RNA Pol II Dynamics Modulate Co-transcriptional Chromatin Modification, CTD Phosphorylation, and Transcriptional Direction.

Eukaryotic genes are marked by conserved post-translational modifications on the RNA pol II C-terminal domain (CTD) and the chromatin template. How the 5'-3' profiles of these marks are established is poorly understood. Using pol II mutants in human cells, we found that slow transcription repositioned specific co-transcriptionally deposited chromatin modifications; histone H3 lysine 36 trimethyl (H3K36me3) shifted within genes toward 5' ends, and histone H3 lysine 4 dimethyl (H3K4me2) extended farther upstream of start sites. Slow transcription also evoked a hyperphosphorylation of CTD Ser2 residues at 5' ends of genes that is conserved in yeast. We propose a "dwell time in the target zone" model to explain the effects of transcriptional dynamics on the establishment of co-transcriptionally deposited protein modifications. Promoter-proximal Ser2 phosphorylation is associated with a longer pol II dwell time at start sites and reduced transcriptional polarity because of strongly enhanced divergent antisense transcription at promoters. These results demonstrate that pol II dynamics help govern the decision between sense and divergent antisense transcription.

RSI1/FLD and its epigenetic target RRTF1 are essential for the retention of infection memory in Arabidopsis thaliana.

Plants can retain a memory of previous pathogen infections to mount a more robust defense response during subsequent infections by developing systemic acquired resistance (SAR). However, the mechanism through which plants develop and retain infection memory is not known. Experiments have shown the association of epigenetic modifications of specific defense-related genes with SAR. RSI1/FLD codes for a histone demethylase and is required for the activation of SAR in Arabidopsis. Here we report the identification of RRTF1 as an epigenetic target of RSI1. RRTF1 expression is higher in pathogen-free distal tissues of the rsi1 mutant. Experiments with loss-of-function and overexpression lines suggest RRTF1 is a negative regulator of basal defense against virulent and avirulent pathogens as well as SAR. Enhanced expression of RRTF1 in a wild-type (WT) background specifically impairs SAR without impacting local resistance. RSI1 is recruited at the RRTF1 locus in a SAR-inducible manner and contributes to H3K4me2 and H3K4me3 demethylation. Introduction of the rrtf1 mutation rescues the loss-of-SAR phenotype of rsi1 plants. However, these plants fail to retain infection memory beyond 7 days post-primary inoculation, whereas WT plants retain memory for at least 11 days. Our results demonstrate that RSI1 and RRTF1 form a functional module for retaining infection memory in Arabidopsis.

lncCPSET1 acts as a scaffold for MLL2/COMPASS to regulate Bmp4 and promote the formation of chicken primordial germ cells.

Primordial germ cells (PGCs) are the ancestors of female and male germ cells. Recent studies have shown that long non-coding RNA (lncRNA) and histone methylation are key epigenetic factors affecting PGC formation; however, their joint regulatory mechanisms have rarely been studied. Here, we explored the mechanism by which lncCPSET1 and H3K4me2 synergistically regulate the formation of chicken PGCs for the first time. Combined with chromatin immunoprecipitation (CHIP) sequencing and RNA-seq of PGCs transfected with the lncCPSET1 overexpression vector, GO annotation and KEGG enrichment analysis revealed that Wnt and TGF-ß signaling pathways were significantly enriched, and Fzd2, Id1, Id4, and Bmp4 were identified as candidate genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that ASH2L, DPY30, WDR5, and RBBP5 overexpression significantly increased the expression of Bmp4, which was up-regulated after lncCPSET1 overexpression as well. It indicated that Bmp4 is a target gene co-regulated by lncCPSET1 and MLL2/COMPASS. Interestingly, co-immunoprecipitation results showed that ASH2L, DPY30 and WDR5 combined and RBBP5 weakly combined with DPY30 and WDR5. lncCPSET1 overexpression significantly increased Dpy30 expression and co-immunoprecipitation showed that interference/overexpression of lncCPSET1 did not affect the binding between the proteins in the complexes, but interference with lncCPSET1 inhibited DPY30 expression, which was confirmed by RNA immunoprecipitation that lncCPSET1 binds to DPY30. Additionally, CHIP-qPCR results showed that DPY30 enriched in the Bmp4 promoter region promoted its transcription, thus promoting the formation of PGCs. This study demonstrated that lncCPSET1 and H3K4me2 synergistically promote PGC formation, providing a reference for the study of the regulatory mechanisms between lncRNA and histone methylation, as well as a molecular basis for elucidating the formation mechanism of PGCs in chickens.

Inhibition of KDM5B participates in immune microenvironment remodeling in pancreatic cancer by inducing STING expression.

OBJECTIVE: This study aims to investigate the effect of lysine demethylase 5B (KDM5B)-mediated dimethyl-lysine 4 histone H3 (H3K4me2) demethylation on immune microenvironment remodeling in pancreatic cancer. METHODS: Pan 02 mouse pancreatic cancer cell lines were cultured and used to establish tumor model in vivo. RT-qPCR and Western blot were used to detect the expression of stimulator of interferon gene (STING) and KDM5B in pancreatic cancer tissues and Pan 02 cells. The specific demethylation domain of KDM5B was detected by isothermal titration calorimetry binding assay. The regulatory roles of KDM5B in cell apoptosis and remodeling of immune microenvironment in vitro and in vivo were explored after loss-of functions in KDM5B. RESULTS: KDM5B was highly expressed but STING was poorly expressed in pancreatic cancer tissues and Pan 02 cells. After knockdown of KDM5B, CD8+ T cells recognized and killed Pan 02 cells, which promoted the infiltration of CD8+ T cells in Pan 02 cells, thus improving the anti-tumor ability. The PHD domain in KDM5B specifically bound to H3K4me2 peptide and inhibition of KDM5B induced STING expression. Knockdown of KDM5B up-regulated STING expression to promote apoptosis, thus regulating the immune microenvironment and inhibiting the growth of tumor in mice. Meanwhile, knockdown of KDM5B and STING simultaneously counteracted the knockdown effect of KDM5B. CONCLUSION: Inhibition of KDM5B can promote the expression of STING through H3K4me2 demethylation, which promoted the recognition and killing of Pan 02 cells by CD8+ T cells, thus improving the anti-tumor ability and regulating the immune microenvironment.

Pygo2 activates BRPF1 via Pygo2-H3K4me2/3 interaction to maintain malignant progression in colon cancer.

Epigenetic alterations have essential roles during colon adenocarcinoma (COAD) progression. As the coactivator of Wnt/b-catenin signaling, Pygopus 2 (Pygo2) binds H3K4me2/3 and participate in chromatin remodeling in multiple cancers. However, It remains unclear whether the Pygo2-H3K4me2/3 association has significance in COAD. We aimed to elucidate the roles of Pygo2 in COAD. Functionally, Pygo2 inhibition attenuated cell proliferation, self-renewal capacities in vitro. Pygo2 overexpression enhanced in vivo tumor growth. Besides, Pygo2 overexpression could also enhance cell migration ability and in vivo distal metastasis. Mechanistically, Pygo2 correlates positively with BRPF1 expressions, one epigenetic reader of histone acetylation. The luciferase reporter assay and Chromatin Immunoprecipitation (ChIP)-qPCR assay were used to find that Pygo2 coordinated with H3K4me2/3 modifications to activate BRPF1 transcriptions via binding to the promoter. Both Pygo2 and BRPF1 expressed highly in tumors and Pygo2 relied on BRPF1 to accelerate COAD progression, including cell proliferation rate, migration abilities, stemness features and in vivo tumor growth. Targeting BPRF1 (GSK5959) is effective to suppress in vitro growth of Pygo2high cell lines, and has mild effect on Pygo2low cells. The subcutaneous tumor model further demonstrated that GSK5959 could effectively suppress the in vivo growth of Pygo2high COAD, but not the Pygo2low subtype. Collectively, our study represented Pygo2/BRPF1 as an epigenetic vulnerability for COAD treatment with predictive significance.

COMPASS core subunits MpSet1 and MpSwd3 regulate Monascus pigments synthesis in Monascus purpureus.

In eukaryotes, methylation of histone H3 at lysine 4 (H3K4me) catalyzed by the complex of proteins associated with Set1 (COMPASS) is crucial for the transcriptional regulation of genes and the development of organisms. In Monascus, the functions of COMPASS in establishing H3K4me remain unclear. This study first identified the conserved COMPASS core subunits MpSet1 and MpSwd3 in Monascus purpureus and confirmed their roles in establishing H3K4me2/3. Loss of MpSet1 and MpSwd3 resulted in slower growth and development and inhibited the formation of cleistothecia, ascospores, and conidia. The loss of these core subunits also decreased the production of extracellular and intracellular Monascus pigments (MPs) by 94.2%, 93.5%, 82.7%, and 82.5%, respectively. In addition, RNA high-throughput sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) showed that the loss of MpSet1 and MpSwd3 altered the expression of 2646 and 2659 genes, respectively, and repressed the transcription of MPs synthesis-related genes. In addition, the ΔMpset1 and ΔMpswd3 strains demonstrated increased sensitivity to cell wall stress with the downregulation of chitin synthase-coding genes. These results indicated that the COMPASS core subunits MpSet1 and MpSwd3 help establish H3K4me2/3 for growth and development, spore formation, and pigment synthesis in Monascus. These core subunits also assist in maintaining cell wall integrity.

BMP4 activates the Wnt-Lin28A-Blimp1-Wnt pathway to promote primordial germ cell formation via altering H3K4me2.

The unique developmental characteristics of chicken primordial germ cells (PGCs) enable them to be used in recovery of endangered bird species, gene editing and the generation of transgenic birds, but the limited number of PGCs greatly limits their application. Studies have shown that the formation of mammalian PGCs is induced by BMP4 signal, but the mechanism underlying chicken PGC formation has not been determined. Here, we confirmed that Wnt signaling activated via BMP4 activates transcription of Lin28A by inducing ß-catenin to compete with LSD1 for binding to TCF7L2, causing LSD1 to dissociate from the Lin28A promoter and enhancing H3K4me2 methylation in this region. Lin28A promotes PGC formation by inhibiting gga-let7a-3p maturation to initiate Blimp1 expression. Interestingly, expression of Blimp1 helped sustain Wnt5A expression by preventing LSD1 binding to the Wnt5A promoter. We thus elucidated a positive feedback pathway involving Wnt-Lin28A-Blimp1-Wnt that ensures PGC formation. In summary, our data provide new insight into the development of PGCs in chickens.

USP7 sustains an active epigenetic program via stabilizing MLL2 and WDR5 in diffuse large B-cell lymphoma.

Activated B-cell-like (ABC)-diffuse large B-cell lymphoma (ABC-DLBCL) is a common subtype of non-Hodgkin's lymphoma with poor prognosis. The survival of ABC-DLBCL relies on constitutive activation of BCR signaling, but the underlying molecular mechanism is not fully addressed. By mining The Cancer Genome Atlas database, we found that the expression of ubiquitin-specific protease 7 (USP7) is significantly elevated in three cancer types including DLBCL. Interestingly, unlike germinal center B-cell-like (GCB)-DLBCL, ABC-DLBCL shows upregulated expression of USP7. Inhibiting the enzymatic activity of USP7 (P22077) has a drastic effect on ABC-DLBCL, but not GCB-DLBCL cells. Compared to GCB-DLBCL, ABC-DLBCL cells show transcriptional upregulation of multiple components of BCR-signaling. USP7 inhibition significantly reduces the expression of upregulated components of BCR signaling. Mechanistically, USP7 inhibition greatly reduces the methylation of histone 3 on lysine 4 (H3K4me2), which is an epigenetic marker for active enhancers. USP7 inhibition greatly reduces the protein level of WDR5 and MLL2, key components of lysine-specific methyltransferase complex (complex of proteins associated with Set1 [COMPASS]). In ABC-DLBCL cells, USP7 stabilizes WDR5 and MLL2. In patients, the expression of USP7 is significantly associated with components of BCR signaling (LYN, SYK, BTK, PLCG2, PRKCB, MALT1, BCL10, and CARD11) and targets of BCR signaling (MYC and IRF4). In summary, we demonstrated an essential role of USP7 in ABC-DLBCL by organizing an oncogenic epigenetic program via stabilization of WDR5 and MLL2. Targeting USP7 might be a novel and efficient approach to treat patients with ABC-DLBCL and it might be better than targeting individual components such as BTK in BCR signaling.

Comprehensive Flow-Cytometric Quality Assessment of Ram Sperm Intended for Gene Banking Using Standard and Novel Fertility Biomarkers.

Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H2DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa.

Narrow H3K4me2 is required for chicken PGC formation.

The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC-related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor-ß). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.

Global epigenetic changes of histone modification under environmental stresses in rice root.

Abiotic stresses are non-living factors with negative morphological and physiological effects on living organisms. Substantial evidence exists that gene expression changes during plant cell growth are regulated by chromatin reconfiguration and histone modification. Several types of histone modifications are dramatically transformed in stress-responsive gene regions under drought stress conditions. Environmental stresses also cause the root apical meristem (RAM) region to decelerate root growth. In this study, we investigated how quantitative changes in epigenetic markers in this region influence rice morphology and physiology. Both iron and salinity treatments changed the epigenetic landscape from euchromatic to heterochromatic according to heterochromatin (H3K9me2) and euchromatin (H3K4me) markers, especially in the proximal meristem region. Moreover, supplementation with external abscisic acid (ABA) was able to mimic the effect of environmental stresses on global epigenetic changes. In contrast, the addition of external auxin (IAA) to rice under saline conditions affected heterochromatin formation without influencing euchromatin transformation. Chromatin dynamics is therefore believed to be directly connected to plant growth regulator signaling. We discuss insights into the role of plant growth regulators: ABA and IAA, peroxide signaling, and their effects on the global epigenetic change of histone modification under abiotic stresses.

Knockdown of KDM1A suppresses tumour migration and invasion by epigenetically regulating the TIMP1/MMP9 pathway in papillary thyroid cancer.

Epigenetic dysregulation plays an important role in cancer. Histone demethylation is a well-known mechanism of epigenetic regulation that promotes or inhibits tumourigenesis in various malignant tumours. However, the pathogenic role of histone demethylation modifiers in papillary thyroid cancer (PTC), which has a high incidence of early lymphatic metastasis, is largely unknown. Here, we detected the expression of common histone demethylation modifiers and found that the histone H3 lysine 4 (H3K4) and H3 lysine 9 (H3K9) demethylase KDM1A (or lysine demethylase 1A) is frequently overexpressed in PTC tissues and cell lines. High KDM1A expression correlated positively with age <55 years and lymph node metastasis in patients with PTC. Moreover, KDM1A was required for PTC cell migration and invasion. KDM1A knockdown inhibited the migration and invasive abilities of PTC cells both in vitro and in vivo. We also identified tissue inhibitor of metalloproteinase 1 (TIMP1) as a key KDM1A target gene. KDM1A activated matrix metalloproteinase 9 (MMP9) through epigenetic repression of TIMP1 expression by demethylating H3K4me2 at the TIMP1 promoter region. Rescue experiments clarified these findings. Altogether, we have uncovered a new mechanism of KDM1A repression of TIMP1 in PTC and suggest that KDM1A may be a promising therapeutic target in PTC.

LincRNAFEZF1-AS1 represses p21 expression to promote gastric cancer proliferation through LSD1-Mediated H3K4me2 demethylation.

BACKGROUND: Although the prognosis of gastric cancer patients have a favorable progression, there are some patients with unusual patterns of locoregional and systemic recurrence. Therefore, a better understanding of early molecular events of the disease is needed. Current evidences demonstrate that long noncoding RNAs (lncRNAs) may be an important class of functional regulators involved in human gastric cancers development. Our previous studies suggest that HOTAIR contributes to gastric cancer development, and the overexpression of HOTAIR predicts a poor prognosis. In this study, we investigated the characteristic of the LncRNA FEZF1-AS1 in gastric cancer. METHODS: QRT-PCR was used to detect the expression of FEZF1-AS1 in gastric cancer tissues and cells. MTT assays, clonogenic survival assays and nude mouse xenograft model were used to examine the tumorigenesis function of FEZF1-AS1 in vitro and in vivo. Bioinformatics analysis were used to select downstream target genes of FEZF1-AS1. Cell cycle analysis, ChIP, RIP,RNA Pulldown assays were examined to dissect molecular mechanisms. RESULTS: In this study, we reported that FEZF1-AS1, a 2564 bp RNA, was overexpressed in gastric cancer, and upregulated FEZF1-AS1 expression indicated larger tumor size and higher clinical stage; additional higher expression of FEZF1-AS1 predicted poor prognosis. Further experiments revealed that knockdown FEZF1-AS1 significantly inhibited gastric cancer cells proliferation by inducing G1 arrest and apoptosis, whereas endogenous expression FEZF1-AS1 promoted cell growth. Additionally, RIP assay and RNA-pulldown assay evidenced that FEZF1-AS1 could epigenetically repress the expression of P21 via binding with LSD1, the first discovered demethylase. ChIP assays demonstrated that LSD1 could directly bind to the promoter of P21, inducing H3K4me2 demethylation. CONCLUSION: In summary, these data demonstrated that FEZF1-AS1 could act as an "oncogene" for gastric cancer partly through suppressing P21 expression; FEZF1-AS1 may be served as a candidate prognostic biomarker and target for new therapies of gastric cancer patients.

Distinct patterns of the histone marks associated with recruitment of the methionine chain-elongation pathway from leucine biosynthesis.

Aliphatic glucosinolates (GLSs) are derived from chain-elongated methionine produced by an iterative three-step process, known to be evolutionarily recruited from leucine biosynthesis. The divergence of homologous genes between two pathways is mainly linked to the alterations in biochemical features. In this study, it was discovered that a distinct pattern of histone modifications is associated with and/or contributes to the divergence of the two pathways. In general, genes involved in leucine biosynthesis were robustly associated with H3k4me2 and H3K4me3. In contrast, despite the considerable abundances of H3K4me2 observed in some of genes involved in methionine chain elongation, H3K4me3 was completely missing. This H3K4m3-depleted pattern had no effect on gene transcription, whereas it seemingly co-evolved with the entire pathway of aliphatic GLS biosynthesis. The results reveal a novel association of the epigenetic marks with plant secondary metabolism, and may help to understand the recruitment of the methionine chain-elongation pathway from leucine biosynthesis.

A microdeletion encompassing PHF21A in an individual with global developmental delay and craniofacial anomalies.

In Potocki-Shaffer syndrome (PSS), the full phenotypic spectrum is manifested when deletions are at least 2.1 Mb in size at 11p11.2. The PSS-associated genes EXT2 and ALX4, together with PHF21A, all map to this region flanked by markers D11S1393 and D11S1319. Being proximal to EXT2 and ALX4, a 1.1 Mb region containing 12 annotated genes had been identified by deletion mapping to explain PSS phenotypes except multiple exostoses and parietal foramina. Here, we report a male patient with partial PSS phenotypes including global developmental delay, craniofacial anomalies, minor limb anomalies, and micropenis. Using microarray, qPCR, RT-qPCR, and Western blot analyses, we refined the candidate gene region, which harbors five genes, by excluding two genes, SLC35C1 and CRY2, which resulted in a corroborating role of PHF21A in developmental delay and craniofacial anomalies. This microdeletion contains the least number of genes at 11p11.2 reported to date. Additionally, we also discuss the phenotypes observed in our patient with respect to those of published cases of microdeletions across the Potocki-Shaffer interval.

H3K4me2 reliably defines transcription factor binding regions in different cells.

Histone modification (HM) patterns are widely applied to identify transcription factor binding regions (TFBRs). However, how frequently the TFBRs overlap with genomic regions enriched with certain types of HMs and which HM marker is more effective to pinpoint the TFBRs have not been systematically investigated. To address these problems, we studied 149 transcription factor (TF) ChIP-seq datasets and 33 HM ChIP-seq datasets in three cell lines. We found that on average about 90% of the TFBRs overlap with the H3K4me2-enriched regions. Moreover, the H3K4me2-enriched regions with stronger signals of H3K4me2 enrichment more likely overlap with the TFBRs than those with weaker signals. In addition, we showed that the H3K4me2-enriched regions together with the H3K27ac-enriched regions can greatly reduce false positive predictions of the TFBRs. Our study sheds light on the comprehensive discovery of the TFBRs using the HeK4me-enriched regions, especially when no good antibody to a TF exists.

LSD1 promotes the FSH responsive follicle formation by regulating autophagy and repressing Wt1 in the granulosa cells.

In a growing follicle, the survival and maturation of the oocyte largely depend on support from somatic cells to facilitate FSH-induced mutual signaling and chemical communication. Although apoptosis and autophagy in somatic cells are involved in the process of FSH-induced follicular development, the underlying mechanisms require substantial study. According to our study, along with FSH-induced antral follicles (AFs) formation, both lysine-specific demethylase 1 (LSD1) protein levels and autophagy increased simultaneously in granulosa cells (GCs) in a time-dependent manner, we therefore evaluated the importance of LSD1 upon facilitating the formation of AFs correlated to autophagy in GCs. Conditional knockout of Lsd1 in GCs resulted in significantly decreased AF number and subfertility in females, accompanied by marked suppression of the autophagy in GCs. On the one hand, depletion of Lsd1 resulted in accumulation of Wilms tumor 1 homolog (WT1), at both the protein and mRNA levels. WT1 prevented the expression of FSH receptor (Fshr) in GCs and thus reduced the responsiveness of the secondary follicles to FSH induction. On the other hand, depletion of LSD1 resulted in suppressed level of autophagy by upregulation of ATG16L2 in GCs. We finally approved that LSD1 contributed to these sequential activities in GCs through its H3K4me2 demethylase activity. Therefore, the importance of LSD1 in GCs is attributable to its roles in both accelerating autophagy and suppressing WT1 expression to ensure the responsiveness of GCs to FSH during AFs formation.

H3K4me2 cooperates with Wnt/TCF7L2 to regulate TDRD1 and promote chicken spermatogonia stem cell formation.

Spermatogonia Stem Cells (SSCs) are the basis of spermatogenesis. In the poultry industry, asthenospermia and azoospermia in roosters seriously reduce economic benefits. In this study, we explored SSCs formation mechanisms in detail. TDRD1, which is a downstream target gene of TCF7L2 and is modified by histone methylation, was screened through multiomics analysis. Functionally, RT-qPCR, flow cytometry, immunohistochemistry, and indirect immunofluorescence results showed that H3K4me2 regulated TDRD1 to promote SSCs formation both in vivo and in vitro. Furthermore, ChIP-qPCR and dual luciferase assays showed that H3K4me2 was enriched in the -800 to 0 bp region of the TDRD1 promoter and positively regulated TDRD1 transcription to promote SSCs formation. Interestingly, in mechanistic terms, dual luciferase assays showed that TDRD1 transcription levels were significantly decreased after co-transfection with dCas9-LSD1-P1/P2/P3 and OETCF7L2, while TDRD1 transcript levels were not significantly altered after transfecting dCas9-LSD1-P4 and OETCF7L2. These results suggested that H3K4me2 enrichment in P1, P2, and P3 of the TDRD1 promoter promotes TDRD1 transcription by reducing enrichment of TCF7L2. This study explored the specific regulatory mechanisms involving the Wnt signaling pathway, H3K4me2, and TDRD1, enriched the regulatory network regulating the formation of SSCs, and laid a theoretical foundation for the specific application of SSCs.

Chromatin Immunostaining of Plant Nuclei.

Recent evidence has demonstrated that specific epigenetic changes are also related to plant growth and development. Immunostaining enables the detection and characterization of chromatin modification, e.g., histone H4 acetylation (H4K5ac), histone H3 methylation (H3K4me2 and H3K9me2), and DNA methylation (5mC) with unique and specific patterns in plant tissues. Here we describe experimental procedures to determine the histone H3 methylation (H3K4me2 and H3K9me2) patterns in 3D-chromatin in whole roots tissue and 2D-chromatin in single nuclei in rice. To analyze both iron and salinity treatments, we show how to test for changes to the epigenetic chromatin landscape using heterochromatin (H3K9me2) and euchromatin (H3K4me) markers for chromatin immunostaining, especially in the proximal meristem region. To elucidate the epigenetic impact of environmental stress and external plant growth regulators, we demonstrate how to apply a combination of salinity, auxin, and abscisic acid treatments. The results of these experiments provide insights into the epigenetic landscape during rice root growth and development.

Structural insight into ASH1L PHD finger recognizing methylated histone H3K4 and promoting cell growth in prostate cancer.

ASH1L is a member of the Trithorax-group protein and acts as a histone methyltransferase for gene transcription activation. It is known that ASH1L modulates H3K4me3 and H3K36me2/3 at its gene targets, but its specific mechanism of histone recognition is insufficiently understood. In this study, we found that the ASH1L plant homeodomain (PHD) finger interacts with mono-, di-, and trimethylated states of H3K4 peptides with comparable affinities, indicating that ASH1L PHD non-selectively binds to all three methylation states of H3K4. We solved nuclear magnetic resonance structures picturing the ASH1L PHD finger binding to the dimethylated H3K4 peptide and found that a narrow binding groove and residue composition in the methylated-lysine binding pocket restricts the necessary interaction with the dimethyl-ammonium moiety of K4. In addition, we found that the ASH1L protein is overexpressed in castrate-resistant prostate cancer (PCa) PC3 and DU145 cells in comparison to PCa LNCaP cells. The knockdown of ASH1L modulated gene expression and cellular pathways involved in apoptosis and cell cycle regulation and consequently induced cell cycle arrest, cell apoptosis, and reduced colony-forming abilities in PC3 and DU145 cells. The overexpression of the C-terminal core of ASH1L but not the PHD deletion mutant increased the overall H3K36me2 level but had no effect on the H3K4me2/3 level. Overall, our study identifies the ASH1L PHD finger as the first native reader that non-selectively recognizes the three methylation states of H3K4. Additionally, ASH1L is required for the deregulation of cell cycle and survival in PCas.