This article reviews the regulation of production of RBCs at several levels. We focus on the regulated expansion of burstforming unit-erythroid erythroid progenitors by glucocorticoids and other factors that occur during chronic anemia, inflammation, and other conditions of stress.We also highlight the rapid production of RBCs by the coordinated regulation of terminal proliferation and differentiation of committed erythroid colony-forming unit-erythroid progenitors by external signals, such as erythropoietin and adhesion to a fibronectin matrix. We discuss the complex intracellular networks of coordinated gene regulation by transcription factors, chromatin modifiers, and miRNAs that regulate the different stages of erythropoiesis. (Blood. 2011;118(24): 6258-6268) IntroductionIn mammals, definitive erythropoiesis first occurs in the fetal liver with progenitor cells from the yolk sac. 1 Within the fetal liver and the adult bone marrow, hematopoietic cells are formed continuously from a small population of pluripotent stem cells that generate progenitors committed to one or a few hematopoietic lineages (Figure 1). In the erythroid lineage, the earliest committed progenitors identified ex vivo are the slowly proliferating burstforming unit-erythroid (BFU-E). Early BFU-E cells divide and further differentiate through the mature BFU-E stage into rapidly dividing colony-forming unit-erythroid (CFU-E). 2 CFU-E progenitors divide 3 to 5 times over 2 to 3 days as they differentiate and undergo many substantial changes, including a decrease in cell size, chromatin condensation, and hemoglobinization, leading up to their enucleation and expulsion of other organelles. 3 In humans, the life span of RBCs is 120 days. Under normal conditions, approximately 1% of RBCs are synthesized each day but RBC production can increase substantially during times of acute or chronic stress, such as acute trauma or hemolysis. Exquisite short-term control of erythropoiesis is regulated by the kidney-derived cytokine erythropoietin (Epo), which is induced under hypoxic conditions and stimulates the terminal proliferation and differentiation of CFU-E progenitors. 4 BFU-E cells respond to many hormones in addition to Epo, including SCF, insulin like growth factor 1 (IGF-1), glucocorticoids (GCs), and IL-3, and IL-6. In cases of chronic erythroid stress, such as hemolysis, the number of CFU-E progenitors is insufficient to produce the needed RBCs, even under high Epo levels, and the body responds by producing more of these progenitors from BFU-E. 5 It is not entirely known which cells in the fetal liver or adult bone marrow produce these and other regulatory cytokines, or how they interact to regulate the division of BFU-E cells and control their self-renewal and their ability to differentiate into more mature CFU-E progenitors.At each stage of RBC production, intracellular signal transduction proteins and transcription factors activated downstream of these hormones interact with a group of DNA-binding and other transcription factors and chromati...

Oncogenic mutations of the MLL histone methyltransferase confer an unusual ability to transform non-self-renewing myeloid progenitors into leukemia stem cells (LSCs) by mechanisms that remain poorly defined. Misregulation of Hox genes is likely to be critical for LSC induction and maintenance but alone it does not recapitulate the phenotype and biology of MLL leukemias, which are clinically heterogeneouspresumably reflecting differences in LSC biology and/or frequency. TALE (three-amino-acid loop extension) class homeodomain proteins of the Pbx and Meis families are also misexpressed in this context, and we thus employed knockout, knockdown, and dominant-negative genetic techniques to investigate the requirements and contributions of these factors in MLL oncoprotein-induced acute myeloid leukemia. Our studies show that induction and maintenance of MLL transformation requires Meis1 and is codependent on the redundant contributions of Pbx2 and Pbx3. Meis1 in particular serves a major role in establishing LSC potential, and determines LSC frequency by quantitatively regulating the extent of self-renewal, differentiation arrest, and cycling, as well as the rate of in vivo LSC generation from myeloid progenitors. Thus, TALE proteins are critical downstream effectors within an essential homeoprotein network that serves a rate-limiting regulatory role in MLL leukemogenesis.[Keywords: Leukemia stem cells; MLL; Meis1; Pbx; TALE homeodomain proteins] Supplemental material is available at http://www.genesdev.org.

Differential expression of Hox genes is associated with normal hematopoiesis, whereas inappropriate maintenance of Hox gene expression, particularly Hoxa7 and Hoxa9, is a feature of leukemias harboring mixed-lineage leukemia (MLL) mutations. To understand the pathogenic roles of Hox genes in MLL leukemias, we assessed the impact of Hoxa7 or Hoxa9 nullizygosity on hematopoietic progenitor compartments and their susceptibility to MLL-induced leukemias. Selective reductions in the absolute numbers of committed progenitors, but not of hematopoietic stem cells, distinguished Hoxa7-and Hoxa9-deficient mice. Megakaryocytic/ erythroid progenitor (MEP) reductions in Hoxa7 ؊/؊ mice correlated with reticulocytosis and thrombocytopenia without anemia. Conversely, Hoxa9 ؊/؊ mice displayed marked lymphopenia and substantial reductions of common lymphoid progenitors (CLPs) and lymphoid precursors, in addition to significant reductions of common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs). In retroviral transduction/transplantation assays, Hoxa7-and Hoxa9-deficient progenitors remained susceptible to transformation by MLL-GAS7, which activates MLL through a dimerization-dependent mechanism. However, Hoxa7 ؊/؊ or Hoxa9 ؊/؊ progenitors were less efficient in generating transformed blast colony-forming units (CFUs) in vitro and induced leukemias with longer disease latencies, reduced penetrance, and less mature phenotypes. Thus, Hoxa7 and Hoxa9 contribute to hematopoietic progenitor homeostasis but are not necessary for MLL-GAS7-mediated leukemogenesis, yet they appear to affect disease latency, penetrance, and phenotypes consistent with their critical roles as downstream targets of MLL fusion proteins. IntroductionThe mixed-lineage leukemia (MLL) gene is a frequent target of chromosomal translocations that result in its structural rearrangement in 10% of adult and 80% of infant acute leukemias. MLL rearrangements induce its promiscuous fusion to more than 30 different partner proteins, resulting in leukemias of different lineages. 1 Others 2 and we 3 have shown that heterologous fusion with transcriptional effector domains constitutes a common mechanism for oncogenic activation of MLL by a subset of its fusion partners. Recently, an alternative pathway was reported showing MLL is oncogenically activated by dimerization domains contributed by another subset of its fusion partners that do not display inherent transactivation properties. 4 Hematopoietic cells transformed by either category of MLL fusion protein consistently express a subset of Hox genes, which are candidate downstream effectors of MLL oncogenic activity. 4,5 These findings are also consistent with studies of infant leukemias showing that expression of specific Hox genes (in particular HoxA9 and HoxA7) and the Hox cofactor Meis1 are up-regulated in MLL leukemias. [6][7][8][9] Hox genes are implicated in normal and abnormal hematopoiesis. 10,11 Differential expression of Hox genes is closely associated with specific lineages and stages of h...

It is unclear how epigenetic changes regulate the induction of erythroid-specific genes during terminal erythropoiesis. Here we use global mRNA sequencing (mRNA-seq) and chromatin immunoprecipitation coupled to high-throughput sequencing (CHIP-seq) to investigate the changes that occur in mRNA levels, RNA polymerase II (Pol II) occupancy, and multiple posttranslational histone modifications when erythroid progenitors differentiate into late erythroblasts. Among genes induced during this developmental transition, there was an increase in the occupancy of Pol II, the activation marks H3K4me2, H3K4me3, H3K9Ac, and H4K16Ac, and the elongation methylation mark H3K79me2. In contrast, genes that were repressed during differentiation showed relative decreases in H3K79me2 levels yet had levels of Pol II binding and active histone marks similar to those in erythroid progenitors. We also found that relative changes in histone modification levels, in particular, H3K79me2 and H4K16ac, were most predictive of gene expression patterns. Our results suggest that in terminal erythropoiesis both promoter and elongation-associated marks contribute to the induction of erythroid genes, whereas gene repression is marked by changes in histone modifications mediating Pol II elongation. Our data map the epigenetic landscape of terminal erythropoiesis and suggest that control of transcription elongation regulates gene expression during terminal erythroid differentiation. (Blood. 2011; 118(16):e128-e138)

Despite advances in defining the critical molecular determinants for leukemia stem cell (LSC) generation and maintenance, little is known about the roles of microRNAs in LSC biology. Here, we identify microRNAs that are differentially expressed in LSC-enriched cell fractions (c-kit + ) in a mouse model of MLL leukemia.Members of the miR-17 family were notably more abundant in LSCs compared with their normal counterpart granulocyte-macrophage progenitors and myeloblast precursors. Expression of miR-17 family microRNAs was substantially reduced concomitant with leukemia cell differentiation and loss of self-renewal, whereas forced expression of a polycistron construct encoding miR-17-19b miRNAs significantly shortened the latency for MLL leukemia development. Leukemias expressing increased levels of the miR-17-19b construct displayed a higher frequency of LSCs, more stringent block of differentiation, and enhanced proliferation associated with reduced expression of p21, a cyclin-dependent kinase inhibitor previously implicated as a direct target of miR-17 microRNAs. Knockdown of p21 in MLL-transformed cells phenocopied the overexpression of the miR-17 polycistron, including a significant decrease in leukemia latency, validating p21 as a biologically relevant and direct in vivo target of the miR-17 polycistron in MLL leukemia. Expression of c-myc, a crucial upstream regulator of the miR-17 polycistron, correlated with miR-17-92 levels, enhanced self-renewal, and LSC potential. Thus, microRNAs quantitatively regulate LSC self-renewal in MLL-associated leukemia in part by modulating the expression of p21, a known regulator of normal stem cell function. Cancer Res; 70(9); 3833-42. ©2010 AACR.