Structural and functional studies on the pituitary-specific transcription factor Pit-1

Abstract

Pit-1 is a pituitary specific transcription factor that plays a central role in the development and maintenance of a number of cell lineages in the anterior pituitary gland. In these cell lineages, Pit-1 is required for the selective expression of the growth hormone (GH), prolactin (PRL) and the b-subunit of the thyroid-stimulating hormone (TSHb). Pit-1 contains a POU DNA-binding domain, which consist of two independent DNA-binding modules (the POU-specific domain (POUs) and the POU-homeodomain (POUhd)), separated by a flexible linker. Loss of Pit-1 function leads to combined pituitary hormone deficiency (CPHD) syndrome, the main feature of which is dwarfism. Chapter II of this thesis describes a clinical case study of a CPHD patient carrying two novel mutations (each on a different allele) in both the POUs and the POUhd. One of these mutations, located in the POUs, substitutes one of the hydrophobic core residues with a charged residue, which leads to a misfolded POUs domain that is unable to bind DNA. The other mutation leads to the deletion of the entire DNA-recognition helix from the POUhd. In the context of the full-length POU-domain, neither of these mutants was capable of high-affinity DNA-binding in vitro or stimulation of the Pit-1 target promoters in vivo. The expression of GH, PRL and TSHb is highly restricted to their respective cell lineage, while Pit-1 is present in all three cell lineages. Therefore, Pit-1 needs to cooperate with other transcription regulators in order to silence or activate its target promoters. The interaction of Pit-1 with two such factors, Ets-1 and GATA-2, is studied in chapter III. Ras-responsive Ets-1 cooperates with Pit-1 to synergistically activate the PRL promoter, which contains composite Ets-1 / Pit-1 recognition sites. However, both proteins can also associate in solution, in absence of DNA. This physical interaction involves the POUhd of Pit-1 and a part of the region III activation domain of Ets-1. Using nuclear magnetic resonance (NMR) monitored protein-protein titrations, we were able to study which residues on the POUhd are likely contacted by the minimal interacting region of Ets-1. The NMR data suggests that Ets-1 binds the POUhd through multiple interacting regions. Interestingly, the affinity of two of these regions on the POUhd may be influenced by post-translational modifications (phosphorylation and possibly acetylation). Since the interaction interface on the POUhd for both DNA and Ets-1 binding overlap to a large extent, it would not be surprising if the DNA binding affinity of the POUhd were somehow affected by Ets-1. However, the study described in chapter IV shows that, at least for the minimal interaction domain of Ets-1, this is not the case. Finally, chapter V investigates the functional relevance of a novel interaction of the conserved region 3 (CR-3) of the 13S splice variant of the adenovirus immediate early gene product (E1A 13S) with the N-terminal activation domain of Pit-1. Transient co-transfection of Pit-1 with E1A 13S showed an inhibitory effect of E1A 13S on Pit-1 mediated stimulation of the GH and PRL promoters.