393 Promoter Analysis of Early Response Genes Induced by Chemokine Receptor Signaling A. B. van 't Wout*, D. J. Westreich, H. He, D. C. Nickle, J. I. Mullins Univ of Washington, Seattle
Background: Most HIV-1 variants can be divided into 2 groups based on their chemokine coreceptor usage, with CCR5 and CXCR4 identified as the coreceptors for HIV-1 R5 and X4 variants, respectively. These variants have biological significance, since the emergence of X4 variants predicts a more rapid disease progression. Signaling through the co-receptors has been shown to occur upon binding of HIV-1, although this signaling is not needed for productive viral infection. Here we studied gene expression patterns resulting from signaling through CCR5 and CXCR4 using cDNA microarrays.
Methods: Cells expressing both CCR5 and CXCR4 were exposed to the chemokine ligands, MIP-1beta or SDF-1alpha. Total RNA and mRNA was extracted from time points between 30 mins and 12 hrs and converted into fluorescently labeled probes. High density cDNA microarrays were used to assess changes in gene expression. Genes were grouped in clusters according to expression patterns. Several publicly available tools for predicting transcription factor binding sites were explored and tested for accuracy.
Results: At least 4 distinct expression patterns were observed in these experiments. The most striking pattern was an early up-regulation of immediate-early genes (IEG), transcription factors including EGR1, TIEG, and SRF and negative regulators NAB1 and NAB2. To determine if all members of this early cluster were regulated by the same IEG signals, we analyzed a 1,003 base pair region upstream of each gene in the cluster for transcription factor binding sites. To control for non-specific findings, we analyzed 1,003 base pair region downstream of each gene. The resulting predicted binding sites were then mapped onto the cluster using parsimony. Surprisingly, this showed that predicted promoter binding sites were not significantly correlated with the observed expression pattern.
Conclusions: Both MIP-1beta and SDF-1alpha induced expression of IEG, consistent with previous findings for other chemokines like RANTES. Early response activation by RANTES resulted from calcium mobilization and induction of the MAP kinase cascade. Several other genes regulated early after stimulation with MIP-1beta and SDF-1alpha appear to be stimulated through additional pathways as their promoter regions did not contain specific sequences associated with the known IEG. We are continuing our analysis of gene expression clusters and transcription factor binding sites to better understand the effects of chemokine signaling.
