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.
Grosveld
Frank
F
Department of Cell Biology, Erasmus University Medical Center, 50 Dr. Molewaterplein, 3015 GE Rotterdam, the Netherlands. f.grosveld@erasmusmc.nl
Rodriguez
Patrick
P
Meier
Natalia
N
Krpic
Sanja
S
Pourfarzad
Farzin
F
Papadopoulos
Petros
P
Kolodziej
Katarzyna
K
Patrinos
George P
GP
Hostert
Arnd
A
Strouboulis
John
J
D000818:Animals; D019921:Biotinylation; D001798:Blood Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D019731:Carbon-Nitrogen Ligases / Q000494:pharmacology; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry; D041905:Erythroid Cells / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000494:pharmacology; D050982:GATA1 Transcription Factor / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D005914:Globins / Q000096:biosynthesis / Q000235:genetics; D006410:Hematopoiesis / Q000235:genetics; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D046911:Macromolecular Substances; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000502:physiology; D025941:Protein Interaction Mapping; D012097:Repressor Proteins / Q000494:pharmacology; D014157:Transcription Factors / Q000302:isolation & purification* / Q000494:pharmacology / Q000502:physiology; D015027:Zebrafish / Q000097:blood / Q000196:embryology
D001798:Blood Proteins; D029968:Escherichia coli Proteins; D050982:GATA1 Transcription Factor; C494689:Gata1 protein, mouse; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D005914:Globins; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli