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1.  Identification of an Enhancer That Increases miR-200b~200a~429 Gene Expression in Breast Cancer Cells 
PLoS ONE  2013;8(9):e75517.
The miR-200b~200a~429 gene cluster is a key regulator of EMT and cancer metastasis, however the transcription-based mechanisms controlling its expression during this process are not well understood. We have analyzed the miR-200b~200a~429 locus for epigenetic modifications in breast epithelial and mesenchymal cell lines using chromatin immunoprecipitation assays and DNA methylation analysis. We discovered a novel enhancer located approximately 5.1kb upstream of the miR-200b~200a~429 transcriptional start site. This region was associated with the active enhancer chromatin signature comprising H3K4me1, H3K27ac, RNA polymerase II and CpG dinucleotide hypomethylation. Luciferase reporter assays revealed the upstream enhancer stimulated the transcription of the miR-200b~200a~429 minimal promoter region approximately 27-fold in breast epithelial cells. Furthermore, we found that a region of the enhancer was transcribed, producing a short, GC-rich, mainly nuclear, non-polyadenylated RNA transcript designated miR-200b eRNA. Over-expression of miR-200b eRNA had little effect on miR-200b~200a~429 promoter activity and its production did not correlate with miR-200b~200a~429 gene expression. While additional investigations of miR-200b eRNA function will be necessary, it is possible that miR-200b eRNA may be involved in the regulation of miR-200b~200a~429 gene expression and silencing. Taken together, these findings reveal the presence of a novel enhancer, which contributes to miR-200b~200a~429 transcriptional regulation in epithelial cells.
PMCID: PMC3783398  PMID: 24086551
2.  Self-Renewal of the Long-Term Reconstituting Subset of Hematopoietic Stem Cells is Regulated by Ikaros 
Stem Cells (Dayton, Ohio)  2009;27(12):3082-3092.
Hematopoietic stem cells (HSCs) are rare, ancestral cells that underlie the development, homeostasis, aging, and regeneration of the blood. Here we show the chromatin-associated protein Ikaros is a crucial self-renewal regulator of the long-term (LT) reconstituting subset of HSCs. Ikaros, and associated family member proteins, are highly expressed in self-renewing populations of stem cells. Ikaros point mutant mice initially develop LT-HSCs with the surface phenotype cKit+Thy1.1(lo)Lin(-/lo)Sca1+Flk2−CD150+ during fetal ontogeny but are unable to maintain this pool, rapidly losing it within two days of embryonic development. A synchronous loss of megakaryocyte/erythrocyte progenitors results, along with a fatal, fetal anemia. At this time, mutation of Ikaros exerts a differentiation defect upon common lymphoid progenitors which cannot be rescued with an ectopic Notch signal in vitro, with hematopoietic cells preferentially committing to the NK lineage. Whilst dispensable for the initial embryonic development of blood, Ikaros is clearly needed for maintenance of this tissue. Achieving successful clinical tissue regeneration necessitates understanding degeneration, and these data provide a striking example by a discrete genetic lesion in the cells underpinning tissue integrity during a pivotal timeframe of organogenesis.
PMCID: PMC3401049  PMID: 19816952
Hematopoietic stem cells; Ikaros; Fetal liver
3.  Evaluation of the Long-Term Reconstituting Subset of Hematopoietic Stem Cells with CD150 
Stem cells (Dayton, Ohio)  2009;27(10):2498-2508.
Blood is a tissue with a high cell turnover rate that is constantly being replenished by bone marrow hematopoietic stem cells (HSCs) seeded during fetal ontogeny from the liver. Here we show that the long-term (LT) reconstituting subset of cKit+Thy1.1(lo)Lin(-/lo)Sca1+Flk2- hematopoietic stem cells is CD150+. HSCs sourced from the fetal liver show long-term, multilineage engraftment from E14.5 onwards, and the CD150 cell surface molecule can readily substitute Thy1.1 as a positive marker of LT-HSCs in this tissue. From both fetal liver and adult bone marrow, cKit+Thy1.1(lo)Lin(-/lo)Sca1+Flk2-CD150+ cells exhibit robust LT competitive engraftment, self-renewal, multilineage differentiation capacity, and an accessible chromatin configuration consistent with high expression of erythroid/megakaryoid genes in purified cell subsets. Our data demonstrate that with appropriate combinations of cell surface markers, stem cells can be accurately isolated to high purity and characterized. This is important for the clarification of lineage relationships and the identification of bona fide regulators of stem cell self-renewal and differentiation, both in normal and neoplastic tissues.
PMCID: PMC2783507  PMID: 19593793
Hematopoietic stem cells; Fetal liver; CD150
4.  A Method to Study the Epigenetic Chromatin States of Rare Hematopoietic Stem and Progenitor Cells; MiniChIP–Chip 
Dynamic chromatin structure is a fundamental property of gene transcriptional regulation, and has emerged as a critical modulator of physiological processes during cellular differentiation and development. Analysis of chromatin structure using molecular biology and biochemical assays in rare somatic stem and progenitor cells is key for understanding these processes but poses a great challenge because of their reliance on millions of cells. Through the development of a miniaturized genome-scale chromatin immunoprecipitation method (miniChIP–chip), we have documented the genome-wide chromatin states of low abundant populations that comprise hematopoietic stem cells and immediate progeny residing in murine bone marrow. In this report, we describe the miniChIP methodology that can be used for increasing an understanding of the epigenetic mechanisms underlying hematopoietic stem and progenitor cell function. Application of this method will reveal the contribution of dynamic chromatin structure in regulating the function of other somatic stem cell populations, and how this process becomes perturbed in pathological conditions.
PMCID: PMC3396287  PMID: 21406121
Miniaturized chromatin immunoprecipitation assays; Microarray technology; Histone modifications; Stem and progenitor cells; Epigenetic regulation; Lineage commitment
5.  JunB protects against myeloid malignancies by limiting hematopoietic stem cell proliferation and differentiation without affecting self-renewal 
Cancer cell  2009;15(4):341-352.
Loss of the JunB/AP-1 transcription factor induces a myeloproliferative disease (MPD) arising from the hematopoietic stem cell (HSC) compartment. Now we show that JunB inactivation deregulates the cell cycle machinery and increases the proliferation of long-term repopulating HSCs (LT-HSCs) without impairing their self-renewal or regenerative potential in vivo. We found that JunB loss destabilizes a complex network of genes and pathways that normally limit myeloid differentiation, leading to impaired responsiveness to both Notch and TGF-β signaling due, in part, to transcriptional deregulation of the Hes1 gene. These results demonstrate that LT-HSC proliferation and differentiation are uncoupled from self-renewal, and establish some of the mechanisms by which JunB normally limits the production of myeloid progenitors hence preventing initiation of myeloid malignancies.
PMCID: PMC2669108  PMID: 19345332

Results 1-5 (5)