14 research outputs found
Characterisation of the leukaemia-associated ETO homologues
Acute myeloid leukemia (AML) is commonly associated with balanced chromosomal translocations. Characteristically, these translocations lead to the fusion of two unrelated genes, resulting in the expression of an aberrant fusion protein. t(8;21) is one of the most common translocations found in patients with AML. It results in the expression of the chimeric protein AML1-ETO. AML1 is a transcription factor of crucial importance during hematopoiesis. The function of the fusion partner eight-twenty-one (ETO) is much less understood. The aim of this thesis was to characterise ETO and its two homologues, myeloid translocation gene 16 (MTG16) and myeloid translocation gene related protein 1 (MTGR1), to elucidate their role in normal and disregulated hematopoiesis. We studied the interaction patterns of the ETO homologues as well as their expression pattern in hematopoietic cells. We also examined the consequences of upregulation or downregulation of the proteins. We found that all the ETO homologues as well as AML1-ETO can interact with each other as determined by IP-Western experiments. We also found that the ETO homologues, but not AML1-ETO can bind to the corepressor SIN3B. The proposed interactions of the ETO homologues might have implications for the onset of leukaemia, since it opens up for an AML1-ETO mediated disturbance of ETO homologue function as well as a regulation of AML1-ETO function by the ETO homologues. Examination of the expression patterns of ETO homologues in hematopoetic cells show that the expression of ETO is restricted to erythroid cells, suggesting a role for ETO in erythropoiesis. MTG16 and MTGR1 are ubiquitously expressed in hematopoietic cells. However, the expression of MTG16 decresases during erythroid and granulocytic differentiation, suggesting a role for MTG16 in early hematopoiesis. Furthermore, the differential expression of the ETO homologues in hematopoetic cells implies a specific function for each protein in hematopoiesis. Attempts to knock-down MTG16 show a discrepancy between RNA levels and protein levels,which could propose a mechanism to keep the expression of MTG16 constant. Finally, overexpression experiments indicate a role for ETO in proliferation and apoptosis
Interactions between the leukaemia-associated ETO homologues of nuclear repressor proteins.
The eight-twenty-one (ETO) homologues, represented by ETO, myeloid transforming gene-related protein 1 (MTGR1) and myeloid transforming gene chromosome 16 (MTG16), are nuclear repressor proteins. ETO is part of the fusion protein acute myeloid leukaemia (AML)1-ETO, resulting from the translocation (8;21). Similarly, MTG16 is disrupted to become part of AML1/MTG16 in t(16;21). The aberrant expression of these chimeras could affect interplay between ETO homologues and contribute to the leukaemogenic process. We investigated possible interactions between the ETO homologues. Ectopic co-expression in COS-cells resulted in heterodimerisation of the various ETO homologues suggesting that they may co-operate. Similarly, the chimeric oncoprotein AML1-ETO interacted with both MTGR1 and MTG16. However, results from cell lines endogenously expressing more than one ETO homologue did not demonstrate co-precipitation. Results from IP-Western and size determination by gel filtration of deletion mutants expressed in COS-cells, indicated an important role of the HHR domain for oligomerisation. A role was also suggested for the Nervy domain in the homologue interactions. Our results suggest that ETO homologues can interact with each other as well as with AML1-ETO, although it is unclear as to what extent these interactions occur in vivo
The Leukemia-associated ETO homologues are differently expressed during hematopoietic differentiation.
The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues-6
Ing with DAPI. The third column shows the localization of individual proteins. The fourth column shows nucleolar localization of the B23 nucleolar marker. The fifth column shows a merge of B23 and individual proteins. hSIN3B (I) and all the ETO homologues (II – IV) show colocalization with B23. AML1-ETO lacks colocalization with B23 (V). Size bars measuring 10 μM are shown in panel II.<p><b>Copyright information:</b></p><p>Taken from "The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues"</p><p>http://www.biomedcentral.com/1471-2199/9/8</p><p>BMC Molecular Biology 2008;9():8-8.</p><p>Published online 19 Jan 2008</p><p>PMCID:PMC2266940.</p><p></p
The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues-2
5) in combination with hSIN3B (S) as described in Materials and Methods. IP and Western were performed with α-V5 (V5 specific) and α-S (hSIN3B specific). () ETO and MTG16 co-precipitated hSIN3B (Lanes 1 and 7 of A respectively). Reciprocal experiments showed that hSIN3B co-precipitated ETO (Lanes 2 and 8 of B respectively). Arrowhead shows the position of ETO in A and MTGR1 in B. MTGR1 failed to co-precipitate hSIN3B (Lane 4 of A). The reciprocal experiment showed that hSIN3B did not precipitate MTGR1 (Lane 5 of B). Lower panels in A and B show input of hSIN3B and ETO homologue in 2% of IP lysate. The positions of the molecular weight markers are indicated at the left.<p><b>Copyright information:</b></p><p>Taken from "The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues"</p><p>http://www.biomedcentral.com/1471-2199/9/8</p><p>BMC Molecular Biology 2008;9():8-8.</p><p>Published online 19 Jan 2008</p><p>PMCID:PMC2266940.</p><p></p
The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues-0
. Middle panels represent negative controls, wherein RT-PCR was carried out without cDNA. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an endogenous control (Lower panels). () Tissue expression of hSIN3B and ETO homologues as measured by real-time PCR. Relative transcript levels were calculated after normalizing with the G3PDH (see materials and methods). Three independent experiments, each in triplicate were performed and error bars show the Standard Deviation (S.D.).<p><b>Copyright information:</b></p><p>Taken from "The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues"</p><p>http://www.biomedcentral.com/1471-2199/9/8</p><p>BMC Molecular Biology 2008;9():8-8.</p><p>Published online 19 Jan 2008</p><p>PMCID:PMC2266940.</p><p></p
The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues-8
Ing. The α-MTG antibody reacts with all ETO homologues. . Localization of hSIN3B and ETO homologues in the nucleolus. The first column of each panel shows the Nomarski index. The second column shows nuclear staining with DAPI. The third column shows the the B23 nucleolar marker. The fourth column shows nucleolar localization of individual proteins. The fifth column shows a merge of B23 and individual protein. hSIN3B (I) and the ETO homologues (II), MTGR1 (III) and MTG16 (IV) showed colocalization with the B23. . Colocalization of hSIN3B and MTG16 or MTGR1 in the nucleolus. Columns are designed as in B. Size bars of 10 μM are shown in panel I of part B and C.<p><b>Copyright information:</b></p><p>Taken from "The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues"</p><p>http://www.biomedcentral.com/1471-2199/9/8</p><p>BMC Molecular Biology 2008;9():8-8.</p><p>Published online 19 Jan 2008</p><p>PMCID:PMC2266940.</p><p></p
The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues-1
On with hSIN3B (S). Cell lysates were analyzed by IP-Western, as described in Materials and Methods. IP and Western were performed with α-E (ETO specific), α-R (MTGR1 specific), α-16 (MTG16 specific) and α-S (hSIN3B specific). () ETO and MTG16 co-precipitated hSIN3B (lane 2 of A and C). Reciprocal experiments showed that hSIN3B co-precipitated ETO and MTG16 (lane 5 of A; lane 6 of C). MTGR1 did not co-precipitate hSIN3B or vice-a-versa (lanes 2 and 5 of B). Arrowheads show the position of hSIN3B corresponding to approximately 135 kDa and ETO corresponding to approximately 75 kDa (lanes 2 and 4). () AML1-ETO failed to co-precipitate hSIN3B (lane 2). The reciprocal experiment with the same lysates showed that hSIN3B did not precipitate AML1-ETO (lane 5). The size of AML1-ETO is about 100 kDa. Control experiments showed that none of the antibodies bound unspecifically. Lower panel shows the input of hSIN3B and ETO homologue in 2% of IP lysate. The positions of the molecular weight markers are indicated at the left.<p><b>Copyright information:</b></p><p>Taken from "The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues"</p><p>http://www.biomedcentral.com/1471-2199/9/8</p><p>BMC Molecular Biology 2008;9():8-8.</p><p>Published online 19 Jan 2008</p><p>PMCID:PMC2266940.</p><p></p
The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues-4
Cental cells as described in Material and Methods. The following antibodies were used: α-hSIN3B (SIN), α-ETO (ETO), α-MTGR1 (R1), α-MTG16 (16) and α-MTG (MTG, reactive with all ETO homologues). Arrowhead shows the position of hSIN3B and ETO homologues. The blots were re-probed with pre-immune serum (PIS) in order to rule out non-specific binding of the antibodies. The blots were probed with α-actin to show equal loading. IP-Western was used to examine the presence of complexes between hSIN3B and the ETO homologues in nuclear extracts. IP with α-MTG pulled down hSIN3B as detected on immunoblotting with α-SIN3B (SIN) (lane 2). In the reverse experiment, IP with α-SIN3B (SIN) pulled down ETO as detected on immunoblotting with α-ETO (ETO) (lane 4). However, IP with α-SIN3B (SIN) did not pull down MTGR1 or MTG16 as no signal was detected upon immunoblotting with α-MTGR1 or MTG16 (lanes 5–8). Lower panel shows input of MTGR1 and MTG16 in 2% of IP lysate. The positions of the molecular weight markers are indicated at the left.<p><b>Copyright information:</b></p><p>Taken from "The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues"</p><p>http://www.biomedcentral.com/1471-2199/9/8</p><p>BMC Molecular Biology 2008;9():8-8.</p><p>Published online 19 Jan 2008</p><p>PMCID:PMC2266940.</p><p></p
The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues-7
Lyzed with immunofluorescence microscopy. ETO and hSIN3B colocalized in nuclear bodies (I), in the matrix of the nucleolus (II) and at the periphery of the nucleolus (III). MTGR1 and hSIN3B were observed separately in nuclear particles and in nuclear bodies respectively (I), but alternatively both were seen at the periphery of nuclear particles (II) or lacked colocalization (III). MTG16 and hSIN3B colocalized in nuclear bodies (I), in the matrix of the nucleolus (II) and at the periphery of the nucleolus (III).AML1-ETO was observed in the nucleus only whereas hSIN3B was seen in the nucleolus (I – III). Size bars of 10 μM are shown in panel I of part A.<p><b>Copyright information:</b></p><p>Taken from "The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues"</p><p>http://www.biomedcentral.com/1471-2199/9/8</p><p>BMC Molecular Biology 2008;9():8-8.</p><p>Published online 19 Jan 2008</p><p>PMCID:PMC2266940.</p><p></p