Dissection of the NF-Y transcriptional activation potential.

A Silvio Di,R Mantovani,C Imbriano

doi:10.1093/nar/27.13.2578

Abstract

NF-Y is a trimeric CCAAT-binding factor with histone fold subunits (NF-YB/NF-YC) and bipartite activation domains located on NF-YA and NF-YC. We reconstituted the NF-Y activation potential in vivo with GAL4 DBD fusions. In the GAL4-YA configuration, activation requires co-expression of the three subunits; with GAL4-YB and GAL4-YC, transfections of the histone fold partners are sufficient, provided that the Q-rich domain of NF-YC is present. Combinations of mutants indicate that the Q-rich domains of NF-YA and NF-YC are redundant in the trimeric complex. Glutamines 101 and 102 of NF-YA are required for activity. We assayed NF-Y on different promoter targets, containing single or multiple GAL4 sites: whereas on a single site NF-Y is nearly as powerful as VP16, on multiple sites neither synergistic nor additive effects are observed. NF-Y activates TATA and Inr core elements and the overall potency is in the same range as other Q-rich and Pro-rich activation domains. These results represent the first in vivo evidence of subunit interactions studies and further support the hypothesis that NF-Y is a general promoter organizer rather than a brute activator.

Highlights

Regulation of gene expression is dictated by DNA regulatory elements found in the proximity of and at a distance from transcribed genes
The NF-YA, NF-YB and NF-YC vectors are derived from the GAL4 fusions by excision of the GAL4 DBD
Hatched boxes refer to the NF-YA and NF-YC Q-rich domains, filled boxes to the homology domains. (B) The reporter vectors were as described [15,16,17] and contain one, two or five GAL4 sites in front of TATA or Inr elements

Summary

Introduction

Regulation of gene expression is dictated by DNA regulatory elements found in the proximity of (promoters) and at a distance from (enhancers) transcribed genes. The core promoter elements (TATA and Initiator) are targets of the general transcription machinery, being involved in the activation of all Pol II transcribed genes. Upstream sequences are a combinatorial puzzle of specific elements that vary among promoters and are recognized by proteins binding DNA with high specificity. In addition to DNA binding, these proteins usually possess a transcriptional activation function. Defined in transfection assays, the activation domains recognize targets in the general transcription apparatus (or holoenzyme). Molecular dissection of different activators led to the discovery of disparate protein stretches: Q-rich, acidic, Pro-rich, Ile-rich etc. Molecular dissection of different activators led to the discovery of disparate protein stretches: Q-rich, acidic, Pro-rich, Ile-rich etc. (for a review see 1)

Methods

Results

Conclusion