Aberrant expression of the human homeobox-containing proto-oncogene TLX1/HOX11 inhibits hematopoietic differentiation programs in a number of murine model systems. Here, we report the establishment of a murine erythroid progenitor cell line, iEBHX1S-4, developmentally arrested by regulatable TLX1 expression. Extinction of TLX1 expression released the iEBHX1S-4 differentiation block, allowing erythropoietin-dependent acquisition of erythroid markers and hemoglobin synthesis. Coordinated activation of erythroid transcriptional networks integrated by the acetyltransferase co-activator CREB-binding protein (CBP) was suggested by bioinformatic analysis of the upstream regulatory regions of several conditionally induced iEBHX1S-4 gene sets. In accord with this notion, CBP-associated acetylation of GATA-1, an essential regulator of erythroid differentiation, increased concomitantly with TLX1 downregulation. Coimmunoprecipitation experiments and glutathione-S-transferase pull-down assays revealed that TLX1 directly binds to CBP, and confocal laser microscopy demonstrated that the two proteins partially colocalize at intranuclear sites in iEBHX1S-4 cells. Notably, the distribution of CBP in conditionally blocked iEBHX1S-4 cells partially overlapped with chromatin marked by a repressive histone methylation pattern, and downregulation of TLX1 coincided with exit of CBP from these heterochromatic regions. Thus, we propose that TLX1-mediated differentiation arrest may be achieved in part through a mechanism that involves redirection of CBP and/or its sequestration in repressive chromatin domains.

The diverged TLX1/HOX11 homeobox gene is frequently transcriptionally activated in T-cell acute lymphoblastic leukemia as a result of two recurrent chromosomal translocations or by as yet unknown mechanisms. Sporadic expression of TLX1 has also been detected in some other human tumors, raising the possibility that TLX1 played a role in the etiology of these malignancies. In this context, it is noteworthy that TLX1 is aberrantly expressed in the human erythroleukemia cell line K562. We previously reported that enforced TLX1 expression immortalizes myeloid progenitors in murine bone marrow and erythroid progenitors originating from murine embryonic stem cell-derived embryoid bodies. Based on these findings, we speculated that dysregulated TLX1 expression contributes to neoplastic transformation by interfering with hematopoietic differentiation programs. Here we carried out genome-wide expression profiling on these model systems to elucidate the mechanism of TLX1-mediated differentiation arrest. Surprisingly, these investigations uncovered a latent erythroid phenotype of the TLX1+ bone marrow progenitor cell lines. Transcriptome comparison with murine GATA-1-null G1E-ER4 erythroblast cells (GEO Accession No. GDS568) showed nonrandom overlap (P = 2.3 × 10−5) with a set of genes that immediately respond to GATA-1 activation. Included among the genes for which a positive correlation was observed were the erythropoietin (Epo) receptor and several functionally-associated downstream signaling components such as Lyn and Dok-1, as well as the SCL and FOG-1 transcription factor genes. We confirmed the biological relevance of these findings by demonstrating that TLX1-immortalized bone marrow progenitors proliferated in response to erythropoietin, synthesizing beta-globin mRNA. We next extended this approach to iEBHX1S-4 cells, an embryoid body-derived cell line generated by conditional (doxycycline-controlled) TLX1 expression (manuscript in preparation). iEBHX1S-4 cells require IL-3 plus stem cell factor for survival and proliferation. However, when doxycycline is removed from the culture medium, the cells undergo Epo-dependent erythroid differentiation characterized by up-regulation of the TER119 surface antigen and hemoglobin synthesis. Interestingly, whereas there was statistically significant overlap of gene expression profiles between constitutive and conditional TLX1+ cells (P = 2.5 × 10−5), there were no significant similarities between the iEBHX1S-4 transcriptome and the G1E-ER4 dataset until 6–24 hours after release of the TLX1 differentiation block. Bioinformatics analysis of the upstream regulatory regions of the genes identified implicated transcriptional networks involving GATA-1 as well as the p53, NF-kappaB and Egr-1 transcription factors. All of these transcription factors are substrates of the acetyltransferase CREB-binding protein (CBP), suggesting that inappropriate TLX1 expression might interfere with CBP activity. In agreement with this notion, acetylation of GATA-1, a key substrate of CBP-modulated erythroid differentiation, was increased upon down-regulation of TLX1 expression in iEBHX1S-4 cells. Experiments to directly confirm TLX1 inhibition of CBP as a central facet of TLX1 transforming function are ongoing, the results of which will be presented.

A flow cytometric procedure has recently been described to isolate hematopoietic stem cells from mouse bone marrow based on the efflux properties of the vital dye Hoechst 33342. The assay defines a subset of cells-termed the "side population" (SP)-by simultaneously measuring fluorescence of the dye at two wavelengths (~450 nm and >670 nm). In this chapter, SP protocols are provided to detect candidate hematopoietic stem cells in mouse bone marrow and human cord blood. In the standard method, SP profiles are readily observed on a stream-in-air cell sorter using 30 mW of 351-356 nm ultraviolet excitation from a krypton-ion laser. Alternatively, SP profiles can be resolved on an analytical flow cytometer with cuvette flow cell using 8 mW of 325-nm ultraviolet excitation from a helium-cadmium laser. The ability to perform the SP assay on an analytical instrument facilitates optimization of staining conditions to identify hematopoietic and other stem cells in a variety of tissues. It is also demonstrated that SP profiles of slightly lower resolution can be obtained on a stream-in-air cell sorter using 100 mW of 407-nm violet excitation from a krypton-ion laser, raising the possibility that with appropriate validation the SP assay could be performed on flow cytometers that are not equipped with ultraviolet lasers.

Reporters based on the green fluorescent protein (GFP) from the jellyfish Aequorea victoria and GFP-like proteins from other marine organisms provide valuable tools to monitor gene transfer and expression noninvasively in living cells. Stable cell lines were generated from the Sp2/0-Ag14 hybridoma that express up to three spectral enhanced versions of GFP, the enhanced cyan fluorescent protein (ECFP), the enhanced green fluorescent protein (EGFP), and the enhanced yellow fluorescent protein (EYFP), and/or a variant of the Discosoma coral red fluorescent protein (DsRed). The panel of lines was used to demonstrate a flow cytometric procedure for simultaneous analysis of all four fluorescent proteins that utilizes dual-laser excitation at 488 nm and 407 nm. Additional schemes for simultaneous detection of two, three or four of these fluorescent proteins are also presented.