H3K27me3 Modification Research Articles

Kaposi's sarcoma-associated herpesvirus terminal repeat regulates inducible lytic gene promoters.

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

Single-cell multi-omics profiling of human preimplantation embryos identifies cytoskeletal defects during embryonic arrest.

Human in vitro fertilized embryos exhibit low developmental capabilities, and the mechanisms that underlie embryonic arrest remain unclear. Here using a single-cell multi-omics sequencing approach, we simultaneously analysed alterations in the transcriptome, chromatin accessibility and the DNA methylome in human embryonic arrest due to unexplained reasons. Arrested embryos displayed transcriptome disorders, including a distorted microtubule cytoskeleton, increased genomic instability and impaired glycolysis, which were coordinated with multiple epigenetic reprogramming defects. We identified Aurora A kinase (AURKA) repression as a cause of embryonic arrest. Mechanistically, arrested embryos induced through AURKA inhibition resembled the reprogramming abnormalities of natural embryonic arrest in terms of the transcriptome, the DNA methylome, chromatin accessibility and H3K4me3 modifications. Mitosis-independent sequential activation of the zygotic genome in arrested embryos showed that YY1 contributed to human major zygotic genome activation. Collectively, our study decodes the reprogramming abnormalities and mechanisms of human embryonic arrest and the key regulators of zygotic genome activation.

Evaluating histone modification analysis of individual preimplantation embryos

BackgroundWe previously reported a modification of the CUT&Tag method (NTU-CAT) that allows genome-wide histone modification analysis in individual preimplantation embryos. In the present study, NTU-CAT was further simplified by taking advantage of the Well-of-the-Well (WOW) system, which enables the processing of multiple embryos in a shorter time with less reagent and cell loss during the procedure (WOW-CUT&Tag, WOW-CAT).ResultsWOW-CAT allowed histone modification profiling from not only a single blastocyst but also from a portion of it. WOW-CAT generated similar H3K4me3 profiles as NTU-CAT, but they were closer to the profiles produced by chromatin immunoprecipitation-sequencing, such as a valley-like trend and relatively lower false positive rates, indicating that WOW-CAT may attenuate the bias of Tn5 transposase to cut open chromatin regions. Simultaneous WOW-CAT of two halves of single blastocysts was conducted to analyze two different histone modifications (H3K4me3 and H3K27ac) within the same embryo. Furthermore, trophectoderm cells were biopsied and subjected to WOW-CAT in anticipation of preimplantation diagnosis of histone modifications. WOW-CAT allowed the monitoring of epigenetic modifications in the main body of the embryo. For example, analysis of H3K4me3 modifications of XIST and DDX3Y in trophectoderm biopsies could be used to sex embryos in combination with quantitative PCR, but without the need for deep sequencing.ConclusionsThese results suggest the applicability of WOW-CAT for flexible epigenetic analysis of individual embryos in preimplantation epigenetic diagnosis.

PCGF6 controls murine Tuft cell differentiation via H3K9me2 modification independently of Polycomb repression

Cell fate is determined by specific transcription programs that are essential for tissue homeostasis and regeneration. The E3-ligases RING1A and B represent the core activity of the Polycomb repressive complex 1 (PRC1) that deposits repressive histone H2AK119 mono-ubiquitination (H2AK119ub1), which is essential for mouse intestinal homeostasis by preserving stem cell functions. However, the specific role of different PRC1 forms, which are defined by the six distinct PCGF1-6 paralogs, remains largely unexplored invivo. We report that PCGF6 regulates mouse intestinal Tuft cell differentiation independently of H2AK119ub1 deposition. We show that PCGF6 chromatin occupancy expands outside Polycomb repressive domains, associating with unique promoter and distal regulatory elements. This occurs in the absence of RING1A/B and involves MGA-mediated E-BOX recognition and specific H3K9me2 promoter deposition. PCGF6 inactivation induces an epithelial autonomous accumulation of Tuft cells that was not phenocopied by RING1A/B loss. This involves direct PCGF6 association with a Tuft cell differentiation program that identified Polycomb-independent properties of PCGF6 in adult tissues homeostasis.

Cfp1 Controls Cardiomyocyte Maturation by Modifying Histone H3K4me3 of Structural, Metabolic, and Contractile Related Genes.

Cardiomyocyte maturation is the final stage of heart development, and abnormal cardiomyocyte maturation will lead to serious heart diseases. CXXC zinc finger protein 1 (Cfp1), a key epigenetic factor in multi-lineage cell development, remains underexplored in its influence on cardiomyocyte maturation. This study investigates the role and mechanisms of Cfp1 in this context. Cardiomyocyte-specific Cfp1 knockout (Cfp1-cKO) mice died within 4 weeks of birth. Cardiomyocytes derived from Cfp1-cKO mice showed an inhibited maturation phenotype, characterized by structural, metabolic, contractile, and cell cycle abnormalities. In contrast, cardiomyocyte-specific Cfp1 transgenic (Cfp1-TG) mice and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) overexpressing Cfp1 displayed a more mature phenotype. Mechanistically, deficiency of Cfp1 led to a reduction in trimethylation on lysine 4 of histone H3 (H3K4me3) modification, accompanied by the formation of ectopic H3K4me3. Furthermore, Cfp1 deletion decreased the level of H3K4me3 modification in adult genes and increased the level of H3K4me3 modification in fetal genes. Collectively, Cfp1 modulates the expression of genes crucial to cardiomyocyte maturation by regulating histone H3K4me3 modification, thereby intricately influencing the maturation process. This study implicates Cfp1 as an important molecule regulating cardiomyocyte maturation, with its dysfunction strongly linked to cardiac disease.

NELF focuses sites of initiation and maintains promoter architecture.

Many factors control the elongation phase of transcription by RNA polymerase II (Pol II), a process that plays an essential role in regulating gene expression. We utilized cells expressing degradation tagged subunits of NELFB, PAF1and RTF1 to probe the effects of depletion of the factors on nascent transcripts using PRO-Seq and on chromatin architecture using DFF-ChIP. Although NELF is involved in promoter proximal pausing, depletion of NELFB had only a minimal effect on the level of paused transcripts and almost no effect on control of productive elongation. Instead, NELF depletion increased the utilization of downstream transcription start sites and caused a dramatic, genome-wide loss of H3K4me3 marked nucleosomes. Depletion of PAF1 and RTF1 both had major effects on productive transcript elongation in gene bodies and also caused initiation site changes like those seen with NELFB depletion. Our study confirmed that the first nucleosome encountered during initiation and early elongation is highly positioned with respect to the major TSS. In contrast, the positions of H3K4me3 marked nucleosomes in promoter regions are heterogeneous and are influenced by transcription. We propose a model defining NELF function and a general role of the H3K4me3 modification in blocking transcription initiation.

The m6A reader YTHDC2 regulates UVB-induced DNA damage repair and histone modification.

Ultraviolet B (UVB) radiation represents a major carcinogen for the development of all skin cancer types. Mechanistically, UVB induces damage to DNA in the form of lesions, including cyclobutane pyrimidine dimers (CPDs). Disruption of the functional repair processes, such as nucleotide excision repair (NER), allows persistence of DNA damage and contributes to skin carcinogenesis. Recent work has implicated m6A RNA methylation and its regulatory proteins as having critical roles in facilitating UVB-induced DNA damage repair. However, the biological functions of the m6A reader YTHDC2 are unknown in this context. Here, we show that YTHDC2 inhibition enhances the repair of UVB-induced DNA damage. We discovered that YTHDC2 inhibition increased the expression of PTEN while it decreased the expression of the PRC2 component SUZ12 and the levels of the histone modification H3K27me3. However, none of these functions were causally linked to the improvements in DNA repair, suggesting that the mechanism utilized by YTHDC2 may be unconventional. Moreover, inhibition of the m6A writer METTL14 reversed the effect of YTHDC2 inhibition on DNA repair while inhibition of the m6A eraser FTO mimicked the effect of YTHDC2 inhibition, indicating that YTHDC2 may regulate DNA repair through the m6A pathway. Finally, compared to normal human skin, YTHDC2 expression was upregulated in human cutaneous squamous cell carcinomas (cSCC), suggesting that it may function as a tumor-promoting factor in skin cancer. Taken together, our findings demonstrate that the m6A reader YTHDC2 plays a role in regulating UVB-induced DNA damage repair and may serve as a potential biomarker in cSCC.

Temporally-coordinated bivalent histone modifications of BCG1 enable fungal invasion and immune evasion

Bivalent histone modifications, including functionally opposite H3K4me3 and H3K27me3 marks simultaneously on the same nucleosome, control various cellular processes by fine-tuning the gene expression in eukaryotes. However, the role of bivalent histone modifications in fungal virulence remains elusive. By mapping the genome-wide landscape of H3K4me3 and H3K27me3 dynamic modifications in Fusarium graminearum (Fg) during invasion, we identify the infection-related bivalent chromatin-marked genes (BCGs). BCG1 gene, which encodes a secreted Fusarium-specific xylanase containing a G/Q-rich motif, displays the highest increase of bivalent modification during Fg infection. We report that the G/Q-rich motif of BCG1 is a stimulator of its xylanase activity and is essential for the full virulence of Fg. Intriguingly, this G/Q-rich motif is recognized by pattern-recognition receptors to trigger plant immunity. We discover that Fg employs H3K4me3 modification to induce BCG1 expression required for host cell wall degradation. After breaching the cell wall barrier, this active chromatin state is reset to bivalency by co-modifying with H3K27me3, which enables epigenetic silencing of BCG1 to escape from host immune surveillance. Collectively, our study highlights how fungal pathogens deploy bivalent epigenetic modification to achieve temporally-coordinated activation and suppression of a critical fungal gene, thereby facilitating successful infection and host immune evasion.

Single-cell joint profiling of multiple epigenetic proteins and gene transcription.

Sculpting the epigenome with a combination of histone modifications and transcription factor occupancy determines gene transcription and cell fate specification. Here, we first develop uCoTarget, utilizing a split-pool barcoding strategy for realizing ultrahigh-throughput single-cell joint profiling of multiple epigenetic proteins. Through extensive optimization for sensitivity and multimodality resolution, we demonstrate that uCoTarget enables simultaneous detection of five histone modifications (H3K27ac, H3K4me3, H3K4me1, H3K36me3, and H3K27me3) in 19,860 single cells. We applied uCoTarget to the in vitro generation of hematopoietic stem/progenitor cells (HSPCs) from human embryonic stem cells, presenting multimodal epigenomic profiles in 26,418 single cells. uCoTarget reveals establishment of pairing of HSPC enhancers (H3K27ac) and promoters (H3K4me3) and RUNX1 engagement priming for H3K27ac activation along the HSPC path. We then develop uCoTargetX, an expansion of uCoTarget to simultaneously measure transcriptome and multiple epigenome targets. Together, our methods enable generalizable, versatile multimodal profiles for reconstructing comprehensive epigenome and transcriptome landscapes and analyzing the regulatory interplay at single-cell level.

The SOX4/EZH2/SLC7A11 signaling axis mediates ferroptosis in calcium oxalate crystal deposition-induced kidney injury

Epigenetic regulation is reported to play a significant role in the pathogenesis of various kidney diseases, including renal cell carcinoma, acute kidney injury, renal fibrosis, diabetic nephropathy, and lupus nephritis. However, the role of epigenetic regulation in calcium oxalate (CaOx) crystal deposition-induced kidney injury remains unclear. Our study demonstrated that the upregulation of enhancer of zeste homolog 2 (EZH2)-mediated ferroptosis facilitates CaOx-induced kidney injury. CaOx crystal deposition promoted ferroptosis in vivo and in vitro. Usage of liproxstatin-1 (Lip-1), a ferroptosis inhibitor, mitigated CaOx-induced kidney damage. Single-nucleus RNA-sequencing, RNA-sequencing, immunohistochemical and western blotting analyses revealed that EZH2 was upregulated in kidney stone patients, kidney stone mice, and oxalate-stimulated HK-2 cells. Experiments involving in vivo EZH2 knockout, in vitro EZH2 knockdown, and in vivo GSK-126 (an EZH2 inhibitor) treatment confirmed the protective effects of EZH2 inhibition on kidney injury and ferroptosis. Mechanistically, the results of RNA-sequencing and chromatin immunoprecipitation assays demonstrated that EZH2 regulates ferroptosis by suppressing solute carrier family 7, member 11 (SLC7A11) expression through trimethylation of histone H3 lysine 27 (H3K27me3) modification. Additionally, SOX4 regulated ferroptosis by directly modulating EZH2 expression. Thus, this study demonstrated that SOX4 facilitates ferroptosis in CaOx-induced kidney injury through EZH2/H3K27me3-mediated suppression of SLC7A11.Graphical

Analysis of histone H3K4me3 modifications in bovine placenta derived from different calf-production methods.

In ruminants, the overgrowth of offspring produced by invitro fertilization (IVF) is a common problem. Abnormal epigenetic modifications caused by environmental factors during the early embryonic period are suspected as an aetiology of overgrowth. In this study, we investigated the genome-wide histone H3K4me3 profiles of bovine placentae that play a pivotal role in foetal development and compared their characteristics between artificial insemination (AI)- and IVF-derived samples. Cotyledons were harvested from the placentae obtained at parturition of 5 AI- and 13 IVF-derived calves, and chromatin immunoprecipitation sequencing was performed for H3K4me3. We confirmed no significant maternal tissue contamination in the samples we used. The revealed H3K4me3 profiles reflected the general characteristics of the H3K4me3 modification, which is abundantly distributed in the promoter region of active genes. By extracting common modifications from multiple samples, the genes involved in placenta-specific biological processes could be enriched. Comparison with the H3K4me3 modifications of blastocyst samples was also effective for enriching the placenta-specific features. Principal component analysis suggested the presence of differential H3K4me3 modifications in AI- and IVF-derived samples. The genes contributing to the difference were related to the developmental biological processes. Imprinted genes such as BEGAIN, ZNF215 and DLX5 were among the extracted genes. Principal component and discriminant analyses using only male samples categorized the samples into three groups based on foetal weight and calf-production methods. To our knowledge, this is the first study to profile the genome-wide histone modifications of bovine foetal placentae and reveal their differential characteristics between different calf-production methods.

CircBRAF promotes the progression of triple-negative breast cancer through modulating methylation by recruiting KDM4B to histone H3K9me3 and IGF2BP3 to mRNA.

Understanding the molecular characteristics of triple-negative breast cancer (TNBC) and developing more tailored treatment approaches is crucial. Circular RNAs (circRNAs), as potential therapeutic targets, remain largely unexplored in TNBC. This study utilized circRNA microarray analysis to determine the expression of circRNAs in TNBC, analyzing nine patient specimens. The characteristics of circBRAF were examined using divergent PCR primers, Sanger sequencing, fluorescence in situ hybridization (FISH) analysis, and the application of RNase and actinomycin D. The biological function of circBRAF in TNBC was further investigated through colony formation, tube formation, and transwell assays. Crucially, the mechanisms underlying the effects of circBRAF on TNBC progression were explored via RNA immunoprecipitation sequencing (RIP-seq) data, MS2 pulldown, RNA sequencing (RNA-seq) analysis, circBRAF knockdown, histone H3K9me3 modification, and Chromatin Isolation by RNA Purification (ChIRP) tests followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We focused particularly on hsa_circ_0007178, produced from exons 4-13 of the oncogene BRAF. Functional experiments revealed that circBRAF is crucial for the development of TNBC, with its knockdown preventing angiogenesis, metastasis, and cell division in vitro. Mechanistically, circBRAF interacts with KDM4B and IGF2BP3, promoting TNBC growth. Interaction of circBRAF with IGF2BP3 increased the expression of VCAN, FN1, CDCA3, or B4GALT3 by controlling mRNA stability through RNA N6-methyladenosine (m6A) modification. Furthermore, circBRAF upregulated the expression of ADAMTS14 and MMP9 through recruitment of KDM4B to enhance respective H3K9me3 modification. Furthermore, overexpression of circBRAF was able to overcome the inhibitory effects of siKDM4B and siIGF2BP3 on cell migration and invasion. Our findings suggest that circBRAF may act as an oncogene in TNBC through its specific interactions with KDM4B and IGF2BP3, implying that circBRAF could serve as a potentially effective novel therapeutic target for TNBC.

EZH2 inhibitors promote β-like cell regeneration in young and adult type 1 diabetes donors

β-cells are a type of endocrine cell found in pancreatic islets that synthesize, store and release insulin. In type 1 diabetes (T1D), T-cells of the immune system selectively destroy the insulin-producing β-cells. Destruction of these cells leads to a lifelong dependence on exogenous insulin administration for survival. Consequently, there is an urgent need to identify novel therapies that stimulate β-cell growth and induce β-cell function. We and others have shown that pancreatic ductal progenitor cells are a promising source for regenerating β-cells for T1D owing to their inherent differentiation capacity. Default transcriptional suppression is refractory to exocrine reaction and tightly controls the regenerative potential by the EZH2 methyltransferase. In the present study, we show that transient stimulation of exocrine cells, derived from juvenile and adult T1D donors to the FDA-approved EZH2 inhibitors GSK126 and Tazemetostat (Taz) influence a phenotypic shift towards a β-like cell identity. The transition from repressed to permissive chromatin states are dependent on bivalent H3K27me3 and H3K4me3 chromatin modification. Targeting EZH2 is fundamental to β-cell regenerative potential. Reprogrammed pancreatic ductal cells exhibit insulin production and secretion in response to a physiological glucose challenge ex vivo. These pre-clinical studies underscore the potential of small molecule inhibitors as novel modulators of ductal progenitor differentiation and a promising new approach for the restoration of β-like cell function.

Normal distribution of H3K9me3 occupancy co-mediated by histone methyltransferase BcDIM5 and histone deacetylase BcHda1 maintains stable ABA synthesis in Botrytis cinerea TB-31.

Abscisic acid (ABA) is a conserved and important "sesquiterpene signaling molecule" widely distributed in different organisms with unique biological functions. ABA coordinates reciprocity and competition between microorganisms and their hosts. In addition, ABA also regulates immune and stress responses in plants and animals. Therefore, ABA has a wide range of applications in agriculture, medicine and related fields. The plant pathogenic ascomycete B. cinerea has been extensively studied as a model strain for ABA production. Nevertheless, there is a relative dearth of research regarding the regulatory mechanism governing ABA biosynthesis in B. cinerea. Here, we discovered that H3K9 methyltransferase BcDIM5 is physically associated with the H3K14 deacetylase BcHda1. Deletion of Bcdim5 and Bchda1 in the high ABA-producing B. cinerea TB-31 led to severe impairment of ABA synthesis. The combined analysis of RNA-seq and ChIP-seq has revealed that the absence of BcDIM5 and BcHda1 has resulted in significant global deficiencies in the normal distribution and level of H3K9me3 modification. In addition, we found that the cause of the decreased ABA production in the ΔBcdim5 and ΔBchda1 mutants was due to cluster gene repression caused by the emergence of hyper-H3K9me3 in the ABA gene cluster. We concluded that the ABA gene cluster is co-regulated by BcDIM5 and BcHda1, which are essential for the normal distribution of the B. cinerea TB-31 ABA gene cluster H3K9me3. This work expands our understanding of the complex regulatory network of ABA biosynthesis and provides a theoretical basis for genetic improvement of high-yielding ABA strains.

Characterization of the distribution and dynamics of chromatin states in the C. elegans germline reveals substantial H3K4me3 remodeling during oogenesis.

Chromatin organization in the C. elegans germline is tightly regulated and critical for germ cell differentiation. Although certain germline epigenetic regulatory mechanisms have been identified, how they influence chromatin structure and ultimately gene expression remains unclear, in part because most genomic studies have focused on data collected from intact worms comprising both somatic and germline tissues. We therefore analyzed histone modification and chromatin accessibility data from isolated germ nuclei representing undifferentiated proliferating and meiosis I populations to define chromatin states. We correlated these states with overall transcript abundance, spatiotemporal expression patterns, and the function of small RNA pathways. Because the essential role of the germline is to transmit genetic information and establish gene expression in the early embryo, we compared epigenetic and transcriptomic profiles from undifferentiated germ cells to those of embryos to define the epigenetic changes during this developmental transition. The active histone modification H3K4me3 shows particularly dynamic remodeling as germ cells differentiate into oocytes, which suggests a mechanism for establishing early transcription of essential genes during zygotic genome activation. This analysis highlights the dynamism of the chromatin landscape across developmental transitions and provides a resource for future investigation into epigenetic regulatory mechanisms in germ cells.

Histone binding of ASF1 is required for fruiting body development but not for genome stability in the filamentous fungus Sordaria macrospora.

Histone chaperones are proteins that are involved in nucleosome assembly and disassembly and can therefore influence all DNA-dependent processes including transcription, DNA replication, and repair. ASF1 is a histone chaperone that is conserved throughout eukaryotes. In contrast to most other multicellular organisms, a deletion mutant of asf1 in the fungus Sordaria macrospora is viable; however, the mutant is sterile. In this study, we could show that the histone-binding ability of ASF1 is required for fertility in S. macrospora, whereas the function of ASF1 in maintenance of genome stability does not require histone binding. We also showed that the histone modifications H3K27me3 and H3K56ac are misregulated in the Δasf1 mutant. Furthermore, we identified a large duplication on chromosome 2 of the mutant strain that is genetically linked to the Δasf1 allele present on chromosome 6, suggesting that viability of the mutant might depend on the presence of the duplicated region.

Poly(I:C) induces anti-inflammatory response against secondary LPS challenge in zebrafish larvae

Poly(I:C) is known as an agonist of the TLR3 receptor which could prime inflammation and elicit the host immune response, which is widely applied as adjuvant or antivirus treatment. However, the negative effects of poly(I:C) on regulating immune response to protect the host from inflammatory diseases remain largely unknown. Here, we establish an in vivo model to pre-treat zebrafish larvae with poly(I:C) at 2 dpf, then challenge them with LPS at 6 dpf, and find that poly(I:C) training could significantly alleviate the LPS challenge-induced septic shock and inflammatory phenotypes. Moreover, the poly(I:C)-trained larvae exhibit decreased number of macrophages, but not neutrophils, after secondary LPS challenge. Furthermore, training the larvae with poly(I:C) could elevate the transcripts of mTOR signaling and heighten the H3K4me3-mediated epigenetic modifications. And interestingly, we find that inhibiting the H3K4me3 modification, rather than mTOR signaling, could recover the number of macrophages in poly(I:C)-trained larvae, which is consistent with the observations of inflammatory phenotypes. Taken together, these results suggest that poly(I:C) training could induce epigenetic rewiring to mediate the anti-inflammatory response against secondary LPS challenge-induced septic shock through decreasing macrophages’ number in vivo, which might expand our understanding of poly(I:C) in regulating fish immune response.

Low H3K27me3 deposition at CYP82E4 determines the nicotinic conversion rate in Nicotiana tabacum

Nicotine conversion is the process by which nornicotine is synthesized from nicotine. The capacity of a plant to carry out this process is represented by the nicotine conversion rate (NCR), which is defined as the percentage of nornicotine content out of the total nicotine + nornicotine content. Nicotine conversion in tobacco is mediated by CYP82E4. Although there are cultivar-specific differences in NCR, these do not correspond to differences in the CYP82E4 promoter or gene body sequences, and little is known about the underlying regulatory mechanism. Here, we found that histone H3 Lysine 27 trimethylation (H3K27me3) was involved in CYP82E4 expression, functioning as a transcriptional repressor. Compared to a high-NCR near-isogenic line, a low-NCR cultivar showed increased levels of the repressive histone modification markers H3K27me3 and H3K9me3 at CYP82E4. Comparison of histone markers between several cultivars with varying NCRs showed that H3K27me3 and H3K9me3 levels were significantly associated with cultivar-specific differences in NCR. Treatment with the H3K27me3 demethylase inhibitor GSK-J4 increased total H3K27me3 levels and enriched H3K27me3 at the CYP82E4 locus; the increased levels of H3K27me3 further inhibited CYP82E4 expression. Knocking out E(z), an indispensable gene for H3K27me3 formation, decreased H3K27me3 levels at CYP82E4, leading to a more than three-fold increase in CYP82E4 expression. Changes in CYP82E4 expression during leaf senescence and chilling stress were also strongly correlated with H3K27me3 levels. These findings reveal a strong correlation between CYP82E4 expression and histone modifications, and demonstrate an instance of histone-mediated alkaloid regulation for the first time.

An intronic LINE-1 regulates IFNAR1 expression in human immune cells

BackgroundDespite their origins as selfish parasitic sequences, some transposons in the human genome have been co-opted to serve as regulatory elements, contributing to the evolution of transcriptional networks. Most well-characterized examples of transposon-derived regulatory elements derive from endogenous retroviruses (ERVs), due to the intrinsic regulatory activity of proviral long terminal repeat regions. However, one subclass of transposable elements, the Long Interspersed Nuclear Elements (LINEs), have been largely overlooked in the search for functional regulatory transposons, and considered to be broadly epigenetically repressed.ResultsWe examined the chromatin state of LINEs by analyzing epigenomic data from human immune cells. Many LINEs are marked by the repressive H3K9me3 modification, but a subset exhibits evidence of enhancer activity in human immune cells despite also showing evidence of epigenetic repression. We hypothesized that these competing forces of repressive and activating epigenetic marks might lead to inducible enhancer activity. We investigated a specific L1M2a element located within the first intron of Interferon Alpha/Beta Receptor 1 (IFNAR1). This element shows epigenetic signatures of B cell-specific enhancer activity, despite being repressed by the Human Silencing Hub (HUSH) complex. CRISPR deletion of the element in B lymphoblastoid cells revealed that the element acts as an enhancer that regulates both steady state and interferon-inducible expression of IFNAR1.ConclusionsOur study experimentally demonstrates that an L1M2a element was co-opted to function as an interferon-inducible enhancer of IFNAR1, creating a feedback loop wherein IFNAR1 is transcriptionally upregulated by interferon signaling. This finding suggests that other LINEs may exhibit cryptic cell type-specific or context-dependent enhancer activity. LINEs have received less attention than ERVs in the effort to understand the contribution of transposons to the regulatory landscape of cellular genomes, but these are likely important, lineage-specific players in the rapid evolution of immune system regulatory networks and deserve further study.

SETDB1 Suppresses Interferon Responses and NK Cell-Mediated Immunosurveillance Specifically in Monocytic AML

Acute myeloid leukemia (AML) with monocytic differentiation (monocytic AML) responds poorly to current treatments, including venetoclax-based therapy. To identify novel therapeutic targets in monocytic AML, we conducted in vivo and in vitro CRISPR/Cas9 library screens using a pooled sgRNA library targeting epigenetic regulators with a mouse monocytic AML model driven by SETBP1 and ASXL1 mutations (cSAM: combined expression of SETBP1 and ASXL1 Mutations). We ranked all the epigenetic genes according to the in vivo specific essentiality, and selected several candidates which were more depleted in mice rather than in cell cultures. The top-ranked gene was Setdb1, which encodes a histone H3 lysine 9 (H3K9) methyltransferase. In addition, several H3K9 trimethylation (H3K9me3) regulators, ATF7IP and TRIM33, were also more important in vivo than in vitro in our screens. ATF7IP is a cofactor of SETDB1 to enhance its enzymatic activity. TRIM33 is also involved in H3K9me3 modification. These data suggest the critical role of the SETDB1-ATF7IP-TRIM33 H3K9 methyltransferase complex in the development of AML in vivo. To validate the results of our screenings and to investigate the role of immune cells in our transplantation assay, we next transplanted control and Setdb1-knockout (KO) cSAM cells into three types of mice with different immune systems: the immunocompetent C57BL/6 mice, the immunodeficient NSG mice lacking T, B and natural killer (NK) cells, and the C57BL/6J- Rag2-/- mice lacking mature T and B cells but possessing NK cells. Although Setdb1 depletion did not inhibit the growth of cSAM cells in NSG mice, it showed the strong growth-inhibitory effect in C57BL/6 mice and Rag2-/- mice (Fig 1A). Since both C57BL/6 mice and Rag2-/- mice have NK cells, these data suggest that SETDB1 promotes leukemogenesis by suppressing NK cell-mediated cytotoxicity in vivo. Mechanistically, loss of Setdb1 induced upregulation of NKG2D ligands, interferon-stimulated genes (ISGs), and endogenous retrovirus (ERV) sequences through H3K9me3 reduction, thereby rendering cSAM cells more susceptible to NK cell-mediated cytotoxicity. Setdb1 depletion also induced myeloid maturation of cSAM cells towards macrophages and downregulation of major histocompatibility complex class I (MHC-I), which contributed to the increased susceptibility of Setdb1-depleted cSAM cells to NK cell-mediated cytotoxicity. Next, we investigated whether SETDB1 inactivation also sensitizes human AML cells to NK cells using the co-culture assay with an IL-2 dependent NK cell line NK-92. Loss of SETDB1 rendered monocytic AML cell lines, NOMO-1, THP-1, OCI-AML3, U937, and NB4, more susceptible to NK-92 cell-mediated cytotoxicity. SETDB1 depletion also induced overactivation of interferon signaling and intrinsic apoptosis in these monocytic AML cells. In contrast, non-monocytic AML cells, such as K562, TF-1, and Kasumi-1, were not sensitive to SETDB1 depletion. SETDB1 depletion neither increased the susceptibility to NK cell-mediated cytotoxicity nor induced apoptosis in non-monocytic AML cells. Subsequent analyses revealed that SETDB1 depletion reduced H3K9me3 at the monocyte-specific enhancer regions with ERV sequences to upregulate NK cell-activating ligands and ISGs. Because the “ERV enhancers” are active only in monocyte lineage, SETDB1 is essential specifically in monocytic AML. Finally, we identified MNDA and its mouse counterpart Ifi203 as biomarkers to predict the sensitivity of each AML to SETDB1 depletion. MNDA is one of the ISGs and is involved in myeloid cell differentiation. Human monocytic AML cells that are dependent on SETDB1 expressed higher MNDA at both RNA and protein levels compared to other subtypes of AML. We also found that Ifi203-high mouse AML cells transformed by MLL-AF9 were sensitive to Setdb1 depletion, whereas Setdb1 was dispensable in Ifi203-low mouse AML cells transformed by RUNX1-RUNX1T1. In summary, our findings reveal the critical and selective role of SETDB1 in monocytic AML. SETDB1 mediates H3K9me3 at the monocyte-specific ERV enhancers to repress the expression of NK cell-activating ligands and ISGs, thereby suppressing immunogenicity and apoptosis in monocytic AML (Fig 1B). SETDB1 as well as its binding partners could be promising therapeutic targets to treat monocytic AMLs that are relatively resistant to the current standard therapies.