The transcription factors E2A and HEB act in concert to induce the expression of FOXO1 in the common lymphoid progenitor.

Recent studies have identified a number of transcriptional regulators, including E proteins, EBF1, FOXO1, and PAX5, that act together to orchestrate the B-cell fate. However, it still remains unclear as to how they are linked at the earliest stages of B-cell development. Here, we show that lymphocyte development in HEB-ablated mice exhibits a partial developmental arrest, whereas B-cell development in E2A(+/-)HEB(-/-) mice is completely blocked at the LY6D(-) common lymphoid progenitor stage. We show that the transcription signatures of E2A- and HEB-ablated common lymphoid progenitors significantly overlap. Notably, we found that Foxo1 expression was substantially reduced in the LY6D(-) HEB- and E2A-deficient cells. Finally, we show that E2A binds to enhancer elements across the FOXO1 locus to activate Foxo1 expression, linking E2A and FOXO1 directly in a common pathway. In summary, the data indicate that the earliest event in B-cell specification involves the induction of FOXO1 expression and requires the combined activities of E2A and HEB.

Factors and networks that underpin early hematopoiesis.

Multiple trajectories have recently been described through which hematopoietic progenitor cells travel prior to becoming lineage-committed effectors. A wide spectrum of transcription factors has recently been identified that modulate developmental progression along such trajectories. Here we describe how distinct families of transcription factors act and are linked together to orchestrate early hematopoiesis.

Multilineage priming of enhancer repertoires precedes commitment to the B and myeloid cell lineages in hematopoietic progenitors.

Recent studies have documented genome-wide binding patterns of transcriptional regulators and their associated epigenetic marks in hematopoietic cell lineages. In order to determine how epigenetic marks are established and maintained during developmental progression, we have generated long-term cultures of hematopoietic progenitors by enforcing the expression of the E-protein antagonist Id2. Hematopoietic progenitors that express Id2 are multipotent and readily differentiate upon withdrawal of Id2 expression into committed B lineage cells, thus indicating a causative role for E2A (Tcf3) in promoting the B cell fate. Genome-wide analyses revealed that a substantial fraction of lymphoid and myeloid enhancers are premarked by the poised or active enhancer mark H3K4me1 in multipotent progenitors. Thus, in hematopoietic progenitors, multilineage priming of enhancer elements precedes commitment to the lymphoid or myeloid cell lineages.

E2A proteins maintain the hematopoietic stem cell pool and promote the maturation of myelolymphoid and myeloerythroid progenitors.

Hematopoiesis is a tightly controlled process maintained by a small pool of hematopoietic stem cells (HSCs). Here, we demonstrate that the LT-HSC, MPP, premegakaryocytic/erythroid, Pre CFU-E, Pre GM, MkP, and granulocyte-macrophage compartments were all significantly reduced in E2A-deficient bone marrow. Despite a severe depletion of erythroid progenitors, the erythrocyte and megakaryocyte compartments were equivalent in E2A-deficient bone marrow as compared with wild-type mice. E2A-deficient HSCs also failed to efficiently maintain the HSC pool on serial transplantation, and we demonstrate that the E2A proteins regulate cell cycle progression of HSCs by regulating the expression of p21(Cip1), p27(Kip1), and the thrombopoietin receptor, known regulators of HSC self-renewal activity. Based on these observations, we propose that the E2A proteins promote the developmental progression of the entire spectrum of early hematopoietic progenitors and to suppress an erythroid specific program of gene expression in alternative cell lineages. Last, the data mechanistically link E2A, cell cycle regulators, and the maintenance of the HSC pool in a common pathway.

Microfluidic sorting of mammalian cells by optical force switching.

Microfluidic-based devices have allowed miniaturization and increased parallelism of many common functions in biological assays; however, development of a practical technology for microfluidic-based fluorescence-activated cell sorting has proved challenging. Although a variety of different physical on-chip switch mechanisms have been proposed, none has satisfied simultaneously the requirements of high throughput, purity, and recovery of live, unstressed mammalian cells. Here we show that optical forces can be used for the rapid (2-4 ms), active control of cell routing on a microfluidic chip. Optical switch controls reduce the complexity of the chip and simplify connectivity. Using all-optical switching, we have implemented a fluorescence-activated microfluidic cell sorter and evaluated its performance on live, stably transfected HeLa cells expressing a fused histone-green fluorescent protein. Recovered populations were verified to be both viable and unstressed by evaluation of the transcriptional expression of two genes, HSPA6 and FOS, known indicators of cellular stress.

Use of moving optical gradient fields for analysis of apoptotic cellular responses in a chronic myeloid leukemia cell model.

To facilitate quantitation of cellular apoptotic responses to various antineoplastic agents, a laser-based technology, Optophoresis, has been developed to provide analysis of cells without any need for labeling or cell processing. Optophoresis is defined as the analysis of the motion of cells, where the motion is either induced or modified by a moving optical gradient field, which produces radiation pressure forces on the cells in an aqueous suspension. Quantitation of the induced motion provides a basis for distinguishing one population of cells from another. One Optophoretic technique, Fast Scan, measures the distribution of distances traversed by a population of cells when exposed to a fast-moving optical gradient. Fast Scan was validated using a cell-based model of chronic myeloid leukemia treated with Gleevec, a specific inhibitor of aberrant Bcr-Abl protein kinase. The Optophoretic measurements were quantitatively comparable to reference assays with regard to drug selectivity and potency and to target specificity, demonstrating the suitability of this technology for pharmaceutical and clinical applications.