Louise N. Winteringham

MADM, a Novel Adaptor Protein That Mediates Phosphorylation of the 14-3-3 Binding Site of Myeloid Leukemia Factor 1

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3ζ and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3ζ via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3ζ, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

Myeloid Leukemia Factor 1 inhibits erythropoietin-induced differentiation, cell cycle exit and p27Kip1 accumulation

Myeloid leukemia factor 1 (MLF1) is a novel oncoprotein involved in translocations associated with acute myeloid leukemia (AML), especially erythroleukemias. In this study, we demonstrate that ectopic expression of Mlf1 prevented J2E erythroleukemic cells from undergoing biological and morphological maturation in response to erythropoietin (Epo). We show that Mlf1 inhibited Epo-induced cell cycle exit and suppressed a rise in the cell cycle inhibitor p27Kip1. Unlike differentiating J2E cells, Mlf1-expressing cells did not downregulate Cul1 and Skp2, components of the ubiquitin E3 ligase complex SCFSkp2 involved in the proteasomal degradation of p27Kip1. In contrast, Mlf1 did not interfere with increases in p27Kip1 and terminal differentiation initiated by thyroid hormone withdrawal from erythroid cells, or cytokine-stimulated maturation of myeloid cells. These data demonstrate that Mlf1 interferes with an Epo-responsive pathway involving p27Kip1 accumulation, which inhibits cell cycle arrest essential for erythroid terminal differentiation.

An integrated expression atlas of miRNAs and their promoters in human and mouse

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.

Myeloid leukemia factor 1 associates with a novel heterogeneous nuclear ribonucleoprotein U-like molecule

Myeloid leukemia factor 1 (MLF1) is an oncoprotein associated with hemopoietic lineage commitment and acute myeloid leukemia. Here we show that Mlf1 associated with a novel binding partner, Mlf1-associated nuclear protein (Manp), a new heterogenous nuclear ribonucleoprotein (hnRNP) family member, related to hnRNP-U. Manp localized exclusively in the nucleus and could redirect Mlf1 from the cytoplasm into the nucleus. The nuclear content of Mlf1 was also regulated by 14-3-3 binding to a canonical 14-3-3 binding motif within the N terminus of Mlf1. Significantly Mlf1 contains a functional nuclear export signal and localized primarily to the nuclei of hemopoietic cells. Mlf1 was capable of binding DNA, and microarray analysis revealed that it affected the expression of several genes, including transcription factors. In summary, this study reveals that Mlf1 translocates between nucleus and cytoplasm, associates with a novel hnRNP, and influences gene expression.