chHDAC11 mRNA Expression During Prenatal and Postnatal Chicken (Gallus gallus) Brain Development

Background: Histone deacetylation plays an essential role in transcriptional regulation of cell cycle progression and other evolutionary processes. Several results confirm the importance of the latest found HDAC11 gene to deacetylate histone core in neurons and their supportive cells in developing the vertebrate Central Nervous System (CNS). Objectives: This study investigates the HDAC11 potential role in early chicken CNS development by studying its mRNA expression profile which may have unique means in studying human subjects. Materials & Methods: Chicken HDAC11 RNAs were reverse transcribed to cDNAs, and the amount of chHDAC11 transcripts was measured by ΔCT mean calculation using the real-time quantitative PCR method. One-way ANOVA and Duncan’s analysis (SigmaStat software version 4.0) were used to test the statistical significance of the results. The levels of significance were set at P≤0.05. Quantitative data are presented as Mean±SD. Results: The amount of HDAC11 mRNAs gradually increases, at least 2-3 times, from as early as day 14 (E14/HH40) of prenatal cortex formation to day P0 (E20=HH45) and continue to increase to day 40 in both cortical and hippocampal regions of the postnatal chicken brain during development (*P≤0.05). HDAC11 mRNA is not only expressed in the postnatal cortex and hippocampi regions but also—for the first time—in the developing brain during the prenatal period. Conclusion: Our results show a possibly important role for the latest found HDAC11 conserved gene in the development of vertebrates’ embryonic brain, which in turn may have a significant impact on understanding human brain development.

Role of Myeloid Tet Methylcytosine Dioxygenase 2 in Pulmonary and Peritoneal Inflammation Induced by Lipopolysaccharide and Peritonitis Induced by Escherichia coli

Tet methylcytosine dioxygenase 2 (Tet2) mediates demethylation of DNA. We here sought to determine the expression and function of Tet2 in macrophages upon exposure to lipopolysaccharide (LPS), and in the host response to LPS induced lung and peritoneal inflammation, and during Escherichia (E.) coli induced peritonitis. LPS induced Tet2 expression in mouse macrophages and human monocytes in vitro, as well as in human alveolar macrophages after bronchial instillation in vivo. Bone marrow-derived macrophages from myeloid Tet2 deficient (Tet2fl/flLysMCre) mice displayed enhanced production of IL-1β, IL-6 and CXCL1 upon stimulation with several Toll-like receptor agonists; similar results were obtained with LPS stimulated alveolar and peritoneal macrophages. Histone deacetylation was involved in the effect of Tet2 on IL-6 production, whilst methylation at the Il6 promoter was not altered by Tet2 deficiency. Tet2fl/flLysMCre mice showed higher IL-6 and TNF levels in bronchoalveolar and peritoneal lavage fluid after intranasal and intraperitoneal LPS administration, respectively, whilst other inflammatory responses were unaltered. E. coli induced stronger production of IL-1β and IL-6 by Tet2 deficient peritoneal macrophages but not in peritoneal lavage fluid of Tet2fl/flLysMCre mice after in vivo intraperitoneal infection. Tet2fl/flLysMCre mice displayed enhanced bacterial growth during E. coli peritonitis, which was associated with a reduced capacity of Tet2fl/flLysMCre peritoneal macrophages to inhibit the growth of E. coli in vitro. Collectively, these data suggest that Tet2 is involved in the regulation of macrophage functions triggered by LPS and during E. coli infection.

Transcriptomic Analysis Reveals the Key Role of Histone Deacetylation-mediated Phytohormones Interaction in Fiber Initiation of Cotton

Background: Histone deacetylation is one of the most important epigenetic modifications and plays diverse roles in plant development. However, the detailed functions and mechanisms of histone deacetylation in fiber development of cotton are still unclear. HDAC inhibitors (HDACi) have been used commonly to study the molecular mechanism underlying histone deacetylation or to facilitate disease therapy in humans through hindering the histone deacetylase catalytic activity. Trichostatin A (TSA) - the most widely used HDACi has been used to determine the role of histone deacetylation on different developmental stages of plants. Results: Here, exogenous TSA was applied in the fiber initiation and elongation in vitro, and the results demonstrated the crucial role of histone deacetylation in fiber initiation regulation. Therefore, we made a transcriptomic analysis to reveal the underlying mechanisms. Through RNA-Seq analysis, the differentially expressed genes were mostly enriched in plant hormone signal transduction,phenylpropanoid biosynthesis, photosynthesis, and carbon metabolism pathways, suggesting the potential role of phytohormone, phenylpropanoid metabolism, and energy metabolism downstream of histone deacetylation in fiber initiation. The phytohormone signal transduction pathways harbor the most differentially expressed genes. Deeper studies showed that some genes promoting auxin, Gibberellic Acid (GA), and Salicylic Acid (SA) signaling were down-regulated, while some genes facilitating Abscisic Acid (ABA) and inhibiting Jasmonic Acid (JA) singling were up-regulated after the TSA treatments. Conclusions: Collectively, we established a model, in which histone deacetylation can regulate some key genes involved in different phytohormone pathways, consequently, promoting the auxin, GA, JA and SA signaling, whereas, repressing the ABA signaling to improve the fiber cell initiation; besides that the genes associated with energy metabolism, phenylpropanoid, and glutathione metabolism were also involved in. The above results provided novel clues to illuminate the underlying mechanisms of epigenetic modifications as well as interactions of different phytohormones in fiber cell differentiation, which is also very valuable for molecular breeding of higher quality cotton.

Inhibition of histone deacetylation with vorinostat does not prevent tunicamycin-mediated acute kidney injury

Endoplasmic reticulum (ER) stress is associated with acute kidney injury (AKI) caused by various mechanisms, including antibiotics, non-steroidal anti-inflammatory drugs, cisplatin, and radiocontrast. Tunicamycin (TM) is a nucleoside antibiotic that induces ER stress and is a commonly used model of AKI. 4-phenylbutyrate (4-PBA) is a chemical chaperone and histone deacetylase (HDAC) inhibitor and has been shown to protect the kidney from ER stress, apoptosis, and structural damage in a tunicamycin model of AKI. The renal protection provided by 4-PBA is attributed to its ability to prevent misfolded protein aggregation and inhibit ER stress; however, the HDAC inhibitor effects of 4-PBA have not been examined in the TM-induced model of AKI. As such, the main objective of this study was to determine if histone hyperacetylation provides any protective effects against TM-mediated AKI. The FDA-approved HDAC inhibitor vorinostat was used, as it has no ER stress inhibitory effects and therefore the histone hyperacetylation properties alone could be investigated. In vitro work demonstrated that vorinostat inhibited histone deacetylation in cultured proximal tubular cells but did not prevent ER stress or protein aggregation induced by TM. Vorinostat induced a significant increase in cell death, and exacerbated TM-mediated total cell death and apoptotic cell death. Wild type male mice were treated with TM (0.5 mg/kg, intraperitoneal injection), with or without vorinostat (50 mg/kg/day) or 4-PBA (1 g/kg/day). Mice treated with 4-PBA or vorinostat exhibited similar levels of histone hyperacetylation. Expression of the pro-apoptotic protein CHOP was induced with TM, and not inhibited by vorinostat. Further, vorinostat did not prevent any renal damage or decline in renal function caused by tunicamycin. These data suggest that the protective mechanisms found by 4-PBA are primarily due to its molecular chaperone properties, and the HDAC inhibitors used did not provide any protection against renal injury caused by ER stress.

Regulation of PXR Function by Coactivator and Corepressor Proteins: Ligand Binding Is Just the Beginning

The pregnane X receptor (PXR, NR1I2) is a nuclear receptor which exerts its regulatory function by heterodimerization with the retinoid-X-receptor α (RXRα, NR2B1) and binding to the promoter and enhancer regions of diverse target genes. PXR is involved in the regulation of drug metabolism and excretion, metabolic and immunological functions and cancer pathogenesis. PXR activity is strongly regulated by the association with coactivator and corepressor proteins. Coactivator proteins exhibit histone acetyltransferase or histone methyltransferase activity or associate with proteins having one of these activities, thus promoting chromatin decondensation and activation of the gene expression. On the contrary, corepressor proteins promote histone deacetylation and therefore favor chromatin condensation and repression of the gene expression. Several studies pointed to clear cell- and ligand-specific differences in the activation of PXR. In this article, we will review the critical role of coactivator and corepressor proteins as molecular determinants of the specificity of PXR-mediated effects. As already known for other nuclear receptors, understanding the complex mechanism of PXR activation in each cell type and under particular physiological and pathophysiological conditions may lead to the development of selective modulators with therapeutic potential.

Biochemical analysis of deacetylase activity of rice sirtuin OsSRT1, a class IV member in plants

The role of plant sirtuins is slowly unwinding. There are only reports of H3K9Ac deacetylation by OsSRT1. This belongs to class IV sirtuin family with a longer C-terminus. Here C-terminus is required for ligand binding and catalysis. OsSRT1 can deacetylate the lys residues at the N terminal tail of both H3 and H4. It can also target the non-histone target, OsPARP1 playing a role in DNA damage repair pathway. Changes in the extent of different histone deacetylation by OsSRT1 is also related with different abiotic stress conditions. NAM and ADP-ribose has negative effect on OsSRT1 deacetylation.

Multiple distinct domains of human XIST are required to coordinate gene silencing and subsequent heterochromatin formation

Background Mammalian dosage compensation is achieved by the inactivation of one X chromosome in XX individuals. In eutheria this process is initiated early in development by the long non-coding RNA XIST. Studies of the initiation of silencing by XIST have focussed on mouse models, so the domains of XIST required to induce silencing in humans, and their relationship with domains required to establish heterochromatin remain to be determined. Methods We have previously established an inducible XIST cDNA in somatic cells and shown it can induce silencing and recruit heterochromatic features. We now assess a series of deletions across the construct for the ability to induce silencing and integrate these results with time-course and chromatin remodelling inhibitor treatments to follow the steps of XIST-induced silencing and heterochromatinization. Discussion We find that in addition to the previously reported necessity of the 5’ A repeat region for XIST-induced silencing, the F repeat region and a non-repetitive region at the 3’ end of the RNA are also required to silence genes. Silencing of genes up to 17Mb from the XIST integration occurs within two days, while formation of a Cot-1 depleted domain is slower, and more dependent on repeat F. The role of Repeat F in both the silencing of actively transcribed genes, the spread of H3K27me3 and the formation of a transcriptionally inert domain suggests a role in a pathway crucial for the spread of XIST across the chromatin to target distal regions of inactivation. Histone deacetylation requires only the A repeat region, with HDAC3 inhibition showing limited effect on silencing, but an impact on H3K27me3 recruitment, and as a result the recruitment of MacroH2A. Global HDAC inhibition impacted silencing in both a distance and dose-dependent fashion. The E repeat region was required for CIZ1 and H4K20me1 recruitment as well as H3K27me3; however, these appeared to act relatively independently. The H3K27me3 mark established by PRC2 integrated silencing and many of the heterochromatic features, while the PRC1 mark ubH2A appeared to be downstream of silencing in these human somatic cells.

Characterization and Study of Gene Expression Profiles of Human Periodontal Mesenchymal Stem Cells in Spheroid Cultures by Transcriptome Analysis

A spheroid is known as a three-dimensional culture model, which better simulates the physiological conditions of stem cells. This study is aimed at identifying genes specifically expressed in spheroid-cultured human periodontal ligament mesenchymal stem cells (hPDLMSCs) using RNA-seq analysis to evaluate their functions. Transcriptome analysis was performed using spheroid and monolayer cultures of hPDLMSCs from four patients. Cluster and Gene Ontology analyses revealed that genes involved in cell-cell adhesion as well as the G2/M and G1/S transitions of mitotic cell cycles were strongly expressed in the monolayer culture group. However, genes involved in the negative regulation of cell proliferation, histone deacetylation, and bone morphogenetic protein signaling were strongly expressed in the spheroid culture group. We focused on the transcription factor nuclear receptor subfamily 4 group A member 2 (NR4A2) among the genes that were strongly expressed in the spheroid culture group and analyzed its function. To confirm the results of the transcriptome analysis, we performed real-time polymerase chain reaction and western blotting analyses. Interestingly, we found that the mRNA and protein expressions of NR4A2 were strongly expressed in the spheroid-cultured hPDLMSCs. Under osteogenic differentiation conditions, we used siRNA to knock down NR4A2 in spheroid-cultured hPDLMSCs to verify its role in osteogenesis. We found that NR4A2 knockdown significantly increased the levels of mRNA expression for osteogenesis-related genes alkaline phosphatase (ALP), Osteopontin (OPN), and type 1 collagen (COL1) (Student’s paired t -test, p < 0.05 ). ALP activity was also significantly increased when compared to the negative control group (Student’s paired t -test, p < 0.05 ). Additionally, spheroid-cultured hPDLMSCs transfected with siNR4A2 were cultured for 12 days, resulting in the formation of significantly larger calcified nodules compared to the negative control group (Student’s paired t -test, p < 0.05 ). On the other hand, NR4A2 knockdown in hPDLMSC spheroid did not affect the levels of chondrogenesis and adipogenesis-related genes under chondrogenic and adipogenic conditions. These results suggest that NR4A2 negatively regulates osteogenesis in the spheroid culture of hPDLMSCs.

Stepwise progression of β-selection during T cell development as revealed by histone deacetylation inhibition

During T cell development, the first step in creating a unique T Cell Receptor (TCR) is the genetic recombination of the TCRβ chain. The quality of this newly recombined gene is assessed at the β-selection checkpoint, and most cells fail this checkpoint and are removed. The coordination of the complex events that combine to control fate at the β-selection checkpoint is not yet understood. We assessed the impact on T cell development of a selective inhibitor to histone deacetylase 6, ACY1215, currently in clinical use. ACY1215 led to bypass of the β-selection checkpoint such that cells in the DN4 stage often lacked expression of TCRβ, and failed to progress to the DP stage. Characterisation of the molecular basis for this bypass revealed a new, pivotal stage in β-selection, the beginning and end of which were defined by the upregulation of the TCR co-receptors, CD28 and CD2 respectively. Within this stage, termed DN3bPre, CD5 and Lef1 are upregulated to reflect pre-TCR signalling. We propose that the progressive expression of CD28, CD5 then CD2 reports and modulates the pre-TCR signal to orchestrate passage through the β-selection checkpoint. By disrupting the functional connection between CD5 and pre-TCR, ACY1215 allows cells to inappropriately bypass the β-selection checkpoint. These findings implicate a refined model of β-selection in which a coordinated increase in expression of pre-TCR, CD5 and Lef1 provides for an escalating test of TCR signalling strength, and culminates in the expression of CD2 to enable exit from the β-selection checkpoint.