The Plant NF-Y DNA Matrix In Vitro and In Vivo

Abstract

Nuclear Factor Y (NF-Y) is an evolutionarily conserved trimer formed by a Histone-Fold Domain (HFD) heterodimeric module shared by core histones, and the sequence-specific NF-YA subunit. In plants, the genes encoding each of the three subunits have expanded in number, giving rise to hundreds of potential trimers. While in mammals NF-Y binds a well-characterized motif, with a defined matrix centered on the CCAAT box, the specificity of the plant trimers has yet to be determined. Here we report that Arabidopsis thaliana NF-Y trimeric complexes, containing two different NF-YA subunits, bind DNA in vitro with similar affinities. We assayed precisely sequence-specificity by saturation mutagenesis, and analyzed genomic DNA sites bound in vivo by selected HFDs. The plant NF-Y CCAAT matrix is different in nucleotides flanking CCAAT with respect to the mammalian matrix, in vitro and in vivo. Our data point to flexible DNA-binding rules by plant NF-Ys, serving the scope of adapting to a diverse audience of genomic motifs.

In Silico Identification and Expression Analysis of Nuclear Factor Y (Nf-Y) Transcription Factors in Cucumber

“…Recently, numerous reports have revealed the important functions of NF-Y genes in mediating abiotic stress tolerance in plants [2,46]. In this study, six stress-responsive elements and nine hormone-responsive elements were observed in the promoters of CsaNF-Y genes ( Figure S1), suggesting that some of the CsaNF-Y genes may also participate in stress responses.…”

Structural determinants for NF‐Y subunit organization and NF‐Y/DNA association in plants

“…Finally, trimerization yielding selectivity for different DNA targets constitutes a third regulation layer. Recently, we have reported the CCAAT matrix for a specific Arabidopsis NF‐Y trimer (AtNF‐Y), assayed through saturation mutagenesis and the analysis of genomic DNA sites bound in vivo (Gnesutta et al ., 2019). The plant NF‐Y CCAAT matrix showed an absolute requirement for the CCAAT pentanucleotide and, even if the AtNF‐Y trimer exhibited higher flexibility with respect to the mammalian matrix, a certain preference for nucleotides flanking the CCAAT was also noticed (Gnesutta et al ., 2019).…”

“…Recently, we have reported the CCAAT matrix for a specific Arabidopsis NF‐Y trimer (AtNF‐Y), assayed through saturation mutagenesis and the analysis of genomic DNA sites bound in vivo (Gnesutta et al ., 2019). The plant NF‐Y CCAAT matrix showed an absolute requirement for the CCAAT pentanucleotide and, even if the AtNF‐Y trimer exhibited higher flexibility with respect to the mammalian matrix, a certain preference for nucleotides flanking the CCAAT was also noticed (Gnesutta et al ., 2019). In addition, the capacity of NF‐YB/NF‐YC dimer to form diverse functional trimeric complexes with other sequence‐specific DNA‐binding modules incredibly expands its potential in the recognition of target DNA elements.…”

“…Second, no structures for plant NF‐Y heterotrimers, nor NF‐COs, are available to settle questions as to the molecular basis of heterotrimeric assemblies in plants, nor for NF‐YA versus CO trimer selectivity (Gnesutta et al ., 2018). Third, structural information is needed to have a complete picture of the residues providing CCAAT box interactions, different preferential binding to nucleotides flanking the CCAAT box (Gnesutta et al ., 2019), and to extend the model to the many plant NF‐Ys and NF‐COs complexes.…”

“…Based on such knowledge, we analysed and generated a set of NF‐YB/NF‐YC trimerization mutants that discriminate between two AtNF‐YAs and CO. Furthermore, the AtNF‐Y/DNA structure identified contacts to CCAAT box flanking bases that contribute to certain specificity, in agreement with recent mutagenesis studies that suggest flexible DNA‐binding rules to recognize a variety of genomic CCAAT motifs (Gnesutta et al ., 2019).…”