IntroductionTranscriptional regulation of major hematopoietic oncogenes and tumor suppressor genes represents a critical step in tumor formation, tumor aggressiveness, and therapy resistance. [1][2][3] In addition, a posttranscriptional inhibitory mechanism involving microRNA (miRNA) binding to the 3Ј-untranslated region of target mRNAs causes transcript degradation or interferes with the translation initiation and has been linked to tumorigenesis. 4,5 Under physiologic conditions, miRNAs regulate developmental processes and cell fate decisions, and tight regulation of their levels represents an important factor in cell and tissue homeostasis. 6 MiR-155, a well-studied miRNA, regulates hematopoietic cell development as documented by murine gene targeting experiments and also by other studies describing its function during immune B-and T-cell response, in production of cytokines and antibodies and in antigen presentation. 7,8 Next, transgenic miR-155 overexpression in the mouse stimulates B-cell proliferation and frequent development of lymphomas. 9 In humans, miR-155 up-regulation has been repeatedly reported in chronic B-cell lymphocytic leukemia (B-CLL), in its solid indolent form of a small lymphocytic lymphoma [10][11][12] and also in aggressive types, including non-Hodgkin 10,13,14 and Hodgkin lymphomas. 13,15 Deregulation of several microRNAs was repeatedly described in B-CLL. 16,17 B-CLL, the most common adult leukemia, is characterized by clonal accumulation of B celllike mature-appearing elements (Ͼ 5000/L) 18 typically coexpressing the CD5, CD19, CD20, and CD23 surface markers. B-CLL represents a heterogeneous disease, the outcome of which may be predicted by the levels of surface protein CD38, intracellular tyrosine kinase ZAP70, or by a status of IgV H somatic hypermutation. 18,19 Cytogenetic alterations of 2 loci that contain the p53 gene (deletion of 17p) and the ATM gene (deletion of 11q) are associated with poor prognosis, shorter duration of remission, and shortest overall survival, 20 whereas normal karyotype or trisomy 12 is considered intermediate risk and the 13q14 deletion is considered a favorable mark. Subsets of B-CLL patients may progress to non-Hodgkin diffuse large B-cell lymphoma by a mechanism that remains largely unknown. Taken together, miR-155 appears to play a central role in B-cell function, and its up-regulation in lymphoproliferative disorders, including B-CLL, may lead to a block of differentiation and accumulation of lymphoid-like cells.Recent studies brought evidence of a context-dependent transcriptional regulation of the MIR155HG. First, oncogenic properties of miR-155 have been demonstrated in breast cancer cells where MIR155HG is up-regulated by transforming growth factor-␤/ Smad pathway involving a Smad response element at the position Ϫ454 nt from the transcription start site (TSS). 21 This regulatory pathway becomes disabled on inhibition of miR-155, resulting in derepression of miR-155 targets (including the RhoA protein) and in decreased cell migration and invasio...

Epigenetic 5-azacitidine (AZA) therapy of high-risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) represents a promising, albeit not fully understood, approach. Hematopoietic transcription factor PU.1 is dynamically regulated by upstream regulatory element (URE), whose deletion causes downregulation of PU.1 leading to AML in mouse. In this study a significant group of the high-risk MDS patients, as well as MDS cell lines, displayed downregulation of PU.1 expression within CD34 þ cells, which was associated with DNA methylation of the URE. AZA treatment in vitro significantly demethylated URE, leading to upregulation of PU.1 followed by derepression of its transcriptional targets and onset of myeloid differentiation. Addition of colony-stimulating factors (CSFs; granulocyte-CSF, granulocyte --macrophage-CSF and macrophage-CSF) modulated AZA-mediated effects on reprogramming of histone modifications at the URE and cell differentiation outcome. Our data collectively support the importance of modifying the URE chromatin structure as a regulatory mechanism of AZA-mediated activation of PU.1 and induction of the myeloid program in MDS.

CCCTC-binding factor (CTCF) can both activate as well as inhibit transcription by forming chromatin loops between regulatory regions and promoters. In this regard, Ctcf binding on non-methylated DNA and its interaction with the Cohesin complex results in differential regulation of the H19/Igf2 locus. Similarly, a role for CTCF has been established in normal hematopoietic development; however its involvement in leukemia remains elusive. Here, we show that Ctcf binds to the imprinting control region of H19/Igf2 in AML blasts. We also demonstrate that Smarca5, which also associates with the Cohesin complex, facilitates Ctcf binding to its target sites on DNA. Furthermore, Smarca5 supports Ctcf functionally and is needed for enhancer-blocking effect at ICR. We next asked whether CTCF and SMARCA5 control the expression of key hematopoiesis regulators. In normally differentiating myeloid cells both CTCF and SMARCA5 together with members of the Cohesin complex are recruited to the SPI1 gene, a key hematopoiesis regulator and leukemia suppressor. Due to DNA methylation, CTCF binding to the SPI1 gene is blocked in AML blasts. Upon AZA-mediated DNA demethylation of human AML blasts, CTCF and SMARCA5 are recruited to the −14.4 Enhancer of SPI1 gene and block its expression. Our data provide new insight into complex SPI1 gene regulation now involving additional key epigenetic factors, CTCF and SMARCA5 that control PU.1 expression at the −14.4 Enhancer.

Hematopoietic transcription factors GATA-1 and PU.1 bind each other on DNA to block transcriptional programs of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells that coexpress GATA-1 and PU.1 are blocked at the blast stage but respond to molecular removal (downregulation) of PU.1 or addition (upregulation) of GATA-1 by inducing terminal erythroid differentiation. To test whether GATA-1 blocks PU.1 in MEL cells, we have conditionally activated a transgenic PU.1 protein fused with the estrogen receptor ligand-binding domain (PUER), resulting in activation of a myeloid transcriptional program. Gene expression arrays identified components of the PU.1-dependent transcriptome negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein α (Cebpa) and core-binding factor, β subunit (Cbfb), which encode two key hematopoietic transcription factors. Inhibition of GATA-1 by small interfering RNA resulted in derepression of PU.1 target genes. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Significant derepression of Cebpa and Cbfb is achieved in MEL cells by either activation of PU.1 or knockdown of GATA-1. Furthermore, transcriptional regulation of these loci by manipulating the levels of PU.1 and GATA-1 involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3K9. Collectively, we show that either activation of PU.1 or inhibition of GATA-1 efficiently reverses the transcriptional block imposed by GATA-1 and leads to the activation of a myeloid transcriptional program directed by PU.1.