Specificity protein 1 (Sp1) maintains basal epithelial expression of the miR-200 family: implications for epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is required for the specification of tissues during embryonic development and is recapitulated during the metastatic progression of tumors. The miR-200 family plays a critical role in enforcing the epithelial state with their expression lost in cells undergoing EMT. EMT can be mediated by activation of the ZEB1 and ZEB2 (ZEB) transcription factors, which repress miR-200 expression via a self-reinforcing double negative feedback loop to promote the mesenchymal state. However, it remains unclear what factors drive and maintain epithelial-specific expression of miR-200 in the absence of EMT-inducing factors. Here, we show that the transcription factor Specificity Protein 1 (Sp1) binds to the miR-200b∼200a∼429 proximal promoter and activates miR-200 expression in epithelial cells. In mesenchymal cells, Sp1 expression is maintained, but its ability to activate the miR-200 promoter is perturbed by ZEB-mediated repression. Reduction of Sp1 expression caused changes in EMT-associated markers in epithelial cells. Furthermore, we observed co-expression of Sp1 and miR-200 during mouse embryonic development wherein miR-200 expression was only lost in regions with high ZEB expression. Together, these findings indicate that miR-200 family members require Sp1 to drive basal expression and to maintain an epithelial state.

Identification of an enhancer that increases miR-200b~200a~429 gene expression in breast cancer cells.

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.

Epigenetic modulation of the miR-200 family is associated with transition to a breast cancer stem-cell-like state.

The miR-200 family is a key regulator of the epithelial-mesenchymal transition, however, its role in controlling the transition between cancer stem-cell-like and non-stem-cell-like phenotypes is not well understood. We utilized immortalized human mammary epithelial (HMLE) cells to investigate the regulation of the miR-200 family during their conversion to a stem-like phenotype. HMLE cells were found to be capable of spontaneous conversion from a non-stem to a stem-like phenotype and this conversion was accompanied by the loss of miR-200 expression. Stem-like cell fractions isolated from metastatic breast cancers also displayed loss of miR-200 indicating similar molecular changes may occur during breast cancer progression. The phenotypic change observed in HMLE cells was directly controlled by miR-200 because restoration of its expression decreased stem-like properties while promoting a transition to an epithelial phenotype. Investigation of the mechanisms controlling miR-200 expression revealed both DNA methylation and histone modifications were significantly altered in the stem-like and non-stem phenotypes. In particular, in the stem-like phenotype, the miR-200b-200a-429 cluster was silenced primarily through polycomb group-mediated histone modifications whereas the miR-200c-141 cluster was repressed by DNA methylation. These results indicate that the miR-200 family plays a crucial role in the transition between stem-like and non-stem phenotypes and that distinct epigenetic-based mechanisms regulate each miR-200 gene in this process. Therapy targeted against miR-200 family members and epigenetic modifications might therefore be applicable to breast cancer.

Hedgehog-responsive candidate cell of origin for diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive tumors of childhood that are almost universally fatal. Our understanding of this devastating cancer is limited by a dearth of available tissue for study and by the lack of a faithful animal model. Intriguingly, DIPGs are restricted to the ventral pons and occur during a narrow window of middle childhood, suggesting dysregulation of a postnatal neurodevelopmental process. Here, we report the identification of a previously undescribed population of immunophenotypic neural precursor cells in the human and murine brainstem whose temporal and spatial distributions correlate closely with the incidence of DIPG and highlight a candidate cell of origin. Using early postmortem DIPG tumor tissue, we have established in vitro and xenograft models and find that the Hedgehog (Hh) signaling pathway implicated in many developmental and oncogenic processes is active in DIPG tumor cells. Modulation of Hh pathway activity has functional consequences for DIPG self-renewal capacity in neurosphere culture. The Hh pathway also appears to be active in normal ventral pontine precursor-like cells of the mouse, and unregulated pathway activity results in hypertrophy of the ventral pons. Together, these findings provide a foundation for understanding the cellular and molecular origins of DIPG, and suggest that the Hh pathway represents a potential therapeutic target in this devastating pediatric tumor.

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.

Epigenetic chromatin states uniquely define the developmental plasticity of murine hematopoietic stem cells.

Heritable epigenetic signatures are proposed to serve as an important regulatory mechanism in lineage fate determination. To investigate this, we profiled chromatin modifications in murine hematopoietic stem cells, lineage-restricted progenitors, and CD4(+) T cells using modified genome-scale mini-chromatin immunoprecipitation technology. We show that genes involved in mature hematopoietic cell function associate with distinct chromatin states in stem and progenitor cells, before their activation or silencing upon cellular maturation. Many lineage-restricted promoters are associated with bivalent histone methylation and highly combinatorial histone modification patterns, which may determine their selective priming of gene expression during lineage commitment. These bivalent chromatin states are conserved in mammalian evolution, with a particular overrepresentation of promoters encoding key regulators of hematopoiesis. After differentiation into progenitors and T cells, activating histone modifications persist at transcriptionally repressed promoters, suggesting that these transcriptional programs might be reactivated after lineage restriction. Collectively, our data reveal the epigenetic framework that underlies the cell fate options of hematopoietic stem cells.

Self-renewal of the long-term reconstituting subset of hematopoietic stem cells is regulated by Ikaros.

Hematopoietic stem cells (HSCs) are rare, ancestral cells that underlie the development, homeostasis, aging, and regeneration of the blood. Here we show that 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 that cannot be rescued with an ectopic Notch signal in vitro, with hematopoietic cells preferentially committing to the NK lineage. Althoughdispensable 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.

Evaluation of the long-term reconstituting subset of hematopoietic stem cells with CD150.

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(-) HSCs is CD150(+). HSCs sourced from the fetal liver show LT, multilineage engraftment from E14.5 onward, 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 show 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.

Automated microfluidic chromatin immunoprecipitation from 2,000 cells.

Chromatin immunoprecipitation (ChIP) is a powerful assay used to probe DNA-protein interactions. Traditional methods of implementing this assay are lengthy, cumbersome and require a large number of cells, making it difficult to study rare cell types such as certain cancer and stem cells. We have designed a microfluidic device to perform sensitive ChIP analysis on low cell numbers in a rapid, automated fashion while preserving the specificity of the assay. Comparing ChIP results for two modified histone protein targets, we showed our automated microfluidic ChIP (AutoChIP) from 2,000 cells to be comparable to that of conventional ChIP methods using 50,000-500,000 cells. This technology may provide a solution to the need for a high sensitivity, rapid, and automated ChIP assay, and in doing so facilitate the use of ChIP for many interesting and valuable applications.

JunB protects against myeloid malignancies by limiting hematopoietic stem cell proliferation and differentiation without affecting self-renewal.

Loss of the JunB/AP-1 transcription factor induces a myeloproliferative disease (MPD) arising from the hematopoietic stem cell (HSC) compartment. Here, 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-beta 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.

Pioneer factor interactions and unmethylated CpG dinucleotides mark silent tissue-specific enhancers in embryonic stem cells.

Recent studies have suggested that, in ES cells, inactive genes encoding early developmental regulators possess bivalent histone modification domains and are therefore poised for activation. However, bivalent domains were not observed at typical tissue-specific genes. Here, we show that windows of unmethylated CpG dinucleotides and putative pioneer factor interactions mark enhancers for at least some tissue-specific genes in ES cells. The unmethylated windows expand in cells that express the gene and contract, disappear, or remain unchanged in nonexpressing tissues. However, in ES cells, they do not always coincide with common histone modifications. Genomic footprinting and chromatin immunoprecipitation demonstrated that transcription factor binding underlies the unmethylated windows at enhancers for the Ptcra and Alb1 genes. After stable integration of premethylated Ptcra enhancer constructs into the ES cell genome, the unmethylated windows readily appeared. In contrast, the premethylated constructs remained fully methylated and silent after introduction into Ptcra-expressing thymocytes. These findings provide initial functional support for a model in which pioneer factor interactions in ES cells promote the assembly of a chromatin structure that is permissive for subsequent activation, and in which differentiated tissues lack the machinery required for gene activation when these ES cell marks are absent. The enhancer marks may therefore represent important features of the pluripotent state.

Epigenetic characterization of hematopoietic stem cell differentiation using miniChIP and bisulfite sequencing analysis.

Hematopoietic stem cells (HSC) produce all blood cell lineages by virtue of their capacity to self-renew and differentiate into progenitors with decreasing cellular potential. Recent studies suggest that epigenetic mechanisms play an important role in controlling stem cell potency and cell fate decisions. To investigate this hypothesis in HSC, we have modified the conventional chromatin immunoprecipitation assay allowing for the analysis of 50,000 prospectively purified stem and progenitor cells. Together with bisulfite sequencing analysis, we found that methylated H3K4 and AcH3 and unmethylated CpG dinucleotides colocalize across defined regulatory regions of lineage-affiliated genes in HSC. These active epigenetic histone modifications either accumulated or were replaced by increased DNA methylation and H3K27 trimethylation in committed progenitors consistent with gene expression. We also observed bivalent histone modifications at a lymphoid-affiliated gene in HSC and downstream transit-amplifying progenitors. Together, these data support a model in which epigenetic modifications serve as an important mechanism to control HSC multipotency.

Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy.

The major myeloid blood cell lineages are generated from hematopoietic stem cells by differentiation through a series of increasingly committed progenitor cells. Precise characterization of intermediate progenitors is important for understanding fundamental differentiation processes and a variety of disease states, including leukemia. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin, and CD41. Our studies revealed a hierarchy of myeloerythroid progenitors with distinct lineage potentials. The global gene expression signatures of these subsets were consistent with their functional capacities, and hierarchical clustering analysis suggested likely lineage relationships. These studies provide valuable tools for understanding myeloid lineage commitment, including isolation of an early erythroid-restricted precursor, and add to existing models of hematopoietic differentiation by suggesting that progenitors of the innate and adaptive immune system can separate late, following the divergence of megakaryocytic/erythroid potential.

The human IL-2 gene promoter can assemble a positioned nucleosome that becomes remodeled upon T cell activation.

Controlled production of the cytokine IL-2 plays a key role in the mammalian immune system. Expression from the gene is tightly regulated with no detectable expression in resting T cells and a strong induction following T cell activation. The IL-2 proximal promoter (+1 to -300) contains many well-defined transcriptional activation elements that respond to T cell stimulation. To determine the role of chromatin structure in the regulation of interleukin-2 gene transcription, nucleosome assembly across the IL-2 promoter region was examined using in vitro chromatin reconstitution assays. The IL-2 promoter assembles a nucleosome that is both translationally and rotationally positioned, spanning some of the major functional control elements. The binding of transcription factors to these elements, with the exception of the architectural protein HMGA1, was occluded by the presence of the nucleosome. Analysis of the chromatin architecture of the IL-2 gene in Jurkat T cells provided evidence for the presence of a similarly positioned nucleosome in vivo. The region encompassed by this nucleosome becomes remodeled following activation of Jurkat T cells. These observations suggest that the presence of a positioned nucleosome across the IL-2 proximal promoter may play an important role in maintaining an inactive gene in resting T cells and that remodeling of this nucleosome is important for gene activation.