Deletion of Mtg16, a Target of t(16;21), Alters Hematopoietic Progenitor Cell Proliferation and Lineage Allocation

Abstract

Chromosomal translocations disrupt master regulatory genes that control cellular proliferation, apoptosis, and the lineage decisions of progenitor cells (25,53). Indeed, a critical component in the development of acute leukemia is the shunting of stem cells or multipotent progenitor cells toward a specific lineage, which also must acquire the ability to self-renew, to give rise to a specific form of acute myeloid leukemia (AML). The myeloid translocation gene on chromosome 16 (MTG16, also known as ETO-2 or CBFA2T3) and myeloid translocation gene on chromosome 8 (MTG8, also known as Eight-Twenty-one or ETO) are disrupted by t(16;21) and t(8;21), respectively. While t(8; 21) accounts for as much as 10 to 15% of de novo AML (11, 12, 16, 17, 30, 43, 44), t(16;21) is relatively rare. Yet, the analysis of genes involved in rare translocations is often highly informative (35). In addition to mutations in acute leukemia, MTG8 was identified as a candidate cancer gene in colorectal carcinoma and mutations in MTG16 were also found in the initial screen (59). Moreover, MTG16 may also be inactivated in breast cancer (31). Thus, the analysis of the normal physiological functions of the MTG family is critical to our understanding of the development of acute leukemia and how mutation of MTG family members contributes to tumorigenesis in other organ systems.The chromosomal translocation fusion proteins created at the breakpoints of t(8;21) and t(16;21) repress the transcription of tumor suppressor genes and genes that are required for hematopoietic differentiation (34). This is achieved by recruiting histone deacetylases and other transcriptional corepressors through their MTG8 or MTG16 sequences. Indeed, MTG family members associate with N-CoR/SMRT; mSin3A/3B; and histone deacetylases 1, 2, and 3 (2,18,23,36,63). MTG family members are recruited by DNA binding factors involved in chromosomal translocations (PLZF and BCL6) and by other regulators of hematopoiesis (e.g., TAL1/SCL, Gfi1, Gfi1b, and HEB) (9,20,39,40,55,68). Thus, the cumulative data suggest that the MTG/ETO family members function as transcriptional corepressors whose activities are coopted by chromosomal translocations to induce leukemia.Gene disruption strategies have been valuable to dissect the regulatory pathways and identify the critical factors that mediate the decision of a stem cell to self-renew and quiesce or to enter the rapidly expanding progenitor cell pool to populate the various hematopoietic cell lineages. Many of these key regulators are DNA binding transcription factors, which control gene expression programs to influence proliferation and differentiation. By contrast, only a limited number of the transcriptional regulators and chromatin remodeling factors that are recruited by DNA binding factors have been pinpointed as contributors to stem cell functions. This is especially true for transcriptional corepressors and gene silencing factors. Although a great deal of information has been gathered about the molecular interactions of the MTG famil...

Mtg16/Eto2 Contributes to Murine T-Cell Development

“…In addition, stem cells lacking E2A showed a considerable defect in competitive repopulation assays (43,47). Likewise, Mtg16 Ϫ/Ϫ mice show defective stress erythropoiesis with few erythropoietic progenitors, as measured by colony forming assays (10), and defects in stem cell competitiveness (M. Fischer et al, unpublished). Chromatin immunoprecipitation assays also suggest that the association between Mtg16 and E proteins plays an important role in the action of these factors during hematopoiesis, as the most robust signal for Mtg16 was found near E-protein binding sites (M .…”

“…Generation of Mtg16-null mice was previously described (10). For studies described here, Mtg16-null mice were backcrossed into the C57BL/6 background for 10 generations.…”

Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification

“…pCMV-Flag-NCoR, pCMVFlag-Sin3a, pCMV-Flag-HDAC3, MIG-myc-MTG16, Gal-TK-luciferase, and Hes1-luciferase have been previously described (3,10,13,23,25,43). To produce GST fusion proteins of MTG16 NHR domains, each domain, with its N-terminal and C-terminal flanking sequences, was isolated from the corresponding pCMV(M2)-Gal-NHR expression plasmid as an XbaI fragment, filled in with Klenow and subcloned into SmaI-linearized pGEX4T3 to generate pGEX4T3-NHR1, pGEX4T3-NHR2, pGEX4T3-NHR3, and pGEX4T3-NHR4.…”

“…ϩ (LSK) hematopoietic progenitors were purified from total bone marrow cells of Mtg16 ϩ/ϩ and Mtg16 Ϫ/Ϫ mice as described previously (10), and mRNA was purified using the 5 Prime PerfectPure kit per the manufacturer's instructions. RNA was reverse transcribed and the resulting cDNA amplified by real-time PCR in a Bio-Rad iCycler instrument, using appropriate Hes1 .0]) supplemented with 3% Casamino Acids, disrupted by sonication, and after 30 min of rocking at 4°C, clarified by centrifugation.…”

“…More recently, mutations or altered expression at MTG loci have been described in nonhematologic malignancies, such as ductal carcinoma of the breast and colorectal cancer, reinforcing their essential contributions to cell growth and development (27,47,49). Mice with constitutive deletion of Mtg16 display defects in hematopoietic stem/progenitor cell functions (10). Inactivation of Mtg16 skews hematopoietic progenitors toward the granulocyte/macrophage lineage and impairs megakaryocytic-erythroid progenitor cell expansion in response to hemolytic stress.…”