T-box factors take on a major role in guiding transcription events during cardiac development, and their combinatorial T-box protein interaction events must be seen as key to determining the individual fate each cell ultimately derives. For example, the combinatorial activities and interactions of Tbx5, Tbx20, Nkx2-5 and Gata4 lead to cardiac chamber differentiation of which a molecular signature is the the upregulation and expression of Nppa
(Natriuretic Precursor Peptide type A
, also known as ANF). Inclusion of Tbx2 and Tbx3 in this network leads to Nppa
suppression, notably in the atrioventricular canal, signifying an apparent suppression of differentiation [8
]. Attempts then to examine the function of T-box factors at the molecular level often turn to techniques that seek to identify interacting protein partners. Perhaps one of the better-known examples was the use of the yeast two-hybrid system by Hiroi and co-workers [13
] in conjunction with the homeodomain-containing transcription factor Nkx2-5. Using Nkx2-5 as bait in a human complementary DNA (cDNA) library screen Tbx5 was identified as interacting partner, the synergistic interaction of which was shown to be a key event in the differentiation of working myocardium [13
]. Mutations which interfere with either the capacity of Tbx5 to bind DNA or its ability to interact with Nkx2-5 can cause human Holt-Oram syndrome [15
]. Similarly, a synergistic interaction between Tbx1 and Nkx2-5 has recently postulated to play a role in another human congenital disease displaying varying heart malformations, DiGeorge syndrome [18
]. Although on its own, in vitro
, Nkx2-5 appears to function as a moderate transcriptional activator, its ultimate activity as an activator or repressor depends on interaction with the T-box factors present in the same cell [12
]. One may then hypothesize that Nkx2-5 acts as a T-box guidance factor instead of a direct transcriptional activator or repressor.
Studying congenital defects can itself represent a ‘natural’ experimental setting to discover and elucidate novel protein interactions. One such study involving isolated septal defects leads to the inclusion of GATA4 as a new synergistic interacting partner of Tbx5, an interaction shown to play a role in the expression of Nppa
]. In our lab we have also demonstrated an in vitro
interaction between GATA4 and Tbx2/3 [unpublished observations], which taken together with the described interaction of Tbx20 with GATA4 [20
] suggests that in the case of overlapping expression of T-box proteins, the stoichiom-etry of T-box proteins and the restricted presence of other positive or negative regulatory factors will ultimately decide timing and direction of cellular fate. In line with this, several other protein interacting partners have been assigned to the Tbx5 list [22
]. The cardiac-enriched MYST family histone acetyltransferase TIP60 and Tbx5 were observed to be mutual interactive cofactors through the TIP60 zinc finger. In transfection assays, TIP60, Tbx5 and Tbx2 activate an enhancer in the SRF
gene required for expression in the developing heart. The zinc-finger-containing protein Sall4, as well as being regulated by Tbx5, takes on a dual role, functioning co-operatively with Tbx5 and Nkx2-5 to upregulate Cx40
) and Fgf10
and antagonistically to downregulate certain genes such as Nppa
. Notably, the presence of Tbx2/3 can completely block the co-operative activity of Sall4 on Tbx5, though the dependence for this activity on DNA binding or protein-protein interaction was not explored. Further, as is observed for the interaction of Gata4 and Nkx2-5 [13
], Sall4 is also unable to associate with the Tbx5 mutants Gly80Arg and Arg237Trp. This may be indicative of a similar mode of interaction, though one must be careful of over-interpreting the effect of point mutations on interactions since even small perturbations in molecular structure can have significant effects on the overall fold of a protein.
TAZ, a WW domain protein, also represents a recently discovered protein interacting partner of Tbx5 [24
]. Postulated to function as a co-activator of Tbx5, TAZ and a related protein, YAP, are hypothesized to play a role in cardiac development and hypertrophy. Although expression patterns of TAZ in the heart are not available, an interesting feature of TAZ is its ability to associate with the histone acetyltransferase proteins p300 and PCAF. Co-transfections of Tbx5, TAZ and p300 or PCAF were shown to upregulate the Nppa
promoter in vitro
. However, the true in vivo
significance of this interaction still needs to be tested. This role of epigenetics in cardiac development can be postulated from the discovery that T-box factors can associate with chromatin-modifying proteins. Chromatin structure and function have been known to have a key role in cell lineage patterning for many years, acetylation or deacetylation, for instance, increasing or decreasing the accessibility of transcription factor binding sites, respectively. In line with this, Tbx5 activity is influenced by the presence of the chromatin remodeling protein BAF60c, shown to potentiate an interaction between Tbx5 and the Baf complex ATPase Brg1 [25
], thereby assisting in upregulation of cardiac differentiation. Likewise, though functioning in a negative fashion, Tbx2 has been shown to recruit and associate with the histone deacetylase HDAC1 [26
Recently, the PDZ-LIM domain protein LMP4 was found to interact with Tbx5 and to repress its transcriptional activity [27
]. LMP4 is localized in the cytoplasm, associated with the actin cytoskeleton. Interaction between Tbx5 and LMP4, which is dynamic and depends on undefined signals, leads to the localization of Tbx5 to actin filaments. LMP4-mediated regulation of Tbx5 nuclear localization is thought to be involved in the transcriptional regulatory activity of Tbx5.
In a review concerning aspects of T-box proteins in respect to cell fate decision, it is interesting to mention the protein-protein interaction of Tbx20 with a marker of the second heart field, Isl1. This Lim/homeodomain protein has so far only been demonstrated to interact directly with Tbx20 [20
]. This interaction, together with a synergistic interaction between Gata4 and Tbx20, plays a role in regulating the expression of Nkx2-5
in the precursors of the outflow tract and right ventricle.
Finally, T-box family members Tbx2 and Tbx5, and Tbx5 and Tbx20, respectively, have also been demonstrated to directly interact with each other [22
] and demonstrate a synergistic role during heart development. Although T-box dimerization has often been postulated, chiefly on the basis of the Xenopus
T protein (Xbra) homodimer crystal structure and DNA binding characteristics of TBX1
], these examples represent the first T-box proteins demonstrated to heterodimerize, forming a synergistically functional complex.