Biomedical subjects

Patrick Rodriguez

Publications and source records attributed to Patrick Rodriguez.

8 recordsLinked to original sources

PubMed2006

The mediator complex functions as a coactivator for GATA-1 in erythropoiesis via subunit Med1/TRAP220.

The Mediator complex forms the bridge between transcriptional activators and RNA polymerase II. Mediator subunit Med1/TRAP220 is a key component of Mediator originally found to associate with nuclear hormone receptors. Med1 deficiency causes lethality at embryonic day 11.5 because of defects in heart and placenta development. Here we show that Med1-deficient 10.5 days postcoitum embryos are anemic but have normal numbers of hematopoietic progenitor cells. Med1-deficient progenitor cells have a defect in forming erythroid burst-forming units (BFU-E) and colony-forming units (CFU-E), but not in forming myeloid colonies. At the molecular level, we demonstrate that Med1 interacts physically with the erythroid master regulator GATA-1. In transcription assays, Med1 deficiency leads to a defect in GATA-1-mediated transactivation. In chromatin immunoprecipitation experiments, we find Mediator components at GATA-1-occupied enhancer sites. Thus, we conclude that Mediator subunit Med1 acts as a pivotal coactivator for GATA-1 in erythroid development.

Animals↗

PubMed2006

Novel binding partners of Ldb1 are required for haematopoietic development.

Ldb1, a ubiquitously expressed LIM domain binding protein, is essential in a number of tissues during development. It interacts with Gata1, Tal1, E2A and Lmo2 to form a transcription factor complex regulating late erythroid genes. We identify a number of novel Ldb1 interacting proteins in erythroleukaemic cells, in particular the repressor protein Eto-2 (and its family member Mtgr1), the cyclin-dependent kinase Cdk9, and the bridging factor Lmo4. MO-mediated knockdowns in zebrafish show these factors to be essential for definitive haematopoiesis. In accordance with the zebrafish results these factors are coexpressed in prehaematopoietic cells of the early mouse embryo, although we originally identified the complex in late erythroid cells. Based on the change in subcellullar localisation of Eto-2 we postulate that it plays a central role in the transition from the migration and expansion phase of the prehaematopoietic cells to the establishment of definitive haematopoietic stem cells.

Adaptor Proteins, Signal Transducing↗

PubMed2006

ETO2 coordinates cellular proliferation and differentiation during erythropoiesis.

The passage from proliferation to terminal differentiation is critical for normal development and is often perturbed in malignancies. To define the molecular mechanisms that govern this process during erythropoiesis, we have used tagging/proteomics approaches and characterized protein complexes nucleated by TAL-1/SCL, a basic helix-loop-helix transcription factor that specifies the erythrocytic lineage. In addition to known TAL-1 partners, GATA-1, E2A, HEB, LMO2 and Ldb1, we identify the ETO2 repressor as a novel component recruited to TAL-1 complexes through interaction with E2A/HEB. Ectopic expression and siRNA knockdown experiments in hematopoietic progenitor cells show that ETO2 actively represses erythroid TAL-1 target genes and governs the expansion of erythroid progenitors. At the onset of erythroid differentiation, a change in the stoichiometry of ETO2 within the TAL-1 complex activates the expression of known erythroid-specific TAL-1 target genes and of Gfi-1b and p21(Cip), encoding two essential regulators of erythroid cell proliferation. These results suggest that the dynamics of ETO2 recruitment within nuclear complexes couple cell proliferation to cell differentiation and determine the onset of terminal erythroid maturation.

Animals↗

PubMed2006

Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads.

Efficient tagging methodologies are an integral aspect of protein complex characterization by proteomic approaches. Owing to the very high affinity of biotin for avidin and streptavidin, biotinylation tagging offers an attractive approach for the efficient purification of protein complexes. The very high affinity of the biotin/(strept)avidin system also offers the potential for the single-step capture of lower abundance protein complexes, such as transcription factor complexes. The identification of short peptide tags that are efficiently biotinylated by the bacterial BirA biotin ligase led to an approach for the single-step purification of transcription factor complexes by specific in vivo biotinylation tagging. A short sequence tag fused N-terminally to the transcription factor of interest is very efficiently biotinylated by BirA coexpressed in the same cells, as was demonstrated by the tagging of the essential hematopoietic transcription factor GATA-1. The direct binding to streptavidin of biotinylated GATA-1 in nuclear extracts resulted in the single-step capture of the tagged factor and associated proteins, which were eluted and identified by mass spectrometry. This led to the characterization of several distinct GATA-1 complexes with other transcription factors and chromatin remodeling cofactors, which are involved in activation and repression of gene targets. Thus, BirA-mediated tagging is an efficient approach for the direct capture and characterization of transcription factor complexes.

Amino Acid Sequence↗

PubMed2005

GATA-1 forms distinct activating and repressive complexes in erythroid cells.

GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes, for example, in erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes, yet it is not clear how both of these functions are mediated. Using a biotinylation tagging/proteomics approach in erythroid cells, we describe distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. Importantly, we show that FOG-1 mediates GATA-1 interactions with the MeCP1 complex, thus providing an explanation for the overlapping functions of these two factors in erythropoiesis. We also show that subsets of GATA-1 gene targets are bound in vivo by distinct complexes, thus linking specific GATA-1 partners to distinct aspects of its functions. Based on these findings, we suggest a model for the different roles of GATA-1 in erythroid differentiation.

Animals↗

PubMed2005

ETO-2 associates with SCL in erythroid cells and megakaryocytes and provides repressor functions in erythropoiesis.

Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.

Animals↗

PubMed2005

Isolation and characterization of hematopoietic transcription factor complexes by in vivo biotinylation tagging and mass spectrometry.

We have described the application of a simple biotinylation tagging approach for the direct purification of tagged transcription factor complexes, based on the use of artificial short peptide tags that are specifically and efficiently biotinylated by the bacterial BirA biotin ligase, which is co-expressed in cells with the tagged factor. We used this approach to initially characterize complexes formed by the hematopoietic transcription factor GATA-1 in erythroid cells. GATA-1 is essential for the erythroid differentiation, its functions encompassing upregulation of erythroid genes, repression of alternative transcription programs, and suppression of cell proliferation. However, it was not clear how all of these GATA-1 functions are mediated. Our work describes, for the first time, distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex, and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. We also provide evidence that distinct GATA-1 complexes are associated with specific GATA-1 functions in erythroid differentiation, for example, GATA-1/Gfi-1b with the suppression of cell proliferation and GATA-1/FOG-1/MeCP1 with the repression of other hematopoietic transcription programs. We next applied the biotinylation tag to Ldb-1, a known partner of GATA-1, and characterized a number of novel interaction partners that are essential in erythroid development, in particular, Eto-2, Lmo4, and CdK9. Last, we are in the process of applying the same technology to characterize the factors that are bound to the suppressed gamma-globin promoter in vivo.

Animals↗

PubMed2003

Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice.

Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.

Animals↗