Isoforms of the TAL1 transcription factor have different roles in hematopoiesis and cell growth.

T-cell acute lymphoblastic leukemia (T-ALL) protein 1 (TAL1) is a central transcription factor in hematopoiesis. The timing and level of TAL1 expression orchestrate the differentiation to specialized blood cells and its overexpression is a common cause of T-ALL. Here, we studied the 2 protein isoforms of TAL1, short and long, which are generated by the use of alternative promoters as well as by alternative splicing. We analyzed the expression of each isoform by deleting an enhancer or insulator, or by opening chromatin at the enhancer location. Our results show that each enhancer promotes expression from a specific TAL1 promoter. Expression from a specific promoter gives rise to a unique 5' UTR with differential regulation of translation. Moreover, our study suggests that the enhancers regulate TAL1 exon 3 alternative splicing by inducing changes in the chromatin at the splice site, which we demonstrate is mediated by KMT2B. Furthermore, our results indicate that TAL1-short binds more strongly to TAL1 E-protein partners and functions as a stronger transcription factor than TAL1-long. Specifically TAL1-short has a unique transcription signature promoting apoptosis. Finally, when we expressed both isoforms in mice bone marrow, we found that while overexpression of both isoforms prevents lymphoid differentiation, expression of TAL1-short alone leads to hematopoietic stem cell exhaustion. Furthermore, we found that TAL1-short promoted erythropoiesis and reduced cell survival in the CML cell line K562. While TAL1 and its partners are considered promising therapeutic targets in the treatment of T-ALL, our results show that TAL1-short could act as a tumor suppressor and suggest that altering TAL1 isoform's ratio could be a preferred therapeutic approach.

Impact of Peptide Sequence on Functional siRNA Delivery and Gene Knockdown with Cyclic Amphipathic Peptide Delivery Agents.

Short-interfering RNA (siRNA) oligonucleotide therapeutics that modify gene expression by accessing RNA-interference (RNAi) pathways have great promise for the treatment of a range of disorders; however, their application in clinical settings has been limited by significant challenges in cellular delivery. Herein, we report a structure-function study using a series of modified cyclic amphipathic cell-penetrating peptides (CAPs) to determine the impact of peptide sequence on (1) siRNA-binding efficiency, (2) cellular delivery and knockdown efficiency, and (3) the endocytic uptake mechanism. Nine cyclic peptides of the general sequence Ac-C[XZ]4CG-NH2 in which X residues are hydrophobic/aromatic (Phe, Tyr, Trp, or Leu) and Z residues are charged/hydrophilic (Arg, Lys, Ser, or Glu) are assessed along with one acyclic peptide, Ac-(WR)4G-NH2. Cyclization is enforced by intramolecular disulfide bond formation between the flanking Cys residues. Binding analyses indicate that strong cationic character and the presence of aromatic residues that are competent to participate in CH-π interactions lead to CAP sequences that most effectively interact with siRNA. CAP-siRNA binding increases in the following order as a function of CAP hydrophobic/aromatic content: His < Phe < Tyr < Trp. Both cationic charge and disulfide-constrained cyclization of CAPs improve uptake of siRNA in vitro. Net neutral CAPs and an acyclic peptide demonstrate less-efficient siRNA translocation compared to the cyclic, cationic CAPs tested. All CAPs tested facilitated efficient siRNA target gene knockdown of at least 50% (as effective as a lipofectamine control), with the best CAPs enabling >80% knockdown. Significantly, gene knockdown efficiency does not strongly correlate with CAP-siRNA internalization efficiency but moderately correlates with CAP-siRNA-binding affinity. Finally, utilization of small-molecule inhibitors and targeted knockdown of essential endocytic pathway proteins indicate that most CAP-siRNA nanoparticles facilitate siRNA delivery through clathrin- and caveolin-mediated endocytosis. These results provide insight into the design principles for CAPs to facilitate siRNA delivery and the mechanisms by which these peptides translocate siRNA into cells. These studies also demonstrate the nature of the relationships between peptide-siRNA binding, cellular delivery of siRNA cargo, and functional gene knockdown. Strong correlations between these properties are not always observed, which illustrates the complexity in the design of optimal next-generation materials for oligonucleotide delivery.

Regulation of alternative splicing by p300-mediated acetylation of splicing factors.

Splicing of precursor mRNA (pre-mRNA) is an important regulatory step in gene expression. Recent evidence points to a regulatory role of chromatin-related proteins in alternative splicing regulation. Using an unbiased approach, we have identified the acetyltransferase p300 as a key chromatin-related regulator of alternative splicing. p300 promotes genome-wide exon inclusion in both a transcription-dependent and -independent manner. Using CD44 as a paradigm, we found that p300 regulates alternative splicing by modulating the binding of splicing factors to pre-mRNA. Using a tethering strategy, we found that binding of p300 to the CD44 promoter region promotes CD44v exon inclusion independently of RNAPII transcriptional elongation rate. Promoter-bound p300 regulates alternative splicing by acetylating splicing factors, leading to exclusion of hnRNP M from CD44 pre-mRNA and activation of Sam68. p300-mediated CD44 alternative splicing reduces cell motility and promotes epithelial features. Our findings reveal a chromatin-related mechanism of alternative splicing regulation and demonstrate its impact on cellular function.

The role of aberrant methylation of trophoblastic stem cell origin in the pathogenesis and diagnosis of placental disorders.

OBJECTIVES: The objective of the study is to investigate the role of methylation levels at promoter regions of placental vascularization genes (VEGF, EGFR, and c-jun) in pathogenesis and diagnosis of placental disorders. METHODS: We analyzed DNA and histone methylation at promoters of VEGF, EGFR, and c-jun via methylation-sensitive high-resolution melting and chromatin immunoprecipitation assay in pregnant women with normal pregnancy in first, second, and third trimesters (n = 30 in each group) and pregnant women with pregnancy complicated with preeclampsia (n = 30) and hydatidiform mole (n = 15). RESULTS: The higher expression of VEGF, EGFR, and c-jun in early pregnancy was observed to be independent of DNA methylation, while it was associated with H3 K9/K27 trimethylations. Also, abnormally higher expression of c-jun in GTDs was associated with lower H3K9me3 level at its promoter. Under preeclampsia conditions, we observed dysregulation of both DNA methylation and H3 trimethylation and subsequent low expression of VEGF, EGFR, and c-jun. Importantly, our promoter methylation data indicated that VEGF may act as novel fetal DNA diagnostic marker for preeclampsia and molar pregnancies in maternal plasma. CONCLUSION: These findings emphasize the importance of dysregulated epigenetic phenomenon behind the pathologies of placental disorders and use of promoter region DNA methylation as an epigenetic marker for these pathological pregnancies. © 2016 John Wiley & Sons, Ltd.

Genotoxicity and immunotoxic effects of 1,2-dichloroethane in Wistar rats.

Dichloroethane is widely used as a solvent, degreasing agent and in a variety of commercial products, and is known for being a ubiquitous contaminant in the environment. Important sources principally include the emissions from industrial processes, improper consumption, storage, and disposal methods. In view of the fact that the mechanism of its genotoxicity has not been satisfactorily elucidated, the acute in vivo toxicological impact is assessed in Rattus norvegicus. A systematic investigation has been made involving the use of conventional methods along with molecular and flow cytometric approaches. The micronucleus and chromosomal aberration frequencies were significantly elevated in bone marrow cells exposed to three concentrations at multiple treatment durations indicating positive time- and dose-response relationships. The mitotic index significantly decreased in similar concentrations in contrast to normal control. Separate studies were performed on blood cells for comet assay. It revealed dichloroethane-induced DNA damage in all exposures readily explainable in a dose- and time-dependent manner. Recent molecular techniques were further employed using leukocytes for the cell apoptosis/cycle and mitochondrial membrane potential employing propidium iodide staining and rhodamine-123, respectively. The effect on mitochondrial membrane permeability, cell cycle phases, and the DNA damage was analyzed through flow cytometry. These indicators revealed dichloroethane treatment decreased the mitochondrial membrane potential, affected the cell cycle, and confirmed the DNA damage, leading to apoptosis of the cells of the immune system responsible for immunotoxic effects of dichloroethane on rat leukocytes.

The Protective Role of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Protein Sigma-1 Receptor in Regulating Endothelial Inflammation and Permeability Associated with Acute Lung Injury.

Earlier studies from our lab identified endoplasmic reticulum (ER) chaperone BiP/GRP78, an important component of MAM, to be a novel determinant of endothelial cell (EC) dysfunction associated with acute lung injury (ALI). Sigma1R (Sig1R) is another unique ER receptor chaperone that has been identified to associate with BiP/GRP78 at the MAM and is known to be a pluripotent modulator of cellular homeostasis. However, it is unclear if Sig1R also plays a role in regulating the EC inflammation and permeability associated with ALI. Our data using human pulmonary artery endothelial cells (HPAECs) showed that siRNA-mediated knockdown of Sig1R potentiated LPS-induced the expression of proinflammatory molecules ICAM-1, VCAM-1 and IL-8. Consistent with this, Sig1R agonist, PRE-084, known to activate Sig1R by inducing its dissociation from BiP/GRP78, blunted the above response. Notably, PRE-084 failed to blunt LPS-induced inflammatory responses in Sig1R-depleted cells, confirming that the effect of PRE-084 is driven by Sig1R. Furthermore, Sig1R antagonist, NE-100, known to inactivate Sig1R by blocking its dissociation from BiP/GRP78, failed to block LPS-induced inflammatory responses, establishing that dissociation from BiP/GRP78 is required for Sig1R to exert its anti-inflammatory action. Unlike Sig1R, the siRNA-mediated knockdown or Subtilase AB-mediated inactivation of BiP/GRP78 protected against LPS-induced EC inflammation. Interestingly, the protective effect of BiP/GRP78 knockdown or inactivation was abolished in cells that were depleted of Sig1R, confirming that BiP/GRP78 knockdown/inactivation-mediated suppression of EC inflammation is mediated via Sig1R. In view of these findings, we determined the in vivo relevance of Sig1R in a mouse model of sepsis-induced ALI. The intraperitoneal injection of PRE-084 mitigated sepsis-induced ALI, as evidenced by a decrease in ICAM-1, IL-6 levels, lung PMN infiltration, and lung vascular leakage. Together, these data evidence a protective role of Sig1R against endothelial dysfunction associated with ALI and identify it as a viable target in terms of controlling ALI in sepsis.

Withania somnifera (L.) Dunal (Ashwagandha); current understanding and future prospect as a potential drug candidate.

Cancer and Neurodegenerative diseases are one of the most dreadful diseases to cure and chemotherapy has found a prime place in cancerous treatments while as different strategies have been tested in neurodegenerative diseases as well. However, due to adverse shortcomings like the resistance of cancerous cells and inefficiency in neurodegenerative disease, plant sources have always found a prime importance in medicinal use for decades, Withania somnifera (L.) Dunal (W. somnifera) is a well-known plant with medicinal use reported for centuries. It is commonly known as winter cherry or ashwagandha and is a prime source of pharmaceutically active compounds withanolides. In recent years research is being carried in understanding the extensive role of W. somnifera in cancer and neurological disorders. W. somnifera has been reported to be beneficial in DNA repair mechanisms; it is known for its cellular repairing properties and helps to prevent the apoptosis of normal cells. This review summarizes the potential properties and medicinal benefits of W. somnifera especially in cancer and neurodegenerative diseases. Available data suggest that W. somnifera is effective in controlling disease progressions and could be a potential therapeutic target benefiting human health status. The current review also discusses the traditional medicinal applications of W. somnifera, the experimental evidence supporting its therapeutical potential as well as obstacles that necessitate being overcome for W. somnifera to be evaluated as a curative agent in humans.

Modulation of dietary folate with age confers selective hepatocellular epigenetic imprints through DNA methylation.

The present study has been designed to determine the effect of folate modulation (deficiency/supplementation) with aging on the promoter methylation of tumor suppressor and proto-oncogenes to understand the underlying mechanism of epigenetic alterations. Folate deficiency was induced for 3 and 5 months in weanling, young and adult groups, and after 3 months of folate deficiency, they were repleted with physiological folate (2 mg/kg diet) and folate oversupplementation (8 mg/kg diet) for another 2 months. The methylation facet in the present study revealed that the combined effect of folate deficiency and aging decreased the methylation index. Folate deficiency with age resulted in the up-regulation of proto-oncogenes (C-MYC and C-JUN) and cell cycle regulator gene Cyclin E as a result of promoter hypomethylation. However, in case of tumor suppressor genes (p53, p15ink4b and p16ink4a), the expression levels were found to be decreased at transcriptional level due to promoter hypermethylation. Upon repletion with physiological folate and folate oversupplementation, we found down-regulation of proto-oncogenes and up-regulation of tumor suppressor genes as a result of promoter hypermethylation and hypomethylation, respectively. Deregulation of these important genes due to folate deficiency may contribute toward the pathogenesis at cellular level.

Identification of regulatory mechanisms of intestinal folate transport in condition of folate deficiency.

Folic acid is an essential micronutrient, deficiency of which can lead to disturbance in various metabolic processes of cell. Folate transport across intestine occurs via the involvement of specialized folate transporters viz. proton coupled folate transporter (PCFT) and reduced folate carrier (RFC), which express at the membrane surfaces. The current study was designed to identify the regulatory mechanisms underlying the effects of folate deficiency (FD) on folate transport in human intestinal cell line as well as in rats and to check the reversibility of such effects. Caco-2 cells were grown for five generations in control and FD medium. Following treatment, one subgroup of cells was shifted on folate sufficient medium and grown for three more generations. Similarly, rats were fed an FD diet for 3 and 5 months, and after 3 months of FD treatment, one group of rats were shifted on normal folate-containing diet. Increase in folate transport and expression of folate transporters were observed on FD treatment. However, when cells and rats were shifted to control conditions after treatment, transport and expression of these genes restored to the control level. FD was found to have no impact on promoter methylation of PCFT and RFC; however, messenger RNA stability of transporters was found to be decreased, suggesting some adaptive response. Overall, increased expression of transporters under FD conditions can be attributed to enhanced rate of transcription of folate transporters and also to the increased binding of specificity protein 1 transcription factor to the RFC promoter only.

Mechanistic insights of intestinal absorption and renal conservation of folate in chronic alcoholism.

Folate mediated one-carbon metabolism is of fundamental importance for various cellular processes, including DNA synthesis and methylation of biological molecules. Due to the exogenous requirement of folate in mammals, there exists a well developed epithelial folate transport system for regulation of normal folate homeostasis. The intestinal and renal folate uptake is tightly and diversely regulated and disturbances in folate homeostasis like in alcoholism have pathological consequences. The study was sought to delineate the regulatory mechanism of folate uptake in intestine and reabsorption in renal tubular cells that could evaluate insights of malabsorption during alcoholism. The folate transporters PCFT and RFC were found to be associated with lipid rafts of membrane surfaces in intestine and kidney. Importantly, the observed lower intestinal and renal folate uptake was associated with decreased levels of folate transporter viz. PCFT and RFC in lipid rafts of intestinal and renal membrane surfaces. The decreased association of folate transporters in lipid rafts was associated with decreased protein and mRNA levels. In addition, immunohistochemical studies showed that alcoholic conditions deranged that localization of PCFT and RFC. These findings could explain the possible mechanistic insights that may result in folate malabsorption during alcoholism.

Recent development in targeting PI3K-Akt-mTOR signaling for anticancer therapeutic strategies.

Cancer is a diverse class of diseases which differ widely in their cause and biology. The aberrant behavior of cancer reflects up regulation of certain oncogenic signaling pathways that promote proliferation, inhibit apoptosis, and enable the cancer to spread and evoke angiogenesis. Phosphoinositide-3-kinase(PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway controls various biological processes that are important for normal functioning of the cell via cell cycle progression, survival, migration, transcription, translation and metabolism. However, PI3K signaling pathway is dysregulated almost in all cancers which is due to the amplification and genetic mutation of PI3K gene, encoding catalytic and regulatory subunit of PI3K isoforms. The current review focuses on the structural features of various PI3K isoforms including Akt and mTOR and their inhibition using specific small molecule inhibitors in an attempt to achieve an attractive target for cancer prevention and chemotherapy.

Understanding histone deacetylases in the cancer development and treatment: an epigenetic perspective of cancer chemotherapy.

Cancer is a pathologic condition that involves genetic and epigenetic events culminating in neoplastic transformation. Alteration in epigenetic events that regulate the transcriptional activity of genes associated with various signaling pathways can influence multiple stages of tumorigenesis. In cancer cells, an imbalance often exists between histone acetyl transferase and histone deacetylase (HDAC) activities, and current research focuses actively on seeking competitive HDAC inhibitors (HDACi) for chemotherapeutic intervention. HDACi are proving useful for cancer prevention and therapy by virtue of their ability to reactivate the expression of epigenetically silenced genes, including those involved in differentiation, cell cycle regulation, apoptosis, angiogenesis, invasion, and metastasis. Furthermore, epidemiological studies suggest that different diets such as intake of cruciferous vegetables may lower the risk of different cancers, and there is growing interest in identifying the specific chemoprotective constituents and mechanistic insights of their action. Interestingly, it has been observed that cancer cells are more sensitive than nontransformed cells to apoptotic induction by some HDACi. Although the mechanistic basis for this sensitivity is unclear, yet HDACi have emerged as important epigenetic target for single and combinatorial chemotherapy. HDACi derived from diverse sources such as microbial, dietary, and synthetic increase acetylation level of cells and bring about anti-proliferative and apoptotic effects specific to cancer cells by way of their role in cell cycle regulation and expression of epigenetically silenced genes.