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1.  Changes in the Expression of miR-381 and miR-495 Are Inversely Associated with the Expression of the MDR1 Gene and Development of Multi-Drug Resistance 
PLoS ONE  2013;8(11):e82062.
Multidrug resistance (MDR) frequently develops in cancer patients exposed to chemotherapeutic agents and is usually brought about by over-expression of P-glycoprotein (P-gp) which acts as a drug efflux pump to reduce the intracellular concentration of the drug(s). Thus, inhibiting P-gp expression might assist in overcoming MDR in cancer chemotherapy. MiRNAome profiling using next-generation sequencing identified differentially expressed microRNAs (miRs) between parental K562 cells and MDR K562 cells (K562/ADM) induced by adriamycin treatment. Two miRs, miR-381 and miR-495, that were strongly down-regulated in K562/ADM cells, are validated to target the 3’-UTR of the MDR1 gene. These miRs are located within a miR cluster located at chromosome region 14q32.31, and all miRs in this cluster appear to be down-regulated in K562/ADM cells. Functional analysis indicated that restoring expression of miR-381 or miR-495 in K562/ADM cells was correlated with reduced expression of the MDR1 gene and its protein product, P-gp, and increased drug uptake by the cells. Thus, we have demonstrated that changing the levels of certain miR species modulates the MDR phenotype in leukemia cells, and propose further exploration of the use of miR-based therapies to overcome MDR.
PMCID: PMC3841137  PMID: 24303078
2.  c-Rel is required for the development of thymic Foxp3+ CD4 regulatory T cells 
The Journal of Experimental Medicine  2009;206(13):3001-3014.
During thymopoiesis, a unique program of gene expression promotes the development of CD4 regulatory T (T reg) cells. Although Foxp3 maintains a pattern of gene expression necessary for T reg cell function, other transcription factors are emerging as important determinants of T reg cell development. We show that the NF-κB transcription factor c-Rel is highly expressed in thymic T reg cells and that in c-rel−/− mice, thymic T reg cell numbers are markedly reduced as a result of a T cell–intrinsic defect that is manifest during thymocyte development. Although c-Rel is not essential for TGF-β conversion of peripheral CD4+CD25− T cells into CD4+Foxp3+ cells, it is required for optimal homeostatic expansion of peripheral T reg cells. Despite a lower number of peripheral T reg cells in c-rel−/− mice, the residual peripheral c-rel−/− T reg cells express normal levels of Foxp3, display a pattern of cell surface markers and gene expression similar to those of wild-type T reg cells, and effectively suppress effector T cell function in culture and in vivo. Collectively, our results indicate that c-Rel is important for both the thymic development and peripheral homeostatic proliferation of T reg cells.
PMCID: PMC2806473  PMID: 19995950
3.  Plasticity of DNA methylation in mouse T cell activation and differentiation 
BMC Molecular Biology  2012;13:16.
Circulating CD4+ T helper cells are activated through interactions with antigen presenting cells and undergo differentiation into specific T helper cell subsets depending on the type of antigen encountered. In addition, the relative composition of the circulating CD4+ T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells.
Here, we report on the highly plastic nature of DNA methylation at the genome-wide level as T cells undergo activation, differentiation and aging. Of particular note were the findings that DNA demethylation occurred rapidly following T cell activation and that all differentiated T cell populations displayed lower levels of global methylation than the non-differentiated population. In addition, T cells from older mice had a reduced level of DNA methylation, most likely explained by the increase in the memory/effector cell fraction. Although significant genome-wide changes were observed, changes in DNA methylation at individual genes were restricted to specific cell types. Changes in the expression of enzymes involved in DNA methylation and demethylation reflect in most cases the changes observed in the genome-wide DNA methylation status.
We have demonstrated that DNA methylation is dynamic and flexible in CD4+ T cells and changes rapidly both in a genome-wide and in a targeted manner during T cell activation, differentiation. These changes are accompanied by parallel changes in the enzymatic complexes that have been implicated in DNA methylation and demethylation implying that the balance between these opposing activities may play a role in the maintaining the methylation profile of a given cell type but also allow flexibility in a cell population that needs to respond rapidly to environmental signals.
PMCID: PMC3386888  PMID: 22642378
DNA demethylation; T cell activation; T cell differentiation; Il2, Csf2
4.  An autocrine TGF-β/ZEB/miR-200 signaling network regulates establishment and maintenance of epithelial-mesenchymal transition 
Molecular Biology of the Cell  2011;22(10):1686-1698.
Epithelial-mesenchymal transition is a form of cellular plasticity that is critical for embryonic development and tumor metastasis. This study shows that a signaling network involving autocrine TGF-β signaling, ZEB transcription factors, and the miR-200 family regulates interconversion between epithelial and mesenchymal states.
 Epithelial-mesenchymal transition (EMT) is a form of cellular plasticity that is critical for embryonic development and tumor metastasis. A double-negative feedback loop involving the miR-200 family and ZEB (zinc finger E-box-binding homeobox) transcription factors has been postulated to control the balance between epithelial and mesenchymal states. Here we demonstrate using the epithelial Madin Darby canine kidney cell line model that, although manipulation of the ZEB/miR-200 balance is able to repeatedly switch cells between epithelial and mesenchymal states, the induction and maintenance of a stable mesenchymal phenotype requires the establishment of autocrine transforming growth factor-β (TGF-β) signaling to drive sustained ZEB expression. Furthermore, we show that prolonged autocrine TGF-β signaling induced reversible DNA methylation of the miR-200 loci with corresponding changes in miR-200 levels. Collectively, these findings demonstrate the existence of an autocrine TGF-β/ZEB/miR-200 signaling network that regulates plasticity between epithelial and mesenchymal states. We find a strong correlation between ZEBs and TGF-β and negative correlations between miR-200 and TGF-β and between miR-200 and ZEBs, in invasive ductal carcinomas, consistent with an autocrine TGF-β/ZEB/miR-200 signaling network being active in breast cancers.
PMCID: PMC3093321  PMID: 21411626
5.  Changes in Chromatin Accessibility Across the GM-CSF Promoter upon T Cell Activation Are Dependent on Nuclear Factor κB Proteins 
Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a key cytokine in myelopoiesis and aberrant expression is associated with chronic inflammatory disease and myeloid leukemias. This aberrant expression is often associated with constitutive nuclear factor (NF)-κB activation. To investigate the relationship between NF-κB and GM-CSF transcription in a chromatin context, we analyzed the chromatin structure of the GM-CSF gene in T cells and the role of NF-κB proteins in chromatin remodeling. We show here that chromatin remodeling occurs across a region of the GM-CSF gene between −174 and +24 upon T cell activation, suggesting that remodeling is limited to a single nucleosome encompassing the proximal promoter. Nuclear NF-κB levels appear to play a critical role in this process. In addition, using an immobilized template assay we found that the ATPase component of the SWI/SNF chromatin remodeling complex, brg1, is recruited to the GM-CSF proximal promoter in an NF-κB–dependent manner in vitro. These results suggest that chromatin remodeling across the GM-CSF promoter in T cells is a result of recruitment of SWI/SNF type remodeling complexes by NF-κB proteins binding to the CD28 response region of the promoter.
PMCID: PMC2193861  PMID: 12591900
transcription; chromatin remodeling; Brg1; cytokine; RelA
7.  The Impact of CpG Island on Defining Transcriptional Activation of the Mouse L1 Retrotransposable Elements 
PLoS ONE  2010;5(6):e11353.
L1 retrotransposable elements are potent insertional mutagens responsible for the generation of genomic variation and diversification of mammalian genomes, but reliable estimates of the numbers of actively transposing L1 elements are mostly nonexistent. While the human and mouse genomes contain comparable numbers of L1 elements, several phylogenetic and L1Xplore analyses in the mouse genome suggest that 1,500–3,000 active L1 elements currently exist and that they are still expanding in the genome. Conversely, the human genome contains only 150 active L1 elements. In addition, there is a discrepancy among the nature and number of mouse L1 elements in L1Xplore and the mouse genome browser at the UCSC and in the literature. To date, the reason why a high copy number of active L1 elements exist in the mouse genome but not in the human genome is unknown, as are the potential mechanisms that are responsible for transcriptional activation of mouse L1 elements.
Methodology/Principal Findings
We analyzed the promoter sequences of the 1,501 potentially active mouse L1 elements retrieved from the GenBank and L1Xplore databases and evaluated their transcription factors binding sites and CpG content. To this end, we found that a substantial number of mouse L1 elements contain altered transcription factor YY1 binding sites on their promoter sequences that are required for transcriptional initiation, suggesting that only a half of L1 elements are capable of being transcriptionally active. Furthermore, we present experimental evidence that previously unreported CpG islands exist in the promoters of the most active TF family of mouse L1 elements. The presence of sequence variations and polymorphisms in CpG islands of L1 promoters that arise from transition mutations indicates that CpG methylation could play a significant role in determining the activity of L1 elements in the mouse genome.
A comprehensive analysis of mouse L1 promoters suggests that the number of transcriptionally active elements is significantly lower than the total number of full-length copies from the three active mouse L1 families. Like human L1 elements, the CpG islands and potentially the transcription factor YY1 binding sites are likely to be required for transcriptional initiation of mouse L1 elements.
PMCID: PMC2894050  PMID: 20613872
8.  Histone Dynamics on the Interleukin-2 Gene in Response to T-Cell Activation 
Molecular and Cellular Biology  2005;25(8):3209-3219.
Several models have been proposed for the mechanism of chromatin remodelling across the promoters of inducible genes in mammalian cells. The most commonly held model is one of cooccupation where histone proteins are modified by acetylation or phosphorylation and nucleosomes are remodelled, allowing the assembly of transcription factor complexes. Using chromatin immunoprecipitation, we observed an apparent decrease of histone acetylation and phosphorylation signals at the proximal promoter region of the inducible interleukin-2 and granulocyte-macrophage colony-stimulating factor genes in response to T-cell activation. We showed that this apparent decrease was due to a loss of histone H3 and H4 proteins corresponding to a decrease in nucleosome occupation of the promoter. This histone loss is reversible; it is dependent on the continual presence of appropriate activating signals and transcription factors and is not dependent on the acetylation status of the histone proteins. These data show for the first time that histone proteins are lost from a mammalian promoter upon activation of transcription and support a model of activation-dependent disassembly and reassembly of nucleosomes.
PMCID: PMC1069623  PMID: 15798206
9.  GM-CSF promoter chromatin remodelling and gene transcription display distinct signal and transcription factor requirements 
Nucleic Acids Research  2005;33(1):225-234.
Granulocyte-macrophage colony stimulating factor (GM-CSF) plays a key role in myeloid cell function and is rapidly and transiently expressed in T cells in response to immune or inflammatory stimuli. Induction of GM-CSF gene expression is accompanied by changes in chromatin structure across the proximal promoter region of the gene. We show that the promoter remodelling and subsequent gene transcription occurs with distinct signal and transcription factor requirements. Activation of the protein kinase C (PKC) signalling pathway is sufficient to induce changes in chromatin structure across the promoter, but both the PKC and calcium signalling pathways are required for efficient gene transcription. Although NFAT transcription factors contribute to GM-CSF gene transcription, they are not required for promoter remodelling. However, the presence of the nuclear factor-κB transcription factor, c-Rel, in the nucleus is strongly correlated with and required for the events of chromatin remodelling.
PMCID: PMC546149  PMID: 15647505

Results 1-9 (9)