A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts

Cellular quiescence is characterized not only by reduced mitotic and metabolic activity but also by altered gene expression. Growing evidence suggests that quiescence is not merely a basal state but is regulated by active mechanisms. To understand the molecular programme that governs reversible cell cycle exit, we focused on quiescence-related gene expression in a culture model of myogenic cell arrest and activation. Here we report the identification of quiescence-induced genes using a gene-trap strategy. Using a retroviral vector, we generated a library of gene traps in C2C12 myoblasts that were screened for arrest-induced insertions by live cell sorting (FACS-gal). Several independent gene- trap lines revealed arrest-dependent induction of betagal activity, confirming the efficacy of the FACS screen.The locus of integration was identified in 15 lines. In three lines,insertion occurred in genes previously implicated in the control of quiescence, i.e. EMSY - a BRCA2--interacting protein, p8/com1 - a p300HAT -- binding protein and MLL5 - a SET domain protein. Our results demonstrate that expression of chromatin modulatory genes is induced in G0, providing support to the notion that this reversibly arrested state is actively regulated.     

Enhancer Activation by Pharmacologic Displacement of LSD1 from GFI1 Induces Differentiation in Acute Myeloid Leukemia

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Specificity of MLL1 and TET3 CXXC domains towards naturally occurring cytosine modifications

CXXC domains have traditionally been considered as CpG specific DNA binding domains that are repelled by cytosine modifications. This view has recently been challenged by the demonstration that CXXC domain of TET3 has relaxed sequence specificity and binds with the highest affinity to symmetric DNA duplex containing 5caCpG. Here, we present a comparative analysis of the MLL1-CXXC and TET3-CXXC sequence specificity and tolerance to cytosine modifications (5-methyl, 5-hydroxymethyl, 5-formyl, 5-carboxyl) in CpG and non-CpG context. For the first time, we take into consideration possible interference from cytosine bases elsewhere in the sequence. We show that despite similar overall structure, MLL1-CXXC has greater sequence and modification specificity than TET3-CXXC. MLL1-CXXC is specific only for CpG and does not tolerate any cytosine modifications. In contrast, TET3-CXXC does not require the CpG context of cytosine bases. Methyl-, formyl- and carboxyl-modifications are tolerated by TET3-CXXC, but only preceding G. Based on our and other data we propose a parsimonious model of MLL1-CXXC and TET3-CXXC DNA binding. This model explains why the binding of modified DNA duplexes by TET3-CXXC requires in some cases a register shift and is therefore context-dependent.     

Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia

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Epigenetic regulation of planarian stem cells by the SET1/MLL family of histone methyltransferases

Chromatin regulation is a fundamental mechanism underlying stem cell pluripotency, differentiation, and the establishment of cell type-specific gene expression profiles. To examine the role of chromatin regulation in stem cells in vivo, we study regeneration in the freshwater planarian Schmidtea mediterranea. These animals possess a high concentration of pluripotent stem cells, which are capable of restoring any damaged or lost tissues after injury or amputation. Here, we identify the S. mediterranea homologs of the SET1/MLL family of histone methyltransferases and COMPASS and COMPASS-like complex proteins and investigate their role in stem cell function during regeneration. We identified six S. mediterranea homologs of the SET1/MLL family (set1, mll1/2, trr-1, trr-2, mll5–1 and mll5–2), characterized their patterns of expression in the animal, and examined their function by RNAi. All members of this family are expressed in the stem cell population and differentiated tissues. We show that set1, mll1/2, trr-1, and mll5–2 are required for regeneration and that set1, trr-1 and mll5–2 play roles in the regulation of mitosis. Most notably, knockdown of the planarian set1 homolog leads to stem cell depletion. A subset of planarian homologs of COMPASS and COMPASS-like complex proteins are also expressed in stem cells and implicated in regeneration, but the knockdown phenotypes suggest that some complex members also function in other aspects of planarian biology. This work characterizes the function of the SET1/MLL family in the context of planarian regeneration and provides insight into the role of these enzymes in adult stem cell regulation in vivo.     

PARPi potentiates with current conventional therapy in MLL leukemia

Acute myeloid leukemias driven by MLL fusion proteins are commonly associated with poor prognosis and refractory treatment. Here, we provide evidence that olaparib can potentiate sensitivity of MLL leukemia cells to conventional chemotherapy and DNMT inhibitors offering new potential therapeutic strategies for MLL rearranged leukemias Using the primary mouse leukemia cells and human MLL-AF9 leukemic cell line, we demonstrate that treatment of MLL-AF9 leukemic cells with DNMT inhibitors or chemotherapies in combination with olaparib results in significant reduction in colony formation or cell growth while the single agent treatments had little impacts. Combining olaparib with DNMT inhibitor induce cell cycle block and apoptosis. Furthermore, we observe a significant increase in proportion of cells with >10 γH2AX DNA damage foci and double stranded breaks when treated with DNMT inhibitors or chemotherapy agents in combination with olaparib, thus providing possible mechanistic insights for the synergism.     

LKB1, Salt-Inducible Kinases,and MEF2C Are Linked Dependencies in Acute Myeloid Leukemia

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