Mechanisms of Nkx6.1 governing beta cell development and function

The NK homeobox transcription factor Nkx6.1 is a critical regulator of the final stages of beta cell differentiation within the developing pancreas. However, very little is known as to how Nkx6.1 directs the phenotypic development of the beta cell or if Nkx6.1 has a role in the mature, insulin-secreting beta cell. Here we elucidate the role of Nkx6.1 by identifying and characterizing three of its native downstream gene targets: the glucagon, insulin , and nkx6.1 genes. Whereas Nkx6.1 appears to act as a repressor of transcription at the glucagon and insulin promoters (a property mapped to its N terminus), Nkx6.1 functions to enhance the transcription of its own gene, perhaps accounting for its persistent expression within the mature beta cell. This transcriptional activation function was mapped to the C terminus of the protein, a region previously suggested to modulate its DNA binding affinity. Interestingly, the repression-versus-activation functions of Nkx6.1 appear to rely at least in part on differential sequence recognition at target promoters. While Nkx6.1 represses transcription through association with conserved TAAT elements, Nkx6.1 activates transcription by binding a non-consensus ATTT element. In an attempt to understand the mechanism by which the C terminus of Nkx6.1 modulates the protein's recognition or affinity for such sequences, we performed numerous biochemical and structural analyses of the protein. Surprisingly, a negatively charged region of the Nkx6.1 C terminus selectively enhances its DNA binding affinity for TAAT-containing DNA sequences, a property that is not attributable to significant structural alterations in the protein in the absence of DNA. Taken together, these data provide a framework by which we can systematically dissect the broad mechanistic role of Nkx6.1 as it relates to beta cell development and function.