Lysine-specific histone demethylase 1 (LSD1), which demethylates mono- or di- methylated histone H3 on lysine 4 (H3K4me1/2), is essential for early embryogenesis and development. Here we show that LSD1 is dispensable for mouse embryonic stem cell (ESC) self-renewal but is required for mouse ESC growth and differentiation. Reintroduction of a catalytically-impaired LSD1 (LSD1MUT) recovers the proliferation capability of mouse ESCs, yet the enzymatic activity of LSD1 is essential to ensure proper differentiation. Indeed, increased H3K4me1 in Lsd1 knockout (KO) mouse ESCs does not lead to major changes in global gene expression programs related to stemness. However, ablation of LSD1 but not LSD1MUT results in decreased DNMT1 and UHRF1 proteins coupled to global hypomethylation. We show that both LSD1 and LSD1MUT control protein stability of UHRF1 and DNMT1 through interaction with HDAC1 and the ubiquitin-specific peptidase 7 (USP7), consequently, facilitating the deacetylation and deubiquitination of DNMT1 and UHRF1. Our studies elucidate a mechanism by which LSD1 controls DNA methylation in mouse ESCs, independently of its lysine demethylase activity.

BACKGROUND: Value analysis of a small-molecule fluorescent probe for methylation detection in different cervical lesions.
METHODS: (1) The grayscale values of distinct lesion tissues were remarkably distinct among the four groups (p < 0.05). The comparison of the grayscale value between the two groups showed that the CA group noticeably exceeded the LSIL and cervicitis groups, and the HSIL group was apparently higher than the LSIL and cervicitis groups (p < 0.05); (2) The mean grayscale values of the enrolled subjects were calculated with 55.21 as the midline, with >55.21 as positive and ≤55.21 as negative.
RESULTS: The results showed that the positive rate of the cervicitis group was 0.00%, the LSIL group 67.74%, the HSIL group 83.33%, and the CA group 100.00%. The results among the four groups were notably distinct (p < 0.05); (3) The comparison among DAPI, probe, bright, and merged images of cervicitis, LSIL, HSIL, and CA indicated that different cervical lesions were with quite various stains.
CONCLUSIONS: The grayscale value, positive rate, and stained picture of distinct cervical lesions were remarkably different. The small-molecule fluorescent probe has a good value in differentiating cervical lesions and can be considered for popularization and application.

In this study, the expression profiles of miR-148a were constructed in eight different ovine tissues, including mammary gland tissue, during six different developmental periods. The effect of miR-148a on the viability, proliferation, and milk fat synthesis of ovine mammary epithelial cells (OMECs) was investigated, and the target relationship of miR-148a with two predicted target genes was verified. The expression of miR-148a exhibited obvious tissue-specific and temporal-specific patterns. miR-148a was expressed in all eight ovine tissues investigated, with the highest expression level in mammary gland tissue (p < 0.05). Additionally, miR-148a was expressed in ovine mammary gland tissue during each of the six developmental periods studied, with its highest level at peak lactation (p < 0.05). The overexpression of miR-148a increased the viability of OMECs, the number and percentage of Edu-labeled positive OMECs, and the expression levels of two cell-proliferation marker genes. miR-148a also increased the percentage of OMECs in the S phase. In contrast, transfection with an miR-148a inhibitor produced the opposite effect compared to the miR-148a mimic. These results indicate that miR-148a promotes the viability and proliferation of OMECs in Small-tailed Han sheep. The miR-148a mimic increased the triglyceride content by 37.78% (p < 0.01) and the expression levels of three milk fat synthesis marker genes in OMECs. However, the miR-148a inhibitor reduced the triglyceride level by 87.11% (p < 0.01). These results suggest that miR-148a promotes milk fat synthesis in OMECs. The dual-luciferase reporter assay showed that miR-148a reduced the luciferase activities of DNA methyltransferase 1 (DNMT1) and peroxisome proliferator-activated receptor gamma coactivator 1-A (PPARGC1A) in wild-type vectors, suggesting that they are target genes of miR-148a. The expression of miR-148a was highly negatively correlated with PPARGC1A (r = -0.789, p < 0.001) in ovine mammary gland tissue, while it had a moderate negative correlation with DNMT1 (r = -0.515, p = 0.029). This is the first study to reveal the molecular mechanisms of miR-148a underlying the viability, proliferation, and milk fat synthesis of OMECs in sheep.

In mammals, global passive demethylation contributes to epigenetic reprogramming during early embryonic development. At this stage, the majority of DNA-methyltransferase 1 (DNMT1) protein is excluded from nucleus, which is considered the primary cause. However, whether the remaining nuclear activity of DNMT1 is regulated by additional mechanisms is unclear. Here, we report that nuclear DNMT1 abundance is finetuned through proteasomal degradation in mouse zygotes. We identify a maternal factor, Pramel15, which targets DNMT1 for degradation via Cullin-RING E3 ligases. Loss of Pramel15 elevates DNMT1 levels in the zygote pronuclei, impairs zygotic DNA demethylation, and causes a stochastic gain of DNA methylation in early embryos. Thus, Pramel15 can modulate the residual level of DNMT1 in the nucleus during zygotic DNA replication, thereby ensuring efficient DNA methylation reprogramming in early embryos.

The human immunodeficiency virus type 1 (HIV-1) reservoir consists of latently infected cells which present a major obstacle to achieving a functional cure for HIV-1. The formation and maintenance of HIV-1 latency have been extensively studied, and latency-reversing agents (LRAs) that can reactivate latent HIV-1 by targeting the involved host factors are developed; however, their clinical efficacies remain unsatisfactory. Therefore, it is imperative to identify novel targets for more potential candidates or better combinations for LRAs. In this study, we utilized CRISPR affinity purification in situ of regulatory elements system to screen for host factors associated with the HIV-1 long terminal repeat region that could potentially be involved in HIV-1 latency. We successfully identified that origin recognition complex 1 (ORC1), the largest subunit of the origin recognition complex, contributes to HIV-1 latency in addition to its function in DNA replication initiation. Notably, ORC1 is enriched on the HIV-1 promoter and recruits a series of repressive epigenetic elements, including DNMT1 and HDAC1/2, and histone modifiers, such as H3K9me3 and H3K27me3, thereby facilitating the establishment and maintenance of HIV-1 latency. Moreover, the reactivation of latent HIV-1 through ORC1 depletion has been confirmed across various latency cell models and primary CD4+ T cells from people living with HIV-1. Additionally, we comprehensively validated the properties of liquid-liquid phase separation (LLPS) of ORC1 from multiple perspectives and identified the key regions that promote the formation of LLPS. This property is important for the recruitment of ORC1 to the HIV-1 promoter. Collectively, these findings highlight ORC1 as a potential novel target implicated in HIV-1 latency and position it as a promising candidate for the development of novel LRAs.
OBJECTIVE: Identifying host factors involved in maintaining human immunodeficiency virus type 1 (HIV-1) latency and understanding their mechanisms prepares the groundwork to discover novel targets for HIV-1 latent infection and provides further options for the selection of latency-reversing agents in the \"shock\" strategy. In this study, we identified a novel role of the DNA replication factor origin recognition complex 1 (ORC1) in maintaining repressive chromatin structures surrounding the HIV-1 promoter region, thereby contributing to HIV-1 latency. This discovery expands our understanding of the non-replicative functions of the ORC complex and provides a potential therapeutic strategy for HIV-1 cure.

BACKGROUND: The treatment of food allergy (FA) needs improvement. The treatment of immune disorders can be improved by regulating epigenetic marks, which is a promising method. The objective of this research is to alleviate experimental FA by employing an inhibitor of DNA methyltransferase-1 (DNMT1).
METHODS: Ovalbumin was used as the specific antigen to establish a mouse model of FA. Intestinal IL-35+ regulatory B cells (Breg cells) were isolated from FA mice, and characterized using immunological approaches.
RESULTS: FA mice had a lower frequency of IL-35+ Breg cells, which was inversely correlated with their FA response. The quantity of IL-35 was lower in intestinal Breg cells from FA mice. Hypermethylation status was detected in the Il35 promoter, which was accompanied with high levels of H3K9me3. Enforced expression of DNMT1 hindered the promoter activity of the IL35 gene. Administration of an inhibitor of DNMT1 (RG108) restored the immune regulatory capacity of FA intestinal Bregs, and effectively suppressed the expression of DNMT1, and attenuated experimental FA.
CONCLUSIONS: The elevated quantity of DNMT1 in intestinal Breg cells compromises the expression of IL-35 and affects the immune regulatory functions, which facilitates the development of FA. The immune regulatory functions of intestinal Breg cells are restored and experimental FA is attenuated by inhibiting DNMT1.

Cervical cancer poses a significant health burden for women globally, and the rapid proliferation of cervical cancer cells greatly worsens patient prognosis. Long non-coding RNAs (lncRNAs) play a crucial role in regulating tumor cell proliferation. However, the involvement of lncRNAs in cervical cancer cell proliferation remains unclear. In this study, we investigated the lncRNA SIX1-1, which was found to be upregulated in cervical cancer tissues and cell lines. Functional assays revealed that knockdown of SIX1-1 inhibited cell proliferation in vitro and reduced tumor growth in vivo. Mechanistically, SIX1-1 was predominantly localized in the nucleus and could bind with DNMT1 protein. The expression of SIX1-1 enhanced the interaction of DNMT1 with RASD1 promoter, leading to the methylation of the promoter and decreased mRNA transcription. Then RASD1 downregulation activated the cAMP/PKA/CREB signaling pathway, promoting cell proliferation. Rescue experiments showed that knockdown of RASD1 restored the inhibited cell proliferation caused by decreased expression of SIX1-1, indicating that RASD1 acted as the functional mediator of SIX1-1. In conclusion, SIX1-1 promoted cervical cancer cell proliferation by modulating RASD1 expression. This suggests that targeting the SIX1-1/RASD1 axis could be a potential antitumor strategy for cervical cancer.

The chemical synthesis of homogeneously ubiquitylated histones is a powerful approach to decipher histone ubiquitylation-dependent epigenetic regulation. Among the various methods, α-halogen ketone-mediated conjugation chemistry has recently been an attractive strategy to generate single-monoubiquitylated histones for biochemical and structural studies. Herein, we report the use of this strategy to prepare not only dual- and even triple-monoubiquitylated histones but also diubiquitin-modified histones. We were surprised to find that the synthetic efficiencies of multi-monoubiquitylated histones were comparable to those of single-monoubiquitylated ones, suggesting that this strategy is highly tolerant to the number of ubiquitin monomers installed onto histones. The facile generation of a series of single-, dual-, and triple-monoubiquitylated H3 proteins enabled us to evaluate the influence of ubiquitylation patterns on the binding of DNA methyltransferase 1 (DNMT1) to nucleosomes. Our study highlights the potential of site-specific conjugation chemistry to generate chemically defined histones for epigenetic studies.

BACKGROUND: Our previous research demonstrated that resveratrol counters DDP-induced ototoxicity by upregulating miR-455-5p, which targets PTEN. This study aimed to elucidate the underlying mechanisms involving GAS5 and DNA methyltransferase 1 (DNMT1) in resveratrol\'s protective action.
METHODS: A luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to study the binding between GAS5 and miR-455-5p, as well as between miR-455-5p and PTEN. HEI-OC1 cells treated with DDP were transfected with vectors for GAS5, si-GAS5, DNMT1, si-DNMT1, and miR-455-5p mimics, as well as PTEN. Subsequently, they were treated with resveratrol and exposed to DDP, both separately and in combination. The distribution of CpG islands in the GAS5 promoter was identified using MethyPrimer, and methylation-specific PCR (MSP) was conducted to determine the methylation levels of GAS5. Chromatin immunoprecipitation (ChIP) was utilized to examine the interaction between DNMT1 and GAS5. The viability of HEI-OC1 cells, catalase (CAT) activity, apoptosis, and ROS levels were assessed using the CCK-8 assay, CAT assay, TUNEL staining, and flow cytometry, respectively. An in vivo mouse model was developed to measure auditory brainstem response (ABR) thresholds, while RT-qPCR and Western blot analysis were employed to evaluate molecular levels.
RESULTS: Our study discovered that GAS5 acts as a sponge for miR-455-5p, thereby increasing PTEN expression in DDP-treated HEI-OC1 cells. This process was reversed upon treatment with resveratrol. Importantly, DNMT1 promoted the methylation of the GAS5 promoter, leading to the suppression of GAS5 expression. This suppression enhanced the effectiveness of resveratrol in combating DDP-induced apoptosis and ROS in HEI-OC1 cells and amplified its protective effect against DDP\'s ototoxicity in vivo.
CONCLUSIONS: Our research emphasizes the significance of the DNMT1/GAS5/miR-455-5p/PTEN axis as a promising new route to boost resveratrol\'s effectiveness against DDP-induced ototoxicity.

BACKGROUND: Papillary thyroid carcinoma (PTC) is a common endocrine malignancy. Studies have indicated that estrogen can regulate the expression of miRNAs in numerous malignancies. MiR-570-3p has been shown to have a regulatory function in various cancers. However, studies of the regulatory function of miR-570-3p and a direct link between estrogen (especially estradiol E2) and miR-570-3p in PTC have not been done.
METHODS: Expression of miR-570-3p and its downstream target DPP4 in PTC tissues and cells was predicted using bioinformatics and validated by qRT-PCR and western blot assays. We then performed a series of gain-and-loss experiments to assess the functional significance of miR-570-3p/DPP4 axis in PTC progression in vitro and in vivo. Additionally, the methylation of the miR-570-3p promoter region was examined via bioinformatics analysis and MSP. Finally, the effects of E2 on PTC progression and the correlation between DNMT1/DNMT3A and EZH2 were predicted by bioinformatic tools and proved by luciferase reporter, ChIP, and co-IP assays.
RESULTS: In PTC tumor tissues and cell lines, there was a lower expression level and a higher methylation level of miR-570-3p compared to normal tissues and cell lines. DPP4 was identified as the downstream target of miR-570-3p. Overexpression of miR-570-3p reduced the proliferative, migratory, and invasive capabilities, and promoted apoptosis, while overexpression of DPP4 reversed these effects in PTC cells. It was also discovered that DNMT1 and DNMT3A increased the CpG methylation level of the miR-570-3p promoter in an EZH2-dependent manner, which led to decreased expression of miR-570-3p. Furthermore, we observed that estrogen (E2) enhanced the methylation of miR-570-3p and suppressed its expression levels, resulting in augmented tumor growth in vivo in PTC.
CONCLUSIONS: Estrogen regulates the EZH2/DNMTs/miR-570-3p/DPP4 signaling pathway to promote PTC progression.