Analysis of the Signals and Mechanisms Mediating Nuclear Trafficking of GATA-4

Abstract

Nucleocytoplasmic transport of GATA-4 is important in maintaining and regulating normal cardiogenesis and heart function. This report investigates the detailed mechanisms of GATA-4 nuclear transport. We characterized a nonclassical nuclear localization signal between amino acids 270 and 324 that actively transports GATA-4 into the nucleus of both HeLa cells and cardiac myocytes. Fine mapping studies revealed four crucial arginine residues within this region that mediate active transport predominately through the nonclassical pathway via interaction with importin ␤. These four residues were also essential for the DNA binding activity of GATA-4 and transcriptional activation of cardiac-specific genes. Interestingly, mutation of these residues not only inhibited DNA binding and gene transcription but also resulted in a preferential accumulation of the GATA-4 protein in distinct subnuclear speckles. A cardiac myocyte-specific, chromosome maintenance region 1-dependent nuclear export signal consisting of three essential leucine residues was also identified. The current study provides detailed information on the nuclear shuttling pathways of GATA-4 that represents an additional mechanism of gene regulation.

Nuclear Receptor-Like Structure and Interaction of Congenital Heart Disease-Associated Factors GATA4 and NKX2-5

“…R264A is able to activate the promoter containing a tandem GATA-site similarly to wtGATA4, in contrast to K299A, which was unable to bind this sequence properly and to activate transcription. These results and their interpretation are consistent with the work of Philips et al [ 43 ], who showed that mutation of R283 and K299 reduces DNA binding to a monomeric GATA element. Alterations in the packing of C- and N-terminal zinc fingers may thus be the cause for the strongly reduced GATA4-NKX2-5 physical interaction detected with these mutants.…”

“…Since all mutations were located outside the antibody recognition site, western blots were used to quantify the mutant protein levels produced in mammalian COS-1 cells. All mutant proteins are expected to localise to the nucleus like native GATA4 since single amino acid substitution in zinc finger area is not sufficient to inhibit the nuclear trafficking [ 43 , 44 ]. Physical interactions with NKX2-5 were assessed in co-immunoprecipitation with N-terminal FLAG-NKX2-5 and analysed by western blots using GATA4 and NKX2-5 antibodies ( Fig 2 ).…”

Genome-Wide Analysis of Histidine Repeats Reveals Their Role in the Localization of Human Proteins to the Nuclear Speckles Compartment

“…Our results show a direct correlation between loss of DNA binding activity and accumulation in nuclear speckles. Similar behavior was recently described for the transcription factor GATA-4, although the subnuclear compartment to which it localized was not identified [67]. Although we cannot ignore that the elimination of the DNA binding domains may result in a conformational change that exposes the His-repeat, we favor a loss of retention in the chromosomal compartment as being responsible for the enrichment in nuclear speckles.…”

SUMOylation Regulates the Transcriptional Repression Activity of FOG-2 and Its Association with GATA-4

“…EGFP-SUMO-1 has been previously described [25] and was kindly provided by Hisato Saitoh (Picower Institute of Medical Research, New York, NY). The brain natriuretic peptide (BNP) reporter construct and pMT3-HA-SUMO-1 have been previously described [19], [26]. FLAG-SENP-1 (Addgene plasmid 17357) and FLAG-SENP-8 (Addgene plasmid 18066) were kindly provided by Edward Yeh (University of Texas, Houston, TX) [27], [28].…”

“…For protein expression, COS-7 cells were grown on 100 mm-diameter Petri dishes and transfected with 1–2 µg of FOG-2 and its derivatives, SUMO-1 or GFP-SUMO-1 expression vectors using Lipofectamine2000 following the manufacturer’s instructions (Invitrogen). HeLa cells used for luciferase assays were cultured in 6-well plates and were transfected with 300 ng of pGL3-Basic-BNP reporter [26], 300 ng of pCS2+GATA-4, 50 to 400 ng of pCS2+FOG-2, pCS2+FOG-2-4KR or SUMO-1-FOG-2-4KR, 50 to 300 ng of GFP-SUMO-1 and 500 ng of pFLAG-SENP-1 and pFLAG-SENP-8. The total amount of DNA was kept constant by adding empty pCS2+ vector.…”