Identification of the DNA sequence to which binds the Zaf A transcriptional activator of zinc homeostasis in Aspergillus fumigatus

Ref ID: 19587

Author:

R Vicentefranqueira1*, H Toledo1, F Leal1, JA Calera1

Author address:

1Instituto de Biologí­a Funcional y Genómica, Salamanca University-CSIC, Salamanca, Spain

Full conference title:

6th Advances Against Aspergillosis 2014

Abstract:

Purpose:
Aspergillus fumigatus virulence depends on many different fungal capacities, including its capability
to uptake zinc from the extreme zinc-limiting environment provided by the lungs of susceptible
individuals. In this fungal pathogen the ZafA transcription factor regulates zinc uptake and zinc
homeostasis under zinc starvation and is essential for virulence. For this reason, we investigated how
ZafA recognizes the promoter regions of genes whose transcription regulates in order to discover
new functions regulated by ZafA that could be essential for fungal virulence.
Methods:
We have hypothesized that ZafA might bind to a DNA consensus sequence located in the promoter
regions of the genes most strongly induced by ZafA such as those involved in zinc uptake (zrfA,
zrfB and zrfC). Thus, we expressed and purified the C-terminus of the ZafA protein that contains
the putative DNA binding domain and used DNA fragments from the zrfA, zrfB and zrfC promoters
to perform EMSA analyses and fluorescent DNase I footprinting assays. In addition, we performed
a statistical analysis of the distribution of the zinc responsive motif at a genome-wide level and
confirmed by northern blot the expression of some selected genes.
Results:
EMSA assays revealed that ZafA does bind to the promoters of the zrfA, zrfB and zrfC genes. By
using a fluorescent DNase I footprinting assay we identified the protected sequences presumably
recognized by ZafA in the promoter regions of these genes. A comparison of all protected regions
revealed a consensus sequence to which ZafA binds or zinc responsive sequence (ZR). A search for
this motif at a genome-wide level revealed that it was present in the promoter regions of 67 coding
sequences. Nevertheless, degenerated versions of the ZR motif in up to two nucleotides indicated
that it was also present in the promoters of 1,440 coding sequences. The statistical analysis of the
distribution of all these ZR motifs at a genome-wide level revealed that the functional ZRs were most
likely located within 1.2 kb upstream of the predicted AUGs start codons, as shown by northern blot
analyses of some selected genes.
Conclusions:
ZafA either up-regulates or down-regulates gene expression through binding to one or more zinc
responsive motifs located at the promoter regions of the genes that regulate. This study has allowed
us to discover new functions regulated by ZafA that could be essential for fungal virulence.

Abstract Number: 112

Conference Year: 2014

Link to conference website: http://www.AAA2014.org

New link: NULL

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