Molecular Analysis of the Interaction between the Hematopoietic Master Transcription Factors GATA-1 and PU.1

Abstract

GATA-1 and PU.1 are transcription factors that control erythroid and myeloid development, respectively. The two proteins have been shown to function in an antagonistic fashion, with GATA-1 repressing PU.1 activity during erythropoiesis and PU.1 repressing GATA-1 function during myelopoiesis. It has also become clear that this functional antagonism involves direct interactions between the two proteins. However, the molecular basis for these interactions is not known, and a number of inconsistencies exist in the literature. We have used a range of biophysical methods to define the molecular details of the GATA-1-PU.1 interaction. A combination of NMR titration data and extensive mutagenesis revealed that the PU.1-Ets domain and the GATA-1 C-terminal zinc finger (CF) form a low affinity interaction in which specific regions of each protein are implicated. Surprisingly, the interaction cannot be disrupted by single alanine substitution mutations, suggesting that binding is distributed over an extended interface. The C-terminal basic tail region of CF appears to be sufficient to mediate an interaction with PU.1-Ets, and neither acetylation nor phosphorylation of a peptide corresponding to this region disrupts binding, indicating that the interaction is not dominated by electrostatic interactions. The CF basic tail shares significant sequence homology with the PU.1 interacting motif from c-Jun, suggesting that GATA-1 and c-Jun might compete to bind PU.1. Taken together, our data provide a molecular perspective on the GATA-1-PU.1 interaction, resolving several issues in the existing data and providing insight into the mechanisms through which these two proteins combine to regulate blood development.

Mathematical modeling of GATA-switching for regulating the differentiation of hematopoietic stem cell

“…(3) In addition, the heterodimer GATA-1-PU.1 has a 3-fold increase of the binding rate constant over GATA-1 to DNA [7]. It was assumed that a 7 = 3 a 4 .…”

“…they stimulate their own production, as well as they are mutually antagonistic, i.e. they repress the production of each other [7-9]. In the erythrocyte/megakaryocite lineage high expression levels of gene GATA-1 and low levels of PU.1 were detected [6,10]; conversely, in the granulocyte/macrophage lineage higher expression levels of PU.1 and low levels of GATA-1 were measured [5,11].…”

Transcriptional control of granulocyte and monocyte development

“…Interaction of c-Jun homodimers with PU.1 depends on integrity of residues in the c-Jun BR and is augmented by contact of PU.1 with C/EBPb bound to DNA nearby (Grondin et al, 2007). The C-terminal zinc finger of GATA-1 competes with the DNA-binding BR of c-Jun for interaction with the Ets domain of PU.1 Liew et al, 2006). This is a potential mechanism to prevent myeloid gene activation in erythroid cells.…”

Signatures of DNA target selectivity by ETS transcription factors

“…For example, Runx1/AML1/CBFa2/PEBP2 positively regulates Ets-1/ DNA binding by displacing and destabilizing an extended inhibitory segment N-terminal to the ETS domain. [38][39][40] In ETS paralogs that are not auto-inhibited, such as PU.1, specific interactions with binding partners that positively regulate (e.g., IRF4/Pip) [41][42][43] or antagonize DNA binding (e.g., GATA-1) 44,45 are known. Another combinatorial strategy that modifies DNA site targeting is to couple binding to intrinsically low-affinity or nonspecific sites to specific interactions with binding partners.…”