Sacchi, N.

Loss of MTG16a (CBFA2T3), a novel rDNA repressor, leads to increased ribogenesis and disruption of breast acinar morphogenesis [J Cell Mol Med., 14 (2010), 1358-70] (Article)

2010-08-01T00:00:00Z

Loss of MTG16a (CBFA2T3), a novel rDNA repressor, leads to increased ribogenesis and disruption of breast acinar morphogenesis (Article)

2010-06-01T00:00:00Z

Human MTG16a (CBFA2T3), a chromatin repressor with nucleolar localization, was described to act as a suppressor of breast tumourigenesis. Here we show that MTG16a is a novel ribosomal gene repressor, which can counteract MYC-driven activation of ribosomal RNA (rRNA) transcription. We also show that either knocking down MTG16a by RNA interference, or sequestering MTG16a outside the nucleolus of human breast epithelial cells, hampers acinar morphogenesis concomitant with up-regulation of rRNA synthesis and increased ribogenesis. This is the first demonstration that loss of MTG16a function in the nucleolus of breast epithelial cells can induce morphological and molecular changes typical of breast cancer initiation. © 2009 The Authors Journal compilation

Novel RNA-binding properties of the MTG chromatin regulatory proteins (Article)

2008-10-24T00:00:00Z

Background: The myeloid translocation gene (MTG) proteins are non-DNA-binding transcriptional regulators capable of interacting with chromatin modifying proteins. As a consequence of leukemia-associated chromosomal translocations, two of the MTG proteins, MTG8 and MTG16, are fused to the DNA-binding domain of AML1, a transcriptional activator crucial for hematopoiesis. The AML1-MTG fusion proteins, as the wild type MTGs, display four conserved homology regions (NHR1-4) related to the Drosophila nervy protein. Structural protein analyses led us to test the hypothesis that specific MTG domains may mediate RNA binding. Results: By using an RNA-binding assay based on synthetic RNA homopolymers and a panel of MTG deletion mutants, here we show that all the MTG proteins can bind RNA. The RNA-binding properties can be traced to two regions: the Zinc finger domains in the NHR4, which mediate Zinc-dependent RNA binding, and a novel short basic region (SBR) upstream of the NHR2, which mediates Zinc-independent RNA binding. The two AML1-MTG fusion proteins, retaining both the Zinc fingers domains and the SBR, also display RNA-binding properties. Conclusion: Evidence has been accumulating that RNA plays a role in transcriptional control. Both wild type MTGs and chimeric AML1-MTG proteins display in vitro RNA-binding properties, thus opening new perspectives on the possible involvement of an RNA component in MTG-mediated chromatin regulation.

A distinct epigenetic signature at targets of a leukemia protein (Article)

2007-02-19T00:00:00Z

Background: Human myelogenous leukemia characterized by either the non random t(8; 21)(q22; q22) or t(16; 21)(q24; q22) chromosome translocations differ for both their biological and clinical features. Some of these features could be consequent to differential epigenetic transcriptional deregulation at AML1 targets imposed by AML1-MTG8 and AML1-MTG16, the fusion proteins deriving from the two translocations. Preliminary findings showing that these fusion proteins lead to transcriptional downregulation of AML1 targets, marked by repressive chromatin changes, would support this hypothesis. Here we show that combining conventional global gene expression arrays with the power of bioinformatic genomic survey of AML1-consensus sequences is an effective strategy to identify AML1 targets whose transcription is epigenetically downregulated by the leukemia-associated AML1-MTG16 protein. Results: We interrogated mouse gene expression microarrays with probes generated either from 32D cells infected with a retroviral vector carrying AML1-MTG16 and unable of granulocyte differentiation and proliferation in response to the granulocyte colony stimulating factor (G-CSF), or from 32D cells infected with the cognate empty vector. From the analysis of differential gene expression alone (using as criteria a p value < 0.01 and an absolute fold change > 3), we were unable to conclude which of the 37 genes downregulated by AML1-MTG16 were, or not, direct AML1 targets. However, when we applied a bioinformatic approach to search for AML1-consensus sequences in the 10 Kb around the gene transcription start sites, we closed on 17 potential direct AML1 targets. By focusing on the most significantly downregulated genes, we found that both the AML1-consensus and the transcription start site chromatin regions were significantly marked by aberrant repressive histone tail changes. Further, the promoter of one of these genes, containing a CpG island, was aberrantly methylated. Conclusion: This study shows that a leukemia-associated fusion protein can impose a distinct epigenetic repressive signature at specific sites in the genome. These findings strengthen the conclusion that leukemia-specific oncoproteins can induce non-random epigenetic changes.

Oligomerization properties of fragile-X mental-retardation protein (FMRP) and the fragile-X-related proteins FXR1P and FXR2P (Article)

1999-01-01T00:00:00Z

The absence of fragile-X mental-retardation protein (FMRP) results in fragile-X syndrome. Two other fragile-X-related (FXR) proteins have been described, FXR1P and FXR2P, which are both very similar in amino acid sequence to FMRP. Interaction between the three proteins as well as with themselves has been demonstrated. The FXR proteins are believed to play a role in RNA metabolism. To characterize a possible functional role of the interacting proteins the complex formation of the FXR proteins was studied in mammalian cells. Double immunofluorescence analysis in COS cells over-expressing either FMRP ISO12/FXR1P or FMRP ISO12/FXR2P confirmed heterotypic interactions. However, Western-blotting studies on cellular homogenates containing physiological amounts of the three proteins gave different indications. Gel-filtration experiments under physiological as well as EDTA conditions showed that the FXR proteins were in complexes of >600 kDa, as parts of messenger ribonuclear protein (mRNP) particles associated with polyribosomes. Salt treatment shifted FMRP, FXR1P and FXR2P into distinct intermediate complexes, with molecular masses between 200 and 300 kDa. Immunoprecipitations of FMRP as well as FXR1P from the dissociated complexes revealed that the vast majority of the FXR proteins do not form heteromeric complexes. Further analysis by [(35)S]methionine labelling in vivo followed by immunoprecipitation indicated that no proteins other than the FXR proteins were present in these complexes. These results suggest that the FXR proteins form homo-multimers preferentially under physiological conditions in mammalian cells, and might participate in mRNP particles with separate functions.