coil region. TRIM28-mutant tumors otherwise lacked WT-typical IGF2 alterations or driver events, except for rare TP53 progression events that occurred with expected frequency. Expression profiling identified TRIM28-mutant tumors as a homogeneous subset of epithelial WTs that mostly present with stage I disease. There was a high prevalence of perilobar nephrogenic rests, putative precursor lesions, that carried the same biallelic TRIM28 alterations in 7/7 cases tested. Importantly, 46% of the TRIM28 mutations were present in blood cells or normal kidney tissue, suggesting germline events or somatic mosaicism, partly supported by family history. Given the high prevalence of predisposing variants in TRIM28-driven WT, we suggest that immunohistochemical testing of TRIM28 be integrated into diagnostic practice as the management of WT in predisposed children differs from that with sporadic tumors. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

E3 ligase TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer by enhancing the recruitment of myeloid-derived suppressor cells.

BACKGROUND: Alterations in several tripartite motif-containing (TRIM) family proteins have been implicated in the pathogenesis of lung cancer. TRIM28, a member of the TRIM E3 ligase family, has been associated with tumorigenesis, cell proliferation, and inflammation. However, little is known about TRIM28 expression and its role in the immune microenvironment of non-small cell lung cancer (NSCLC). METHODS: We assessed the clinical significance of TRIM28 in tissue microarrays and TCGA cohorts. We investigated the function of TRIM28 in syngeneic mouse tumor models, the KrasLSL-G12D/+; Tp53fl/fl (KP) mouse model, and humanized mice. Immune cell composition was analyzed using flow cytometry and immunohistochemistry. RESULTS: Our findings revealed a positive correlation between TRIM28 expression and the infiltration of suppressive myeloid-derived suppressor cells (MDSCs) in NSCLC. Moreover, silencing TRIM28 enhanced the efficacy of anti-PD-1 immunotherapy by reshaping the inflamed tumor microenvironment. Mechanistically, we demonstrated that TRIM28 could physically interact with receptor-interacting protein kinase 1 (RIPK1) and promote K63-linked ubiquitination of RIPK1, which is crucial for sustaining activation of the NF-κB pathway. Mutagenesis of the E3 ligase domain corroborated the essential role of E3 ligase activity in TRIM28-mediated NF-κB activation. Further experiments revealed that TRIM28 could upregulate the expression of CXCL1 by activating NF-κB signaling. CXCL1 could bind to CXCR2 on MDSCs and promote their migration to the tumor microenvironment. TRIM28 knockdown increased responsiveness to anti-PD-1 therapy in immunocompetent mice, characterized by increased CD8+T tumor-infiltrating lymphocytes and decreased MDSCs. CONCLUSION: The present study identified TRIM28 as a promoter of chemokine-driven recruitment of MDSCs through RIPK1-mediated NF-κB activation, leading to the suppression of infiltrating activated CD8+T cells and the development of anti-PD-1 resistance. Understanding the regulation of MDSC recruitment and function by TRIM28 provides crucial insights into the association between TRIM28 signaling and the development of an immunosuppressive tumor microenvironment. These insights may inform the development of combination therapies to enhance the effectiveness of immune checkpoint blockade therapy in NSCLC.

Children with Chronic Immune Thrombocytopenia Exhibit High Expression of Human Endogenous Retroviruses TRIM28 and SETDB1.

Chronic immune thrombocytopenia (CITP) is an autoimmune disease whose underlying biologic mechanisms remain elusive. Human endogenous retroviruses (HERVs) derive from ancestral infections and constitute about 8% of our genome. A wealth of clinical and experimental studies highlights their pivotal pathogenetic role in autoimmune diseases. Epigenetic mechanisms, such as those modulated by TRIM28 and SETDB1, are involved in HERV activation and regulation of immune response. We assessed, through a polymerase chain reaction real-time Taqman amplification assay, the transcription levels of pol genes of HERV-H, HERV-K, and HERV-W; env genes of Syncytin (SYN)1, SYN2, and HERV-W; as well as TRIM28 and SETDB1 in whole blood from 34 children with CITP and age-matched healthy controls (HC). The transcriptional levels of all HERV sequences, with the exception of HERV-W-env, were significantly enhanced in children with CITP as compared to HC. Patients on eltrombopag treatment exhibited lower expression of SYN1, SYN2, and HERV-W-env as compared to untreated patients. The mRNA concentrations of TRIM28 and SETDB1 were significantly higher and were positively correlated with those of HERVs in CITP patients. The over-expressions of HERVs and TRIM28/SETDB1 and their positive correlations in patients with CITP are suggestive clues of their contribution to the pathogenesis of the disease and support innovative interventions to inhibit HERV and TRIM28/SETDB1 expressions in patients unresponsive to standard therapies.

TRIM28 modulates nuclear receptor signaling to regulate uterine function.

Estrogen and progesterone, acting through their cognate receptors the estrogen receptor α (ERα) and the progesterone receptor (PR) respectively, regulate uterine biology. Using rapid immunoprecipitation and mass spectrometry (RIME) and co-immunoprecipitation, we identified TRIM28 (Tripartite motif containing 28) as a protein which complexes with ERα and PR in the regulation of uterine function. Impairment of TRIM28 expression results in the inability of the uterus to support early pregnancy through altered PR and ERα action in the uterine epithelium and stroma by suppressing PR and ERα chromatin binding. Furthermore, TRIM28 ablation in PR-expressing uterine cells results in the enrichment of a subset of TRIM28 positive and PR negative pericytes and epithelial cells with progenitor potential. In summary, our study reveals the important roles of TRIM28 in regulating endometrial cell composition and function in women, and also implies its critical functions in other hormone regulated systems.

Acetylation-Mimic Mutation of TRIM28-Lys304 to Gln Attenuates the Interaction with KRAB-Zinc-Finger Proteins and Affects Gene Expression in Leukemic K562 Cells.

TRIM28/KAP1/TIF1ß is a crucial epigenetic modifier. Genetic ablation of trim28 is embryonic lethal, although RNAi-mediated knockdown in somatic cells yields viable cells. Reduction in TRIM28 abundance at the cellular or organismal level results in polyphenism. Posttranslational modifications such as phosphorylation and sumoylation have been shown to regulate TRIM28 activity. Moreover, several lysine residues of TRIM28 are subject to acetylation, but how acetylation of TRIM28 affects its functions remains poorly understood. Here, we report that, compared with wild-type TRIM28, the acetylation-mimic mutant TRIM28-K304Q has an altered interaction with Krüppel-associated box zinc-finger proteins (KRAB-ZNFs). The TRIM28-K304Q knock-in cells were created in K562 erythroleukemia cells by CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein nuclease 9) gene editing method. Transcriptome analysis revealed that TRIM28-K304Q and TRIM28 knockout K562 cells had similar global gene expression profiles, yet the profiles differed considerably from wild-type K562 cells. The expression levels of embryonic-related globin gene and a platelet cell marker integrin-beta 3 were increased in TRIM28-K304Q mutant cells, indicating the induction of differentiation. In addition to the differentiation-related genes, many zinc-finger-proteins genes and imprinting genes were activated in TRIM28-K304Q cells; they were inhibited by wild-type TRIM28 via binding with KRAB-ZNFs. These results suggest that acetylation/deacetylation of K304 in TRIM28 constitutes a switch for regulating its interaction with KRAB-ZNFs and alters the gene regulation as demonstrated by the acetylation mimic TRIM28-K304Q.

TRIM28 negatively regulates the RLR signaling pathway by targeting MAVS for degradation via K48-linked polyubiquitination.

Mitochondrial antiviral signaling (MAVS) protein is a core signaling adapter in the retinoid acid-inducible gene-I-like receptor (RLR) signaling pathway that recruits downstream signaling factors, ultimately leading to the activation of type Ⅰ interferons. However, the mechanisms that modulate the RLR signaling pathway by manipulating MAVS are not fully understood. Previous studies suggested that tripartite motif 28 (TRIM28) participates in regulating innate immune signaling pathways by inhibiting the expression of immune-related genes at the transcriptional level. In this study, we characterized TRIM28 as a negative regulator of the RLR signaling pathway in a MAVS-dependent manner. Overexpression of TRIM28 inhibited the MAVS-induced production of type Ⅰ interferons and proinflammatory cytokines, while knocking down TRIM28 exerted the opposite effect. Mechanistically, TRIM28 targeted MAVS for proteasome-mediated degradation via K48-linked polyubiquitination. The RING domain of TRIM28, especially the cysteine residues at positions 65 and 68, was critical for the suppressive effect of TRIM28 on MAVS-mediated RLR signaling, while each of the C-terminal domains of TRIM28 contributed to its interaction with MAVS. Further investigation revealed that TRIM28 transferred ubiquitin chains to the K7, K10, K371, K420, and K500 residues of MAVS. Together, our results reveal a previously uncharacterized mechanism involving TRIM28 in fine-tuning innate immune responses and provide new insights into the mechanisms by which MAVS is regulated, which contribute to the understanding of the molecular mechanisms underlying immune homeostasis maintenance.

A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis.

The long interspersed element 1 (LINE-1 or L1) integration is affected by many cellular factors through various mechanisms. Some of these factors are required for L1 amplification, while others either suppress or enhance specific steps during L1 propagation. Previously, TRIM28 has been identified to suppress transposable elements, including L1 expression via its canonical role in chromatin remodeling. Here, we report that TRIM28 through its B box domain increases L1 retrotransposition and facilitates shorter cDNA and L1 insert generation in cultured cells. Consistent with the latter, we observe that tumor specific L1 inserts are shorter in endometrial, ovarian, and prostate tumors with higher TRIM28 mRNA expression than in those with lower TRIM28 expression. We determine that three amino acids in the B box domain that are involved in TRIM28 multimerization are critical for its effect on both L1 retrotransposition and cDNA synthesis. We provide evidence that B boxes from the other two members in the Class VI TRIM proteins, TRIM24 and TRIM33, also increase L1 retrotransposition. Our findings could lead to a better understanding of the host/L1 evolutionary arms race in the germline and their interplay during tumorigenesis.