• hElp3 Directly Modulates the Expression of HSP70 Gene in HeLa Cells via HAT Activity

      Li, Fen; Ma, Jixian; Ma, Yu; Hu, Yanyan; Tian, Shujuan; White, Richard E.; Han, Guichun; Department of Pharmacology and Toxicology (2011-12-21)
      Human Elongator complex, which plays a key role in transcript elongation in vitro assay, is incredibly similar in either components or function to its yeast counterpart. However, there are only a few studies focusing on its target gene characterization in vivo. We studied the effect of down-regulation of the human elongation protein 3 (hELP3) on the expression of HSP70 through antisense strategy. Transfecting antisense plasmid p1107 into HeLa cells highly suppressed hELP3 expression, and substantially reduced expression of HSP70 mRNA and protein. Furthermore, chromatin immunoprecipitation assay (ChIP Assay) revealed that hElp3 participates in the transcription elongation of HSPA1A in HeLa cells. Finally, complementation and ChIP Assay in yeast showed that hElp3 can not only complement the growth and slow activation of HSP70 (SSA3) gene transcription, but also directly regulates the transcription of SSA3. On the contrary, these functions are lost when the HAT domain is deleted from hElp3. These data suggest that hElp3 can regulate the transcription of HSP70 gene, and the HAT domain of hElp3 is essential for this function. These findings now provide novel insights and evidence of the functions of hELP3 in human cells.
    • Modulation of indoleamine 2, 3-dioxygenase 1 expression by activated Tcells in breast cancer is controlled by epigenetic mechanisms

      Noonepalle, Satish Kumar Reddy; Department of Biochemistry and Molecular Biology (2015-09)
      Tumor infiltrating lymphocytes (TILs) secrete cytokines that modulate immune responses at the tumor microenvironment. Tumor suppressor activity of interferon gamma (IFNy) cytokine also activates expression of immune suppressive factors such as IDO and PD-L1 in tumor cells. However, there is still much to learn about how tumor cells counter the immune cells at the gene expression level. In this study, RNA-seq analysis of breast cancer cells after in-vitro co-culture with anti-CD3/CD28 activated human T -cells revealed that the IFNy induced immune response gene signature is common to both triple negative breast cancer (TNBC) MDA-MB-231 and estrogen receptor positive (ER+) MCF7 cells. However, IDOl expression was differentially upregulated with significantly higher expression in MDA-MB-231 compared to MCF7 cells. Analysis of the TCGA breast invasive carcinoma dataset revealed subtype specific mRNA expression and IDOl promoter DNA methylation. We observed that IDOl mRNA expression and promoter methylation followed inverse correlation. TNBC/Basal subtype was hypomethylated at the IDOl promoter with higher mRNA expression compared to the ER+ subtype that was hypermethylated with relatively lower IDOl mR.NA expression. The IDOl promoter methylation was confirmed by pyrosequencing analysis of a panel of breast cancer cell lines and patient tumors. IFNy treatment of MDA-MB-231 and MCF7 breast cancer cells revealed no difference in terms of upstream signaling and IDOl mRNA stability. Treatment with demethylating agent, 5-azadeoxycytidine, synergistically up-regulated IDOl mRNA expression in ER+ MCF7 cells highlighting that CpG methylation controls !DO 1 gene expression. We also found a positive correlation between !DO I and CDBA expression and better relapse free survival in TNBC/basal subtype patients suggesting that !DO 1 expression is driven by intrinsic immune surveillance of TILs. These findings provide evidence that !DO 1 promoter methylation regulates anti-immune responses by tumor cells towards TILs and it could be used as a predictive biomarker for IDO inhibitor-based immunotherapy of breast cancer.
    • NF-Y Recruits Both Transcription Activator and Repressor to Modulate Tissue- and Developmental Stage-Specific Expression of Human γ-Globin Gene

      Zhu, Xingguo; Wang, Yongchao; Pi, Wenhu; Liu, Hui; Wickrema, Amittha; Tuan, Dorothy; Department of Biochemistry and Molecular Biology (2012-10-10)
      The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.
    • Novel Use of Stem-loop DNA in SELEX

      Coe, Genevieve; Department of Chemistry and Physics (Augusta University, 2015-05)
      Aptamers are composed of oligonucleotides and are most recognized for their ability to function much like antibodies. Unlike antibodies, aptamers can be generated in vitro, have a longer shelf life and are potentially faster to synthesize. The current method of aptamer generation is through a cycle known as SELEX, which stands for Systematic Evolution of Ligands through Exponential Enrichment. The SELEX method traditionally uses a library of single-stranded DNA or RNA as a starting material, which is incubated with a target. The nucleotides that bind to the target are separated from the unbound nucleotides, eluted from the target and amplified by PCR. The amplified nucleotides go through several more cycles of SELEX until aptamers with a high binding affinity for the target are produced. Before the double-stranded products from PCR can be used in another cycle they have to be separated into single-strands. The process of regenerating single-stranded DNA from double-stranded DNA is often time consuming and results in a low product yield, decreasing the efficiency of the SELEX process. In order to improve the efficiency of aptamer generation, we explored the use of a novel starting material based on its potential to make SELEX a more automated process. Stem-loop DNA has both double-stranded and single-stranded DNA portions due to its unique secondary structure. It has the ability to retain the single-stranded portion of its special conformation after PCR amplification. After characterization of a specifically designed stem-loop, we incorporated the use of stem-loop DNA as a starting material in SELEX to potentially bypass the regeneration of single-strand DNA in SELEX. The success of this modified approach to SELEX could result in a more efficient method for the generation of aptamers.
    • Uracils at nucleotide position 9â 11 are required for the rapid turnover of miR-29 family

      Zhang, Zhuo; Zou, Jun; Wang, Guo-Kun; Zhang, Jun-Tao; Huang, Shuang; Qin, Yong-Wen; Jing, Qing; Department of Biochemistry and Molecular Biology (2011-02-1)
      MicroRNAs are endogenous small RNA molecules that regulate gene expression. Although the biogenesis of microRNAs and their regulation have been thoroughly elucidated, the degradation of microRNAs has not been fully understood. Here by using the pulseâ chase approach, we performed the direct measurement of microRNA lifespan. Five representative microRNAs demonstrated a general feature of relatively long lifespan. However, the decay dynamic varies considerably between these individual microRNAs. Mutation analysis of miR-29b sequence revealed that uracils at nucleotide position 9â 11 are required for its rapid decay, in that both specific nucleotides and their position are critical. The effect of uracil-rich element on miR-29b decay dynamic occurs in duplex but not in single strand RNA. Moreover, analysis of published data on microRNA expression profile during development reveals that a substantial subset of microRNAs with the uracil-rich sequence tends to be down-regulated compared to those without the sequence. Among them, Northern blotting shows that miR-29c and fruit fly bantam possess a relatively rapid turnover rate. The effect of uracil-rich sequence on microRNA turnover depends on the sequence context. The present work indicates that microRNAs contain sequence information in the middle region besides the sequence element at both ends.