Co-transcriptional genome surveillance by HUSH is coupled to termination machinery.

The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and-via its component MPP8-with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.

The Histone Methyltransferase SETDB1 Controls T Helper Cell Lineage Integrity by Repressing Endogenous Retroviruses.

Upon activation, naive CD4+ T cells differentiate into distinct T cell subsets via processes reliant on epigenetically regulated, lineage-specific developmental programs. Here, we examined the function of the histone methyltransferase SETDB1 in T helper (Th) cell differentiation. Setdb1-/- naive CD4+ T cells exhibited exacerbated Th1 priming, and when exposed to a Th1-instructive signal, Setdb1-/- Th2 cells crossed lineage boundaries and acquired a Th1 phenotype. SETDB1 did not directly control Th1 gene promoter activity but relied instead on deposition of the repressive H3K9me3 mark at a restricted and cell-type-specific set of endogenous retroviruses (ERVs) located in the vicinity of genes involved in immune processes. Refined bioinformatic analyses suggest that these retrotransposons regulate Th1 gene cis-regulatory elements or act as Th1 gene enhancers. Thus, H3K9me3 deposition by SETDB1 ensures Th cell lineage integrity by repressing a repertoire of ERVs that have been exapted into cis-regulatory modules to shape and control the Th1 gene network.

DAXX safeguards heterochromatin formation in embryonic stem cells.

Genomes comprise a large fraction of repetitive sequences folded into constitutive heterochromatin, which protect genome integrity and cell identity. De novo formation of heterochromatin during preimplantation development is an essential step for preserving the ground-state of pluripotency and the self-renewal capacity of embryonic stem cells (ESCs). However, the molecular mechanisms responsible for the remodeling of constitutive heterochromatin are largely unknown. Here, we identify that DAXX, an H3.3 chaperone essential for the maintenance of mouse ESCs in the ground state, accumulates in pericentromeric regions independently of DNA methylation. DAXX recruits PML and SETDB1 to promote the formation of heterochromatin, forming foci that are hallmarks of ground-state ESCs. In the absence of DAXX or PML, the three-dimensional (3D) architecture and physical properties of pericentric and peripheral heterochromatin are disrupted, resulting in de-repression of major satellite DNA, transposable elements and genes associated with the nuclear lamina. Using epigenome editing tools, we observe that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX is crucial for the maintenance and 3D organization of the heterochromatin compartment and protects ESC viability.

YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia†.

Genomic integrity is critical for sexual reproduction, ensuring correct transmission of parental genetic information to the descendant. To preserve genomic integrity, germ cells have evolved multiple DNA repair mechanisms, together termed as DNA damage response. The RNA N6-methyladenosine is the most abundant mRNA modification in eukaryotic cells, which plays important roles in DNA damage response, and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) is a well-acknowledged N6-methyladenosine reader protein regulating the mRNA decay and stress response. Despite this, the correlation between YTHDF2 and DNA damage response in germ cells, if any, remains enigmatic. Here, by employing a Ythdf2-conditional knockout mouse model as well as a Ythdf2-null GC-1 mouse spermatogonial cell line, we explored the role and the underlying mechanism for YTHDF2 in spermatogonial DNA damage response. We identified that, despite no evident testicular morphological abnormalities under the normal circumstance, conditional mutation of Ythdf2 in adult male mice sensitized germ cells, including spermatogonia, to etoposide-induced DNA damage. Consistently, Ythdf2-KO GC-1 cells displayed increased sensitivity and apoptosis in response to DNA damage, accompanied by the decreased SET domain bifurcated 1 (SETDB1, a histone methyltransferase) and H3K9me3 levels. The Setdb1 knockdown in GC-1 cells generated a similar phenotype, but its overexpression in Ythdf2-null GC-1 cells alleviated the sensitivity and apoptosis in response to DNA damage. Taken together, these results demonstrate that the N6-methyladenosine reader YTHDF2 promotes DNA damage repair by positively regulating the histone methyltransferase SETDB1 in spermatogonia, which provides novel insights into the mechanisms underlying spermatogonial genome integrity maintenance and therefore contributes to safe reproduction.

Genome-Scale Analysis of Cellular Restriction Factors That Inhibit Transgene Expression from Adeno-Associated Virus Vectors.

Adeno-associated virus (AAV) vectors are one of the leading platforms for gene delivery for the treatment of human genetic diseases, but the antiviral cellular mechanisms that interfere with optimal transgene expression are incompletely understood. Here, we performed two genome-scale CRISPR screens to identify cellular factors that restrict transgene expression from recombinant AAV vectors. Our screens revealed several components linked to DNA damage response, chromatin remodeling, and transcriptional regulation. Inactivation of the Fanconi anemia gene FANCA; the human silencing hub (HUSH)-associated methyltransferase SETDB1; and the gyrase, Hsp90, histidine kinase, and MutL (GHKL)-type ATPase MORC3 led to increased transgene expression. Moreover, SETDB1 and MORC3 knockout improved transgene levels of several AAV serotypes as well as other viral vectors, such as lentivirus and adenovirus. Finally, we demonstrated that the inhibition of FANCA, SETDB1, or MORC3 also enhanced transgene expression in human primary cells, suggesting that they could be physiologically relevant pathways that restrict AAV transgene levels in therapeutic settings. IMPORTANCE Recombinant AAV (rAAV) vectors have been successfully developed for the treatment of genetic diseases. The therapeutic strategy often involves the replacement of a defective gene by the expression of a functional copy from the rAAV vector genome. However, cells possess antiviral mechanisms that recognize and silence foreign DNA elements thereby limiting transgene expression and its therapeutic effect. Here, we utilize a functional genomics approach to uncover a comprehensive set of cellular restriction factors that inhibit rAAV-based transgene expression. Genetic inactivation of selected restriction factors increased rAAV transgene expression. Hence, modulation of identified restriction factors has the potential to enhance AAV gene replacement therapies.

The crucial role of SETDB1 in structural and functional transformation of epithelial cells during regeneration after intestinal ischemia reperfusion injury.

Su (var) 3-9, enhancer of seste, trithorax (SET)-domain bifurcated histone lysine methyltransferase (SETDB1) plays a crucial role in maintaining intestinal stem cell homeostasis; however, its physiological function in epithelial injury is largely unknown. In this study, we investigated the role of SETDB1 in epithelial regeneration using an intestinal ischemia/reperfusion injury (IRI) mouse model. Jejunum tissues were sampled after 75 min of ischemia followed by 3, 24, and 48 h of reperfusion. Morphological evaluations were performed using light microscopy and electron microscopy, and the involvement of SETDB1 in epithelial remodeling was investigated by immunohistochemistry. Expression of SETDB1 was increased following 24 h of reperfusion and localized in not only the crypt bottom but also in the transit amplifying zone and part of the villi. Changes in cell lineage, repression of cell adhesion molecule expression, and decreased histone H3 methylation status were detected in the crypts at the same time. Electron microscopy also revealed aberrant alignment of crypt nuclei and fusion of adjacent villi. Furthermore, increased SETDB1 expression and epithelial remodeling were confirmed with loss of stem cells, suggesting SETDB1 affects epithelial cell plasticity. In addition, crypt elongation and increased numbers of Ki-67 positive cells indicated active cell proliferation after IRI; however, the expression of PCNA was decreased compared to sham mouse jejunum. These morphological changes and the aberrant expression of proliferation markers were prevented by sinefungin, a histone methyltransferase inhibitor. In summary, SETDB1 plays a crucial role in changes in the epithelial structure after IRI-induced stem cell loss.

SetDB1 and Su(var)3-9 are essential for late stages of larval development of Drosophila melanogaster.

Methylation of H3K9 histone residue is a marker of gene silencing in eukaryotes. Three enzymes responsible for adding this modification - G9a, SetDB1/Egg, and Su(var)3-9 - are known in Drosophila. To understand how simultaneous mutations of SetDB1 and Su(var)3-9 may affect the fly development, appropriate combinations were obtained. Double mutants egg; Su(var)3-9 displayed pronounced embryonic lethality, slower larval growth and died before or during metamorphosis. Analysis of transcription in larval salivary glands and wing imaginal disks indicated that the effect of double mutation is tissue-specific. In salivary gland chromosomes, affected genes display low H3K9me2 enrichment and are rarely bound by SetDB1 or Su(var)3-9. We suppose that each of these enzymes directly or indirectly controls its own set of gene targets in different organs, and double mutation results in an imbalanced developmental program. This also indicates that SetDB1 and Su(var)3-9 may affect transcription via H3K9-independent mechanisms. Unexpectedly, in double and triple mutants, amount of di- and tri-methylated H3K9 is drastically reduced, but not completely absent. We hypothesize that this residual methylation implies the existence of additional H3K9-specific methyltransferase in Drosophila.

SETDB1 deletion causes DNA demethylation and upregulation of multiple zinc-finger genes.

BACKGROUND: SETDB1 (SET domain bifurcated-1) is a histone H3-lysine 9 (H3K9)-specific methyltransferase that mediates heterochromatin formation and repression of target genes. Despite the assumed functional link between DNA methylation and SETDB1-mediated H3K9 trimethylations, several studies have shown that SETDB1 operates autonomously of DNA methylation in a region- and cell-specific manner. This study analyzes SETDB1-null HAP1 cells through a linked methylome and transcriptome analysis, intending to explore genes controlled by SETDB1-involved DNA methylation. METHODS AND RESULTS: We investigated SETDB1-mediated regulation of DNA methylation and gene transcription in human HAP1 cells using reduced-representation bisulfite sequencing (RRBS) and RNA sequencing. While two-thirds of differentially methylated CpGs (DMCs) in genic regions were hypomethylated in SETDB1-null cells, we detected a plethora of C2H2-type zinc-finger protein genes (C2H2-ZFP, 223 of 749) among the DMC-associated genes. Most C2H2-ZFPs with DMCs in their promoters were found hypomethylated in SETDB1-KO cells, while other non-ZFP genes with promoter DMCs were not. These C2H2-ZFPs with DMCs in their promoters were significantly upregulated in SETDB1-KO cells. Similarly, C2H2-ZFP genes were upregulated in SETDB1-null 293T cells, suggesting that SETDB1's function in ZFP gene repression is widespread. There are several C2H2-ZFP gene clusters on chromosome 19, which were selectively hypomethylated in SETDB1-KO cells. CONCLUSIONS: SETDB1 collectively and specifically represses a substantial fraction of the C2H2-ZFP gene family. Through the en-bloc silencing of a set of ZFP genes, SETDB1 may help establish a panel of ZFP proteins that are expressed cell-type specifically and thereby can serve as signature proteins for cellular identity.

SETDB1: Progress and prospects in cancer treatment potential and inhibitor research.

SET domain bifurcated methyltransferase 1 (SETDB1) serves as a histone lysine methyltransferase, catalyzing the di- and tri-methylation of histone H3K9. Mounting evidence indicates that the abnormal expression or activity of SETDB1, either through amplification or mutation, plays a crucial role in tumorigenesis and progression. This is particularly evident in the context of tumor immune evasion and resistance to immune checkpoint blockade therapy. Furthermore, there is a robust association between SETDB1 dysregulation and an unfavorable prognosis across various types of tumors. The oncogenic role of SETDB1 primarily arises from its methyltransferase function, which contributes to the establishment of a condensed and transcriptionally inactive heterochromatin state. This results in the inactivation of genes that typically hinder cancer development and silencing of retrotransposons that could potentially trigger an immune response. These findings underscore the substantial potential for SETDB1 as an anti-tumor therapeutic target. Nevertheless, despite significant strides in recent years in tumor biology research, challenges persist in SETDB1-targeted therapy. To better facilitate the development of anti-tumor therapy targeting SETDB1, we have conducted a comprehensive review of SETDB1 in this account. We present the structure and function of SETDB1, its role in various tumors and immune regulation, as well as the advancements made in SETDB1 antagonists. Furthermore, we discuss the challenges encountered and provide perspectives for the development of SETDB1-targeted anti-tumor therapy.

The critical role of SETDB1-mediated CCND1/PI3K/AKT pathway via p53-RS di-methylation at K370 in the proliferation of WRL68 cells induced by nicotine.

Constituents of cigarette smoke are known to be carcinogens. Additionally, there is mounting evidence that the liver is an organ susceptible to tobacco carcinogenicity. Nicotine, the primary constituent of tobacco, plays a role in cancer progression. In our previous study, it was found that nicotine enhances the proliferation of a human normal fetal hepatic (WRL68) cell due to the activation of p53 mutation at Ser249 (p53-RS)/STAT1/CCND1 signaling pathway. Here, we further elucidated the mechanism of regulating this pathway. Firstly, dose-dependent increase of SETDB1 protein level in WRL68 cells upon exposure to nicotine (1.25, 2.5, and 5 µM), significantly enhanced cellular proliferation. In addition, the upregulation of SETDB1 protein was necessary for the nuclear translocation of p53-RS to establish a ternary complex with STAT1 and SETDB1, which facilitated p53-RS di-methylation at K370 (p53-RS/K370me2). After that, the activation of CCND1/PI3K/AKT pathway was initiated when STAT1 stability was enhanced by p53-RS/K370me2, ultimately resulting in cell proliferation. Altogether, the study revealed that the increase in SETDB1 expression could potentially have a significant impact on the activation of CCND1/PI3K/AKT pathway through p53-RS/K370me2, leading to the proliferation of WRL68 cells induced by nicotine, which could contribute to hepatocellular carcinoma for smokers. Besides, the results of this study provided a foundation for the development of anticancer therapies for cancers associated with tobacco use.

Neural stemness unifies cell tumorigenicity and pluripotent differentiation potential.

Neural stemness is suggested to be the ground state of tumorigenicity and pluripotent differentiation potential. However, the relationship between these cell properties is unclear. Here, by disrupting the neural regulatory network in neural stem and cancer cells and by serial transplantation of cancer cells, we show that tumorigenicity and pluripotent differentiation potential are coupled cell properties unified by neural stemness. We show that loss of neural stemness via inhibition of SETDB1, an oncoprotein with enriched expression in embryonic neural cells during vertebrate embryogenesis, led to neuronal differentiation with reduced tumorigenicity and pluripotent differentiation potential in neural stem and cancer cells, whereas enhancement of neural stemness by SETDB1 overexpression caused the opposite effects. SETDB1 maintains a regulatory network comprising proteins involved in developmental programs and basic cellular functional machineries, including epigenetic modifications (EZH2), ribosome biogenesis (RPS3), translation initiation (EIF4G), and spliceosome assembly (SF3B1); all of these proteins are enriched in embryonic neural cells and play active roles in cancers. In addition, SETDB1 represses the transcription of genes promoting differentiation and cell cycle and growth arrest. Serial transplantation of cancer cells showed that neural stemness, tumorigenicity, and pluripotent differentiation potential were simultaneously enhanced; these effects were accompanied by increased expression of proteins involved in developmental programs and basic machineries, including SETDB1 and the abovementioned proteins, as well as by increased alternative splicing events. These results indicate that basic machineries work together to define a highly proliferative state with pluripotent differentiation potential and also suggest that neural stemness unifies tumorigenicity and differentiation potential.

Expressions of Type I and III Interferons, Endogenous Retroviruses, TRIM28, and SETDB1 in Children with Respiratory Syncytial Virus Bronchiolitis.

Interferons (IFNs) and IFN-stimulated genes (ISGs) play essential roles for the control of viral infections. Their expression in infants with respiratory syncytial virus (RSV) bronchiolitis is poorly defined. Human endogenous retroviruses (HERVs) represent 8% of our genome and modulate inflammatory and immune reactions. TRIM28 and SETDB1 participate in the epigenetic regulation of genes involved in the immune response, including IFNs and HERVs. No study has explored the expression of HERVs, TRIM28, and SETDB1 during RSV bronchiolitis. We assessed, through a PCR real-time Taqman amplification assay, the transcription levels of six IFN-I ISGs, four IFNλs, the pol genes of HERV-H, -K, and -W families, the env genes of Syncytin (SYN)1 and SYN2, and of TRIM28/SETDB1 in whole blood from 37 children hospitalized for severe RSV bronchiolitis and in healthy children (HC). The expression of most IFN-I ISGs was significantly higher in RSV+ patients than in age-matched HC, but it was inhibited by steroid therapy. The mRNA concentrations of IFN-λs were comparable between patients and age-matched HC. This lack of RSV-driven IFN-III activation may result in the defective protection of the airway mucosal surface leading to severe bronchiolitis. The expression of IFN-III showed a positive correlation with age in HC, that could account for the high susceptibility of young children to viral respiratory tract infections. The transcription levels of every HERV gene were significantly lower in RSV+ patients than in HC, while the expressions of TRIM28/SETDB1 were overlapping. Given the negative impact of HERVs and the positive effects of TRIM28/SETDB1 on innate and adaptive immune responses, the downregulation of the former and the normal expression of the latter may contribute to preserving immune functions against infection.

SetDB1 and Su(var)3-9 play non-overlapping roles in somatic cell chromosomes of Drosophila melanogaster.

We explored functional roles of two H3K9-specific histone methyltransferases of Drosophila melanogaster, SetDB1 (also known as Eggless) and Su(var)3-9. Using the DamID approach, we generated the binding profile for SetDB1 in Drosophila salivary gland chromosomes, and matched it to the profile of Su(var)3-9. Unlike Su(var)3-9, SetDB1 turned out to be an euchromatic protein that is absent from repeated DNA compartments, and is largely restricted to transcription start sites (TSSs) and 5' untranslated regions (5'UTRs) of ubiquitously expressed genes. Significant SetDB1 association is also observed at binding sites for the insulator protein CP190. SetDB1 and H3K9 di- and tri-methylated (me2 and me3)-enriched sites tend to display poor overlap. At the same time, SetDB1 has a clear connection with the distribution of H3K27me3 mark. SetDB1 binds outside the domains possessing this modification, and about half of the borders of H3K27me3 domains are decorated by SetDB1 together with actively transcribed genes. On the basis of poor correlation between the distribution of SetDB1 and H3K9 methylation marks, we speculate that, in somatic cells, SetDB1 may contribute to the methylation of a broader set of chromosomal proteins than just H3K9. In addition, SetDB1 can be expected to play a role in the establishment of chromatin functional domains.

Hyperglycaemia aggravates periodontal inflamm-aging by promoting SETDB1-mediated LINE-1 de-repression in macrophages.

AIM: To explore whether hyperglycaemia plays a role in periodontal inflamm-aging by inducing phenotypical transformation of macrophages, as well as the potential mechanism via SET domain-bifurcated histone lysine methyltransferase 1 (SETDB1). MATERIALS AND METHODS: A hyperglycaemic mouse model was established using streptozotocin injection. The alveolar bone was analysed using micro-computed tomography. Periodontal inflamm-aging was detected using western blotting, quantitative real-time PCR and immunohistochemical analysis. In vitro, RAW 264.7 macrophages were incubated with various doses of glucose. siRNA or overexpression plasmids were used to determine the regulatory mechanism of SETDB1 in macrophage senescence and inflamm-aging under hyperglycaemic conditions. Expression and distribution of SETDB1 and long interspersed element 1 (LINE-1) in gingival tissues of patients with or without diabetes were detected using immunofluorescent staining. RESULTS: SETDB1 expression in the periodontal tissues of patients and mice with diabetes was down-regulated compared with that in non-diabetic controls. SETDB1 deficiency induced senescence-like phenotypical changes in macrophages, which aggravated periodontal inflamm-aging in diabetic mice. Furthermore, metformin treatment rejuvenated SETDB1 activity and alleviated the hyperglycaemia-induced periodontal inflamm-aging. CONCLUSIONS: The findings of this study show that SETDB1 regulates senescence-like phenotypical switching of macrophages and is a potential candidate for the treatment of diabetes-induced periodontal inflamm-aging.

Atf7ip Inhibits Osteoblast Differentiation via Negative Regulation of the Sp7 Transcription Factor.

Epigenetic modifications are critical for cell differentiation and growth. As a regulator of H3K9 methylation, Setdb1 is implicated in osteoblast proliferation and differentiation. The activity and nucleus localization of Setdb1 are regulated by its binding partner, Atf7ip. However, whether Atf7ip is involved in the regulation of osteoblast differentiation remains largely unclear. In the present study, we found that Atf7ip expression was upregulated during the osteogenesis of primary bone marrow stromal cells and MC3T3-E1 cells, and was induced in PTH-treated cells. The overexpression of Atf7ip impaired osteoblast differentiation in MC3T3-E1 cells regardless of PTH treatment, as measured by the expression of osteoblast differentiation markers, Alp-positive cells, Alp activity, and calcium deposition. Conversely, the depletion of Atf7ip in MC3T3-E1 cells promoted osteoblast differentiation. Compared with the control mice, animals with Atf7ip deletion in the osteoblasts (Oc-Cre;Atf7ipf/f) showed more bone formation and a significant increase in the bone trabeculae microarchitecture, as reflected by µ-CT and bone histomorphometry. Mechanistically, Atf7ip contributed to the nucleus localization of Setdb1 in MC3T3-E1, but did not affect Setdb1 expression. Atf7ip negatively regulated Sp7 expression, and through specific siRNA, Sp7 knockdown attenuated the enhancing role of Atf7ip deletion in osteoblast differentiation. Through these data, we identified Atf7ip as a novel negative regulator of osteogenesis, possibly via its epigenetic regulation of Sp7 expression, and demonstrated that Atf7ip inhibition is a potential therapeutic measure for enhancing bone formation.

SETDB1 interactions with PELP1 contributes to breast cancer endocrine therapy resistance.

BACKGROUND: Methyltransferase SETDB1 is highly expressed in breast cancer (BC), however, the mechanisms by which SETDB1 promotes BC progression to endocrine therapy resistance remains elusive. In this study, we examined the mechanisms by which SETDB1 contribute to BC endocrine therapy resistance. METHODS: We utilized therapy sensitive (MCF7 and ZR75), therapy resistant (MCF7-TamR, MCF7-FR, MCF7-PELP1cyto, MCF7-SETDB1) estrogen receptor alpha positive (ER+)BC models and conducted in vitro cell viability, colony formation, 3-dimensional cell growth assays to investigate the role of SETDB1 in endocrine resistance. RNA-seq of parental and SETDB1 knock down ER+ BC cells was used to identify unique pathways. SETDB1 interaction with PELP1 was identified by yeast-two hybrid screen and confirmed by immunoprecipitation and GST-pull down assays. Mechanistic studies were conducted using Western blotting, reporter gene assays, RT-qPCR, and in vitro methylation assays. Xenograft assays were used to establish the role of PELP1 in SETDB1 mediated BC progression. RESULTS: RNA-seq analyses showed that SETDB1 regulates expression of a subset of estrogen receptor (ER) and Akt target genes that contribute to endocrine therapy resistance. Importantly, using yeast-two hybrid screen, we identified ER coregulator PELP1 as a novel interacting protein of SETDB1. Biochemical analyses confirmed SETDB1 and PELP1 interactions in multiple BC cells. Mechanistic studies confirmed that PELP1 is necessary for SETDB1 mediated Akt methylation and phosphorylation. Further, SETDB1 overexpression promotes tamoxifen resistance in BC cells, and PELP1 knockdown abolished these effects. Using xenograft model, we provided genetic evidence that PELP1 is essential for SETDB1 mediated BC progression in vivo. Analyses of TCGA datasets revealed SETDB1 expression is positively correlated with PELP1 expression in ER+ BC patients. CONCLUSIONS: This study suggests that the PELP1/SETDB1 axis play an important role in aberrant Akt activation and serves as a novel target for treating endocrine therapy resistance in breast cancer.

The role of the histone methyltransferase SET domain bifurcated 1 during palatal development.

The histone methyltransferase SET domain bifurcated 1 (SETDB1) catalyzes the trimethylation of lysine 9 of histone H3, thereby regulating gene expression. In this study, we used conditional knockout mice, where Setdb1 was deleted only in neural crest cells (Setdb1fl/fl,Wnt1-Cre + mice), to clarify the role of SETDB1 in palatal development. Setdb1fl/fl,Wnt1-Cre + mice died shortly after birth due to a cleft palate with full penetration. Reduced palatal mesenchyme proliferation was seen in Setdb1fl/fl,Wnt1-Cre + mice, which might be a possible mechanism of cleft palate development. Quantitative RT-PCR and in situ hybridization showed that expression of the Pax9, Bmp4, Bmpr1a, Wnt5a, and Fgf10 genes, known to be important for palatal development, were markedly decreased in the palatal mesenchyme of Setdb1fl/fl,Wnt1-Cre + mice. Along with these phenomena, SMAD1/5/9 phosphorylation was decreased by the loss of Setdb1. Our results demonstrated that SETDB1 is indispensable for palatal development partially through its proliferative effect. Taken together with previous reports that PAX9 regulates BMP signaling during palatal development which implies that loss of Setdb1 may be involved in the cleft palate development by decreasing SMAD-dependent BMP signaling through Pax9.

Distinct functions and temporal regulation of methylated histone H3 during early embryogenesis.

During the first hours of embryogenesis, formation of higher-order heterochromatin coincides with the loss of developmental potential. Here, we examine the relationship between these two events, and we probe the processes that contribute to the timing of their onset. Mutations that disrupt histone H3 lysine 9 (H3K9) methyltransferases reveal that the methyltransferase MET-2 helps terminate developmental plasticity, through mono- and di-methylation of H3K9 (me1/me2), and promotes heterochromatin formation, through H3K9me3. Although loss of H3K9me3 perturbs formation of higher-order heterochromatin, embryos are still able to terminate plasticity, indicating that the two processes can be uncoupled. Methylated H3K9 appears gradually in developing C. elegans embryos and depends on nuclear localization of MET-2. We find that the timing of H3K9me2 and nuclear MET-2 is sensitive to rapid cell cycles, but not to zygotic genome activation or cell counting. These data reveal distinct roles for different H3K9 methylation states in the generation of heterochromatin and loss of developmental plasticity by MET-2, and identify the cell cycle as a crucial parameter of MET-2 regulation.

Histone methyltransferase SETDB1 inhibits TGF-ß-induced epithelial-mesenchymal transition in pulmonary fibrosis by regulating SNAI1 expression and the ferroptosis signaling pathway.

The epithelial-mesenchymal transition (EMT) is an important pathological process in the occurrence of pulmonary fibrosis. Changes in histone methylation modifications of key genes play an important role in this process. As a histone methyltransferase, the regulatory mechanism and role of SET domain bifurcated 1 (SETDB1) in pulmonary fibrosis remain unclear. We found that SETDB1 inhibited EMT and that cells attenuated the expression of SETDB1 to relieve this inhibition during transforming growth factor-ß (TGF-ß)-induced EMT. Silencing SETDB1 expression significantly enhanced the mesenchymal phenotype induced by TGF-ß and the expression and deposition of fibronectin and significantly reduced the expression of E-cadherin. The decrease in E-cadherin expression and the induction of EMT led to increased lipid reactive oxygen species (ROS) and ferrous ions, which induced ferroptosis. Chromatin immunoprecipitation (ChIP) results showed that SETDB1 regulates the expression of Snai1 by catalyzing the histone H3 lysine 9 trimethylation (H3K9me3) of Snai1, the main transcription factor that initiates the process of EMT, and thus, indirectly regulates E-cadherin. Surprisingly, when examining the effect of overexpressed SETDB1 on EMT, we found that overexpressed SETDB1 alleviated EMT and also caused ferroptosis. We suggest that the overexpression of SETDB1 partially reverses the mesenchymal phenotype to an epithelial state, while those cells that fail to reverse are depleted by ferroptosis. In conclusion, the histone methylase SETDB1 regulates Snai1 epigenetically, driving EMT gene reprogramming and ferroptosis in response to TGF-ß. However, there are unexplored links between the epigenetic reprogramming and transcriptional processes that regulate EMT in a TGF-ß-dependent manner.

SETDB1 promotes gastric carcinogenesis and metastasis via upregulation of CCND1 and MMP9 expression.

SETDB1 is a histone lysine methyltransferase that has critical roles in cancers. However, its potential role in gastric cancer (GC) remains obscure. Here, we mainly investigate the clinical significance and the possible role of SETDB1 in GC. We find that SETDB1 expression is upregulated in GC tissues and its high-level expression was a predictor of poor prognosis in patients. Overexpression of SETDB1 promoted cell proliferation and metastasis, while SETDB1 suppression had an opposite effect both in vitro and in vivo. Mechanistically, SETDB1 was shown to interact with ERG to promote the transcription of cyclin D1 (CCND1) and matrix metalloproteinase 9 (MMP9) through binding to their promoter regions. In addition, the expression of SETDB1 was also enhanced by the transcription factor TCF4 at the transcriptional level in GC. Furthermore, SETDB1 expression was found to be induced by Helicobacter pylori (H. pylori) infection in a TCF4-dependent manner. Taken together, our results indicate that SETDB1 is aberrantly overexpressed in GC and plays key roles in gastric carcinogenesis and metastasis via upregulation of CCND1 and MMP9. Our work also suggests that SETDB1 could be a potential oncogenic factor and a therapeutic target for GC. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.