Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene produce chimeric proteins that cause abnormal expression of a subset of HOX genes and leukemia development. Here, we show that MLL normally regulates expression of mir-196b, a hematopoietic microRNA located within the HoxA cluster , in a pattern similar to that of the surrounding 5 Hox genes, Hoxa9 and Hoxa10, during embryonic stem (ES) cell differentiation. Within the hematopoietic lineage, mir-196b is most abundant in short-term hematopoietic stem cells and is down-regulated in more differentiated hematopoietic cells. Leukemogenic MLL fusion proteins cause overexpression of mir-196b, while treatment of MLL-AF9 transformed bone marrow cells with mir-196-specific antagomir abrogates their replating potential in methylcellulose. This demonstrates that mir-196b function is necessary for MLL fusion-mediated im-mortalization. Furthermore, overexpres-sion of mir-196b was found specifically in patients with MLL associated leukemias as determined from analysis of 55 primary leukemia samples. Overexpression of mir-196b in bone marrow progenitor cells leads to increased proliferative capacity and survival, as well as a partial block in differentiation. Our results suggest a mechanism whereby increased expression of mir-196b by MLL fusion proteins significantly contributes to leukemia development. (Blood. 2009;113:3314-3322) Introduction The Mixed Lineage Leukemia (MLL) gene is commonly involved in chromosome translocations that cause leukemia. 1,2 MLL-associated leukemias can be myeloid, lymphoid or biphenotypic, depending on the partner gene to which MLL is fused. 3 There have been more than 60 different MLL fusion partners isolated to date and, in most cases, overexpression of a subset of HOX genes is a hallmark of the disease. 4 HOX genes are transcription factors that play an important role during development and hematopoiesis. 5,6 Humans have 13 paralogous groups of HOX genes clustered on 4 different chromosomes. Expression of HOX genes is spatially and temporally regulated with 3 genes expressed earlier and having a more anterior boundary of expression. 5 Similarly, expression of HOX genes is tightly regulated during hemato-poiesis. Genes located at the 3 end of the cluster are down-regulated as CD34 cells become lineage-specific progenitors while 5 genes, like HOXA10, are turned off only after cells progress to the more differentiated CD34 stage. 7 MLL regulates expression of some of the HOX genes at the chromatin level by binding to the promoters and recruiting various transcriptional regulators. 8-10 However, what happens at the molecular level in the presence of the leukemogenic fusion proteins to cause HOX overexpression is still poorly understood. Among 6800 genes analyzed by expression microarrays, overex-pression of HOXA9 was the most correlative marker of poor prognosis in acute myeloid leukemia patients. 11 Immortalization of bone marrow progenitors by the MLL fusion protein MLL-ENL is dependent on the presence of Hoxa9 and Hoxa7ge...

The cellular mechanisms that modulate the redox state of p53 tumor suppressor remain unclear, although its DNA binding function is known to be strongly inhibited by oxidative and nitrosative stresses. We show that human p53 is subjected to a new and reversible posttranslational modification, namely, S-glutathionylation in stressed states, including DNA damage. First, a rapid and direct incorporation of biotinylated GSH or GSSG into the purified recombinant p53 protein was observed. The modified p53 had a significantly weakened ability to bind its consensus DNA sequence. Reciprocal immunoprecipitations and a GST overlay assay showed that p53 in tumor cells was marginally glutathionylated; however, the level of modification increased greatly after oxidant and DNA-damaging treatments. GSH modification coexisted with the serine phophorylations in activated p53, and the thiol-conjugated protein was present in nuclei. When tumor cells treated with camptothecin or cisplatin were subsequently exposed to glutathione-enhancing agents, p53 underwent dethiolation accompanied by detectable increases in the level of p21waf1 expression, relative to the DNA-damaging drugs alone. Mass spectrometry of GSH-modified p53 protein identified cysteines 124, 141, and 182, all present in the proximal DNA-binding domain, as the sites of glutathionylation. Biotinylated maleimide also reacted rapidly with Cys141, implying that this is the most reactive cysteine on the p53 surface. The glutathionylatable cysteines were found to exist in a negatively charged microenvironment in cellular p53. Molecular modeling studies located Cys124 and -141 at the dimer interface of p53 and showed glutathionylation of either residue would inhibit p53-DNA association and also interfere with protein dimerization. These results show for the first time that shielding of reactive cysteines contributes to a negative regulation for human p53 and imply that such an inactivation of the transcription factor may represent an acute defensive response with significant consequences for oncogenesis.