Histone H3 lysine 27 crotonylation mediates gene transcriptional repression in chromatin.

Histone lysine acylation, including acetylation and crotonylation, plays a pivotal role in gene transcription in health and diseases. However, our understanding of histone lysine acylation has been limited to gene transcriptional activation. Here, we report that histone H3 lysine 27 crotonylation (H3K27cr) directs gene transcriptional repression rather than activation. Specifically, H3K27cr in chromatin is selectively recognized by the YEATS domain of GAS41 in complex with SIN3A-HDAC1 co-repressors. Proto-oncogenic transcription factor MYC recruits GAS41/SIN3A-HDAC1 complex to repress genes in chromatin, including cell-cycle inhibitor p21. GAS41 knockout or H3K27cr-binding depletion results in p21 de-repression, cell-cycle arrest, and tumor growth inhibition in mice, explaining a causal relationship between GAS41 and MYC gene amplification and p21 downregulation in colorectal cancer. Our study suggests that H3K27 crotonylation signifies a previously unrecognized, distinct chromatin state for gene transcriptional repression in contrast to H3K27 trimethylation for transcriptional silencing and H3K27 acetylation for transcriptional activation.

BRD4-PRC2 represses transcription of T-helper 2-specific negative regulators during T-cell differentiation.

BRD4 is a well-recognized transcriptional activator, but how it regulates gene transcriptional repression in a cell type-specific manner has remained elusive. In this study, we report that BRD4 works with Polycomb repressive complex 2 (PRC2) to repress transcriptional expression of the T-helper 2 (Th2)-negative regulators Foxp3 and E3-ubiqutin ligase Fbxw7 during lineage-specific differentiation of Th2 cells from mouse primary naïve CD4+ T cells. Brd4 binds to the lysine-acetylated-EED subunit of the PRC2 complex via its second bromodomain (BD2) to facilitate histone H3 lysine 27 trimethylation (H3K27me3) at target gene loci and thereby transcriptional repression. We found that Foxp3 represses transcription of Th2-specific transcription factor Gata3, while Fbxw7 promotes its ubiquitination-directed protein degradation. BRD4-mediated repression of Foxp3 and Fbxw7 in turn promotes BRD4- and Gata3-mediated transcriptional activation of Th2 cytokines including Il4, Il5, and Il13. Chemical inhibition of the BRD4 BD2 induces transcriptional de-repression of Foxp3 and Fbxw7, and thus transcriptional downregulation of Il4, Il5, and Il13, resulting in inhibition of Th2 cell lineage differentiation. Our study presents a previously unappreciated mechanism of BRD4's role in orchestrating a Th2-specific transcriptional program that coordinates gene repression and activation, and safeguards cell lineage differentiation.

Class IIa HDAC4 and HDAC7 cooperatively regulate gene transcription in Th17 cell differentiation.

Class II histone deacetylases (HDACs) are important in regulation of gene transcription during T cell development. However, our understanding of their cell-specific functions is limited. In this study, we reveal that class IIa Hdac4 and Hdac7 (Hdac4/7) are selectively induced in transcription, guiding the lineage-specific differentiation of mouse T-helper 17 (Th17) cells from naive CD4+ T cells. Importantly, Hdac4/7 are functionally dispensable in other Th subtypes. Mechanistically, Hdac4 interacts with the transcription factor (TF) JunB, facilitating the transcriptional activation of Th17 signature genes such as Il17a/f. Conversely, Hdac7 collaborates with the TF Aiolos and Smrt/Ncor1-Hdac3 corepressors to repress transcription of Th17 negative regulators, including Il2, in Th17 cell differentiation. Inhibiting Hdac4/7 through pharmacological or genetic methods effectively mitigates Th17 cell-mediated intestinal inflammation in a colitis mouse model. Our study uncovers molecular mechanisms where HDAC4 and HDAC7 function distinctively yet cooperatively in regulating ordered gene transcription during Th17 cell differentiation. These findings suggest a potential therapeutic strategy of targeting HDAC4/7 for treating Th17-related inflammatory diseases, such as ulcerative colitis.

The Short Isoform of BRD4 Promotes HIV-1 Latency by Engaging Repressive SWI/SNF Chromatin-Remodeling Complexes.

BET proteins commonly activate cellular gene expression, yet inhibiting their recruitment paradoxically reactivates latent HIV-1 transcription. Here we identify the short isoform of BET family member BRD4 (BRD4S) as a corepressor of HIV-1 transcription. We found that BRD4S was enriched in chromatin fractions of latently infected T cells, and it was more rapidly displaced from chromatin upon BET inhibition than the long isoform. BET inhibition induced marked nucleosome remodeling at the latent HIV-1 promoter, which was dependent on the activity of BRG1-associated factors (BAF), an SWI/SNF chromatin-remodeling complex with known repressive functions in HIV-1 transcription. BRD4S directly bound BRG1, a catalytic subunit of BAF, via its bromodomain and extraterminal (ET) domain, and this isoform was necessary for BRG1 recruitment to latent HIV-1 chromatin. Using chromatin immunoprecipitation sequencing (ChIP-seq) combined with assay for transposase-accessible chromatin coupled to high-throughput sequencing (ATAC-seq) data, we found that the latent HIV-1 promoter phenotypically resembles endogenous long terminal repeat (LTR) sequences, pointing to a select role of BRD4S-BRG1 complexes in genomic silencing of invasive retroelements.

Distinct Roles of Brd2 and Brd4 in Potentiating the Transcriptional Program for Th17 Cell Differentiation.

The BET proteins are major transcriptional regulators and have emerged as new drug targets, but their functional distinction has remained elusive. In this study, we report that the BET family members Brd2 and Brd4 exert distinct genomic functions at genes whose transcription they co-regulate during mouse T helper 17 (Th17) cell differentiation. Brd2 is associated with the chromatin insulator CTCF and the cohesin complex to support cis-regulatory enhancer assembly for gene transcriptional activation. In this context, Brd2 binds the transcription factor Stat3 in an acetylation-sensitive manner and facilitates Stat3 recruitment to active enhancers occupied with transcription factors Irf4 and Batf. In parallel, Brd4 temporally controls RNA polymerase II (Pol II) processivity during transcription elongation through cyclin T1 and Cdk9 recruitment and Pol II Ser2 phosphorylation. Collectively, our study uncovers both separate and interdependent Brd2 and Brd4 functions in potentiating the genetic program required for Th17 cell development and adaptive immunity.

HIPK2 directs cell type-specific regulation of STAT3 transcriptional activity in Th17 cell differentiation.

SignificanceSTAT3 (signal transducer and activator of transcription 3) is a master transcription factor that organizes cellular responses to cytokines and growth factors and is implicated in inflammatory disorders. STAT3 is a well-recognized therapeutic target for human cancer and inflammatory disorders, but how its function is regulated in a cell type-specific manner has been a major outstanding question. We discovered that Stat3 imposes self-directed regulation through controlling transcription of its own regulator homeodomain-interacting protein kinase 2 (Hipk2) in a T helper 17 (Th17) cell-specific manner. Our validation of the functional importance of the Stat3-Hipk2 axis in Th17 cell development in the pathogenesis of T cell-induced colitis in mice suggests an approach to therapeutically treat inflammatory bowel diseases that currently lack a safe and effective therapy.

Artificial intelligence and machine learning-aided drug discovery in central nervous system diseases: State-of-the-arts and future directions.

Neurological disorders significantly outnumber diseases in other therapeutic areas. However, developing drugs for central nervous system (CNS) disorders remains the most challenging area in drug discovery, accompanied with the long timelines and high attrition rates. With the rapid growth of biomedical data enabled by advanced experimental technologies, artificial intelligence (AI) and machine learning (ML) have emerged as an indispensable tool to draw meaningful insights and improve decision making in drug discovery. Thanks to the advancements in AI and ML algorithms, now the AI/ML-driven solutions have an unprecedented potential to accelerate the process of CNS drug discovery with better success rate. In this review, we comprehensively summarize AI/ML-powered pharmaceutical discovery efforts and their implementations in the CNS area. After introducing the AI/ML models as well as the conceptualization and data preparation, we outline the applications of AI/ML technologies to several key procedures in drug discovery, including target identification, compound screening, hit/lead generation and optimization, drug response and synergy prediction, de novo drug design, and drug repurposing. We review the current state-of-the-art of AI/ML-guided CNS drug discovery, focusing on blood-brain barrier permeability prediction and implementation into therapeutic discovery for neurological diseases. Finally, we discuss the major challenges and limitations of current approaches and possible future directions that may provide resolutions to these difficulties.

Viral metagenomics reveals two novel anelloviruses in feces of experimental rats.

BACKGROUND: Rodents are widely distributed and are the natural reservoirs of a diverse group of zoonotic viruses. Thus, analyzing the viral diversity harbored by rodents could assist efforts to predict and reduce the risk of future emergence of zoonotic viral diseases. Rodents are commonly used in animal testing, particularly mice and rats. Experimental rats are important animal models, and a history of pathogenic infections in these animals will directly affect the animal trial results. The pathogenicity of Anellovirus (AV) remains poorly understood due to the lack of a suitable model cell line or animal to support the viral cycle. This study aimed to discover possible anelloviruses from the virome in feces of experimental rats by viral metagenomic technique. METHODS: Fecal samples were collected from 10 commercial SD rats and pooled into a sample pool and then subjected to libraries construction which was then sequenced on Illumina MiSeq platform. The sequenced reads were analyzed using viral metagenomic analysis pipeline and two novel anelloviruses (AVs) were identified from fecal sample of experimental rats. The prevalence of these two viruses was investigated by conventional PCR. RESULTS: The complete genomic sequence of these two AVs were determined and fully characterized, with strain name ratane153-zj1 and ratane153-zj2. The circular genomes of ratane153-zj1 and ratane153-zj2 are 2785 nt and 1930 nt in length, respectively, and both include three ORFs. Ratane153-zj1 closely clustered with members within the genus Wawtorquevirus and formed a separate branch based on the phylogenetic tree constructed over the amino acid sequence of ORF1 of the two AVs identified in this study and other related AVs. While the complete amino acid sequences of ORF1 of ratane153-zj2 (nt 335 to 1390) had the highest sequence identity with an unclassified AV (GenBank No. ATY37438) from Chinchilla lanigera, and they clustered with one AV (GenBank No. QYD02305) belonging to the genus Etatorquevirus from Lynx rufus. Conventional PCR with two sets of specific primers designed based on the two genomes, respectively, showed that they were detectable at a low frequency in cohorts of experimental rats. CONCLUSION: Our study expanded the genome diversity of AVs and provided genetic background information of viruses existed in experimental rats.

Spatially constrained tandem bromodomain inhibition bolsters sustained repression of BRD4 transcriptional activity for TNBC cell growth.

The importance of BET protein BRD4 in gene transcription is well recognized through the study of chemical modulation of its characteristic tandem bromodomain (BrD) binding to lysine-acetylated histones and transcription factors. However, while monovalent inhibition of BRD4 by BET BrD inhibitors such as JQ1 blocks growth of hematopoietic cancers, it is much less effective generally in solid tumors. Here, we report a thienodiazepine-based bivalent BrD inhibitor, MS645, that affords spatially constrained tandem BrD inhibition and consequently sustained repression of BRD4 transcriptional activity in blocking proliferation of solid-tumor cells including a panel of triple-negative breast cancer (TNBC) cells. MS645 blocks BRD4 binding to transcription enhancer/mediator proteins MED1 and YY1 with potency superior to monovalent BET inhibitors, resulting in down-regulation of proinflammatory cytokines and genes for cell-cycle control and DNA damage repair that are largely unaffected by monovalent BrD inhibition. Our study suggests a therapeutic strategy to maximally control BRD4 activity for rapid growth of solid-tumor TNBC cells.

Generation of tryptamine derivatives through biotransformation by Diaporthe sp.

Three new tryptamine derivatives diaporols T-V (1-3) were isolated by adding tryptamine into the culture of Diaporthe sp., a fungus obtained from the leaves of Rhizophora stylosa. The structures of these compounds were elucidated by NMR spectroscopy and high resolution mass spectroscopic data. Among them, compound 1 showed moderate cytotoxic activity against SW480 cancer cell with IC50 9.84 µM.

[Clinical application of the modified pediatric nutritional risk screening tool].

OBJECTIVE: To study the clinical application of the modified nutritional risk screening tool and nutrition assessment in pediatric patients in China, and to provide a theoretical basis for establishing a standardized nutritional management process for pediatric patients. METHODS: A retrospective analysis was performed for the nutritional risk screening and nutrition assessment data of 16 249 hospitalized children. According to the degree of nutritional risk, the children were divided into a high nutritional risk group with 588 children, a moderate nutritional risk group with 4 330 children, and a non-nutritional risk group with 11 331 children. Nutrition assessment results were compared between groups. The composition of nutritional risk screening scores and the impact of nutritional risk screening on the rate of nutrition support therapy were analyzed. RESULTS: The incidence rate of nutritional risk was 30.27% (4 918/16 249), and the incidence rates of malnutrition and overnutrition were 27.37% (4 448/16 249) and 11.29% (1 834/16 249), respectively. Nutrition assessment results were significantly correlated with nutritional risk (≥ 5 years old:rs=0.313, P < 0.05; < 5 years old:rs=-0.304, P < 0.05). There was a significant difference in the composition of scoring items between the groups with different nutritional risks (P < 0.05). With the implementation of nutritional risk screening, there was a gradual increase in the rate of nutrition support therapy year by year (P < 0.05). CONCLUSIONS: There is a high incidence rate of nutritional risk in hospitalized children. The use of the modified pediatric nutritional risk screening tool can promote the implementation of standardized nutritional management.

In silico design and molecular basis for the selectivity of Olinone toward the first over the second bromodomain of BRD4.

Bromodomains (BrDs), a conserved structural module in chromatin-associated proteins, are well known for recognizing ε-N-acetyl lysine residues on histones. One of the most relevant BrDs is BRD4, a tandem BrD containing protein (BrD1 and BrD2) that plays a critical role in numerous diseases including cancer. Growing evidence shows that the two BrDs of BRD4 have different biological functions; hence selective ligands that can be used to study their functions are of great interest. Here, as a follow-up of our previous work, we first provide a detailed characterization study of the in silico rational design of Olinone as part of a series of five tetrahydropyrido indole-based compounds as BRD4 BrD1 inhibitors. Additionally, we investigated the molecular basis for Olinone's selective recognition by BrD1 over BrD2. Molecular dynamics simulations, free energy calculations, and conformational analyses of the apo-BRD4-BrD1|2 and BRD4-BrD1|2/Olinone complexes showed that Olinone's selectivity is facilitated by five key residues: Leu92 in BrD1|385 in BrD2 of ZA loop, Asn140|433, Asp144|His437 and Asp145|Glu438 of BC loop, and Ile146|Val49 of helix C. Furthermore, the difference in hydrogen bonds number and in mobility of the ZA and BC loops of the acetyl-lysine binding site between BRD4 BrD1/Olinone and BrD2/Olinone complexes also contribute to the difference in Olinone's binding affinity and selectivity toward BrD1 over BrD2. Altogether, our computer-aided molecular design techniques can effectively guide the development of small-molecule BRD4 BrD1 inhibitors, explain their selectivity origin, and further open doors to the design of new therapeutically improved derivatives.

Lacrimal sac bacteriology and susceptibility pattern in infants with congenital nasolacrimal duct obstruction in the 1st year of life: a cross-sectional study.

BACKGROUND: Congenital nasolacrimal duct obstruction (CNLDO) is one of the main causes of epiphora in infants, and antibiotics are usually used as a conservative therapy in the first year. Yet, little is known about the bacteriology of the occluded lacrimal drainage system in this group of patients. The aim of this study was to evaluate the microbiology of lacrimal sac (LS) in Chinese children with CNLDO in their first year of life. METHODS: Patients with CNLDO between May 1, 2017 and August 31, 2018 at a tertiary care children's hospital were enrolled. The study recruited infants who received lacrimal probing under 1 year old, and refluxed discharge from LS was collected. Samples were cultured and susceptibility test was performed for positive culture. RESULTS: Thirty-two patients with CNLDO were included. The ratio of male to female was 23:9. The mean age was 6.7 ± 2.4 (1.7-12) months. Positive cultures was identified in 87.5% of the sample, and presented 38 strains of bacteria. Mixed infection was identified in 10 (31.3%) children. Gram-positive bacteria accounted for 60.5% of all the strains, with Streptococcus (50%) being the most frequent species, whereas Haemophilus (21.1%) and Neisseriae (13.2%) were most common isolates for Gram-negative organisms. Methicillin-resistant Staphylococcus aureus (MRSA) was detected in 2 infants whose symptoms resolved by a routine probing. No difference of bacteriology pattern was detected between patients under 6 months old and those beyond. The pathogens were highly sensitive to chloramphenicol (88%) and levofloxacin (84%), but resistant to erythromycin (40%) and sulfamethoxazole (32%). CONCLUSIONS: Infants with CNLDO under 1 year of age presented predominance of Streptococcus as Gram-positive organism, and Haemophilus as Gram-negative organism. Levofloxacin was an active topical antibiotic agent with few chance of resistance especially for Chinese children. These findings could help clinicians choose optimal medicine for CNLDO as the conservative treatments.

BET N-terminal bromodomain inhibition selectively blocks Th17 cell differentiation and ameliorates colitis in mice.

T-helper 17 (Th17) cells have important functions in adaptor immunity and have also been implicated in inflammatory disorders. The bromodomain and extraterminal domain (BET) family proteins regulate gene transcription during lineage-specific differentiation of naïve CD4+ T cells to produce mature T-helper cells. Inhibition of acetyl-lysine binding of the BET proteins by pan-BET bromodomain (BrD) inhibitors, such as JQ1, broadly affects differentiation of Th17, Th1, and Th2 cells that have distinct immune functions, thus limiting their therapeutic potential. Whether these BET proteins represent viable new epigenetic drug targets for inflammatory disorders has remained an unanswered question. In this study, we report that selective inhibition of the first bromodomain of BET proteins with our newly designed small molecule MS402 inhibits primarily Th17 cell differentiation with a little or almost no effect on Th1 or Th2 and Treg cells. MS402 preferentially renders Brd4 binding to Th17 signature gene loci over those of housekeeping genes and reduces Brd4 recruitment of p-TEFb to phosphorylate and activate RNA polymerase II for transcription elongation. We further show that MS402 prevents and ameliorates T-cell transfer-induced colitis in mice by blocking Th17 cell overdevelopment. Thus, selective pharmacological modulation of individual bromodomains likely represents a strategy for treatment of inflammatory bowel diseases.

[Expression analysis of tPA/gGH double genes in transfected goat mammary epithelial cells].

tPA is a thrombolytic agent widely used in clinical settings. While double gene co-integration into organisms can produce synergistic effects and improved expression levels of the target gene, there are few reports detailing the co-integration of the tPA and gGH genes and an increased expression level of tPA. In order to study this, we obtained monoclonal goat mammary epithelial cell lines with tPA/gGH double gene integration and we analyzed the tPA expression level of single and double gene integration cells. We constructed a mammary gland-specific expression vector PCL25/gGH by using the ß-casein gene as the regulatory sequence. The tPA and gGH genes were co-transfected into goat mammary epithelial cells by electrotransfection. Resistant cell lines were screened by G418, and transgenic monoclonal cell lines were obtained by PCR detection. tPA expression was induced by prolactin and subsequently, the cell induction solution was assayed after 48 hours by ELISA and Western blotting. The results show that a total of 142 resistant monoclonal cells were obtained including 53 tPA monogenic integration cell lines and 34 tPA/gGH double gene integration cell lines. The rate of double gene integration was 23.9% (34/142). A total of 29 cells were detected to be able to express tPA, of which 12 were single-gene-expressing cells and the corresponding expression rate was 22.6% (12/53). There were 17 double-gene- expressing cells with a corresponding expression rate of 50.0% (17/34). The expression level of tPA in single-gene cells was 7.5-52.0 µg/mL, while in double-gene cells was 40-360 µg/mL, which was significantly greater than that in single- gene cells. The goat mammary epithelial cell lines with tPA/gGH gene integration were successfully obtained by electrotransfection, and we proved that the expression level of tPA in the double gene integration cell lines with tPA/gGH gene integration was significantly increased. Our findings lay the foundation for the additional study of highly expressed transgenic goats and other animals with determination of scientific and clinical utility.

BET inhibitors reduce cell size and induce reversible cell cycle arrest in AML.

Inhibitors of the bromodomain and extraterminal domain family (BETi) offer a new approach to treat hematological malignancies, with leukemias containing mixed lineage leukemia rearrangements being especially sensitive due to a reliance on the regulation of transcription elongation. We explored the mechanism of action of BETi in cells expressing the t(8;21), and show that these compounds reduced the size of acute myeloid leukemia cells, triggered a rapid but reversible G0 /G1 arrest, and with time, cause cell death. Meta-analysis of PRO-seq data identified ribosomal genes, which are regulated by MYC, were downregulated within 3 hours of addition of the BETi. This reduction of MYC regulated metabolic genes coincided with the loss of mitochondrial respiration and large reductions in the glycolytic rate. In addition, gene expression analysis showed that transcription of BCL2 was rapidly affected by BETi but this did not cause dramatic increases in cell death. Cell cycle arrest, lowered metabolic activity, and reduced BCL2 levels suggested that a second compound was needed to push these cells over the apoptotic threshold. Indeed, low doses of the BCL2 inhibitor, venetoclax, in combination with the BETi was a potent combination in t(8;21) containing cells. Thus, BET inhibitors that affect MYC and BCL2 expression should be considered for combination therapy with venetoclax.

A New Quinoline BRD4 Inhibitor Targets a Distinct Latent HIV-1 Reservoir for Reactivation from Other "Shock" Drugs.

Upon HIV-1 infection, a reservoir of latently infected resting T cells prevents the eradication of the virus from patients. To achieve complete depletion, the existing virus-suppressing antiretroviral therapy must be combined with drugs that reactivate the dormant viruses. We previously described a novel chemical scaffold compound, MMQO (8-methoxy-6-methylquinolin-4-ol), that is able to reactivate viral transcription in several models of HIV latency, including J-Lat cells, through an unknown mechanism. MMQO potentiates the activity of known latency-reversing agents (LRAs) or "shock" drugs, such as protein kinase C (PKC) agonists or histone deacetylase (HDAC) inhibitors. Here, we demonstrate that MMQO activates HIV-1 independently of the Tat transactivator. Gene expression microarrays in Jurkat cells indicated that MMQO treatment results in robust immunosuppression, diminishes expression of c-Myc, and causes the dysregulation of acetylation-sensitive genes. These hallmarks indicated that MMQO mimics acetylated lysines of core histones and might function as a bromodomain and extraterminal domain protein family inhibitor (BETi). MMQO functionally mimics the effects of JQ1, a well-known BETi. We confirmed that MMQO interacts with the BET family protein BRD4. Utilizing MMQO and JQ1, we demonstrate how the inhibition of BRD4 targets a subset of latently integrated barcoded proviruses distinct from those targeted by HDAC inhibitors or PKC pathway agonists. Thus, the quinoline-based compound MMQO represents a new class of BET bromodomain inhibitors that, due to its minimalistic structure, holds promise for further optimization for increased affinity and specificity for distinct bromodomain family members and could potentially be of use against a variety of diseases, including HIV infection.IMPORTANCE The suggested "shock and kill" therapy aims to eradicate the latent functional proportion of HIV-1 proviruses in a patient. However, to this day, clinical studies investigating the "shocking" element of this strategy have proven it to be considerably more difficult than anticipated. While the proportion of intracellular viral RNA production and general plasma viral load have been shown to increase upon a shock regimen, the global viral reservoir remains unaffected, highlighting both the inefficiency of the treatments used and the gap in our understanding of viral reactivation in vivo Utilizing a new BRD4 inhibitor and barcoded HIV-1 minigenomes, we demonstrate that PKC pathway activators and HDAC and bromodomain inhibitors all target different subsets of proviral integration. Considering the fundamental differences of these compounds and the synergies displayed between them, we propose that the field should concentrate on investigating the development of combinatory shock cocktail therapies for improved reservoir reactivation.

Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a.

Expression of the INK4b/ARF/INK4a tumor suppressor locus in normal and cancerous cell growth is controlled by methylation of histone H3 at lysine 27 (H3K27me) as directed by the Polycomb group proteins. The antisense noncoding RNA ANRIL of the INK4b/ARF/INK4a locus is also important for expression of the protein-coding genes in cis, but its mechanism has remained elusive. Here we report that chromobox 7 (CBX7) within the polycomb repressive complex 1 binds to ANRIL, and both CBX7 and ANRIL are found at elevated levels in prostate cancer tissues. In concert with H3K27me recognition, binding to RNA contributes to CBX7 function, and disruption of either interaction impacts the ability of CBX7 to repress the INK4b/ARF/INK4a locus and control senescence. Structure-guided analysis reveals the molecular interplay between noncoding RNA and H3K27me as mediated by the conserved chromodomain. Our study suggests a mechanism by which noncoding RNA participates directly in epigenetic transcriptional repression.

Inhibitor of CBP Histone Acetyltransferase Downregulates p53 Activation and Facilitates Methylation at Lysine 27 on Histone H3.

Tumor suppressor p53-directed apoptosis triggers loss of normal cells, which contributes to the side-effects from anticancer therapies. Thus, small molecules with potential to downregulate the activation of p53 could minimize pathology emerging from anticancer therapies. Acetylation of p53 by the histone acetyltransferase (HAT) domain is the hallmark of coactivator CREB-binding protein (CBP) epigenetic function. During genotoxic stress, CBP HAT-mediated acetylation is essential for the activation of p53 to transcriptionally govern target genes, which control cellular responses. Here, we present a small molecule, NiCur, which blocks CBP HAT activity and downregulates p53 activation upon genotoxic stress. Computational modeling reveals that NiCur docks into the active site of CBP HAT. On CDKN1A promoter, the recruitment of p53 as well as RNA Polymerase II and levels of acetylation on histone H3 were diminished by NiCur. Specifically, NiCur reduces the levels of acetylation at lysine 27 on histone H3, which concomitantly increases the levels of trimethylation at lysine 27. Finally, NiCur attenuates p53-directed apoptosis by inhibiting the Caspase 3 activity and cleavage of Poly (ADP-ribose) polymerase (PARP) in normal gastrointestinal epithelial cells. Collectively, NiCur demonstrates the potential to reprogram the chromatin landscape and modulate biological outcomes of CBP-mediated acetylation under normal and disease conditions.

[The Thyroid Hormone Receptor Mediated Luciferase Reporter Gene Assays Screening for EDCs].

OBJECTIVE: This study in order to use report gene assay based on the thyroid hormone receptor (TR) α/ß from human origin for screening endocrine disruptors chemicals (EDCs), evaluating the thyroid hormone activity of Bisphenol (BPA), 1-Naphthaleny methyl carbamate and 1-naphthol (1-NAP). METHODS: Using Rhesus monkey kidney cells (LLC-MK2) as transfection cell to establish the gene report assay system based on pGL-3-promega and pGL4.27 of TRα/ß through the method of transient transfection. Using T3 and T4 as positive subjects to evaluation the effectiveness of two detection systems and detect the thyroid hormone activity of BPA, 1-Naphthaleny methyl carbamate, 1-NAP. RESULTS: The TRß LLC-MK2 report gene assay based on pGL3-promega, the minimum detectable limit of T3 is 1.216×10-11 mol/L, the largest induction multiple was shown at 7.482×10-6 mol/L, the expression multiple of induced Lucifrerase was 5.98-fold that of the vehicle control, the EC50 was 3.327×10-8 mol/L; The minimum detectable limit of T4 was 1.622×10-8 mol/L, the largest induction Luc expression was 3.4-fold of vehicle control, the EC50 was 2.213×10-7 mol/L. The TRß LLC-MK2 report gene assay based on pGL4.27, the minimum detectable limit of T3 was 9.863×10-12 mol/L, the largest induction Luc expression as shown at 1.671×10-6 mol/L, resulting in 8.57-fold of vehicle control, the EC50 is 3.327×10-8 mol/L. The minimum detectable limit of T4 was 1.349×10-9 mol/L, the largest induction Luc expression was 4.6-fold of vehicle control, the EC50 is 4.074×10-7 mol/L. There was no thyroid hormone activity by using TRß report gene assay to evaluate BPA, 1-Naphthaleny methyl carbamate or 1-NAP, but 1-Naphthaleny methyl carbamate and 1-NAP have some degree receptor antagonism. CONCLUSIONS: The TRß LLC-MK2 report gene assay based on pGL3- promega and pGL4.27 show highly sensitive (pGL4.27 relatively higher), can be used to screen for EDCS and test chemical thyroid hormone activity effectively.