GATA-3 Dominant Negative Mutant

Abstract

The GATA family of transcription factors regulates a wide variety of genes, including those involved in differentiation of erythrocytes and T lymphocytes. We report here the creation of a dominant negative mutant of GATA-3, KRR, which effectively blocks wild-type GATA-1, GATA-2, and GATA-3 transactivation when co-expressed in transient assays. KRR was generated by site-directed mutagenesis while investigating a putative activation domain of GATA-3, located between its two zinc fingers. The GATA-3 KRR mutation does not affect expression, nuclear translocation, or the ability to bind to a consensus GATA sequence. KRR can suppress the activity of the minimal T cell receptor (TCR) α and β enhancers by 12- and 3.4-fold, respectively. However, KRR did not have a significant effect on the activity of larger TCR-α and -β enhancer fragments. Thus, functional redundancy in the TCR-α and -β enhancers can compensate for the loss of GATA-3 activity. The GATA family of transcription factors regulates a wide variety of genes, including those involved in differentiation of erythrocytes and T lymphocytes. We report here the creation of a dominant negative mutant of GATA-3, KRR, which effectively blocks wild-type GATA-1, GATA-2, and GATA-3 transactivation when co-expressed in transient assays. KRR was generated by site-directed mutagenesis while investigating a putative activation domain of GATA-3, located between its two zinc fingers. The GATA-3 KRR mutation does not affect expression, nuclear translocation, or the ability to bind to a consensus GATA sequence. KRR can suppress the activity of the minimal T cell receptor (TCR) α and β enhancers by 12- and 3.4-fold, respectively. However, KRR did not have a significant effect on the activity of larger TCR-α and -β enhancer fragments. Thus, functional redundancy in the TCR-α and -β enhancers can compensate for the loss of GATA-3 activity.