Global effects of vitamin A deficiency on gene expression in rat liver: evidence for hypoandrogenism.

Vitamin A (retinol) metabolites are ligands for transcription factors that regulate many genes. The liver is the main storage depot for retinol and plays a role in vitamin A homeostasis. To better understand the effects of vitamin A deficiency on liver gene expression, we produced retinol deficiency in male rats by feeding a diet low in retinol for 53 days after weaning and examined the effects on gene expression in liver using Affymetrix oligonucleotide microarrays. We detected expression of 41% of the 8799 probe sets represented on the RGU-34A GeneChips. Vitamin A deficiency resulted in major changes in liver gene expression: 805 genes (22% of all genes detected) differed at P

Reproducibility of oligonucleotide arrays using small samples.

BACKGROUND: Low RNA yields from small tissue samples can limit the use of oligonucleotide microarrays (Affymetrix GeneChips). Methods using less cRNA for hybridization or amplifying the cRNA have been reported to reduce the number of transcripts detected, but the effect on realistic experiments designed to detect biological differences has not been analyzed. We systematically explore the effects of using different starting amounts of RNA on the ability to detect differential gene expression. RESULTS: The standard Affymetrix protocol can be used starting with only 2 micrograms of total RNA, with results equivalent to the recommended 10 micrograms. Biological variability is much greater than the technical variability introduced by this change. A simple amplification protocol described here can be used for samples as small as 0.1 micrograms of total RNA. This amplification protocol allows detection of a substantial fraction of the significant differences found using the standard protocol, despite an increase in variability and the 5' truncation of the transcripts, which prevents detection of a subset of genes. CONCLUSIONS: Biological differences in a typical experiment are much greater than differences resulting from technical manipulations in labeling and hybridization. The standard protocol works well with 2 micrograms of RNA, and with minor modifications could allow the use of samples as small as 1 micrograms. For smaller amounts of starting material, down to 0.1 micrograms RNA, differential gene expression can still be detected using the single cycle amplification protocol. Comparisons of groups of four arrays detect many more significant differences than comparisons of three arrays.

Global effect of PEG-IFN-alpha and ribavirin on gene expression in PBMC in vitro.

Using oligonucleotide microarrays, we have examined the expression of 22,000 genes in peripheral blood cells treated with pegylated interferon-alpha2b (PEG-IFN-alpha) and ribavirin. Treatment with ribavirin had very little effect on gene expression, whereas treatment with PEG-IFN-alpha had a dramatic effect, modulating the expression of approximately 1000 genes (at p < 0.001). In addition to genes previously reported to be induced by type I or type II IFNs, many novel genes were found to be upregulated, including transcription factors, such as ATF3, ATF4, properdin, a key regulator of the complement pathway, a homeobox gene (HESX1), and an RNA editing enzyme (apobec3). Chemokines CXCL10 and CXCL11 were upregulated, whereas CXCL5 was downregulated. Cytokines interleukin-15 (IL-15) and IL-18 were also significantly induced, whereas IL-1alpha and IL-1beta were downregulated. Most other interleukins were not affected. The results of the microarrays were confirmed by kinetic real-time PCR. These data indicate that IFN treatment causes upregulation of genes associated with the stress response, apoptosis, and signaling, and an equal number of genes are downregulated, including those associated with protein synthesis, specific cytokines and chemokines and other biosynthetic functions.

A retroviral repetitive element confers tissue-specificity to the human alcohol dehydrogenase 1C (ADH1C) gene.

The human ADH1A, ADH1B, and ADH1C genes encode alcohol dehydrogenases (ADHs) that metabolize ethanol. They evolved by recent tandem duplications and have similar proximal cis-acting elements, but differ in tissue-specificity. We hypothesized that distal cis-acting elements confer tissue-specificity. In this article, we identify multiple cis-acting elements in the ADH1C upstream region. Negative elements in the fragments from bp -1,078 to -622 and from bp -3,957 to -2,651 decreased transcription activity to 41 and 14%, respectively. A tissue-specific regulatory element in the region between bp -1,503 and -1,053 stimulated transcription sixfold in H4IIE-C3 hepatoma cells but reduced transcription to 23% in HeLa cells. This regulatory element was mapped to a repetitive sequence that is similar to the U3 repeat within the long terminal repeat of human endogenous retrovirus ERV9. The 30-fold difference in expression between two cell lines demonstrates that this upstream U3 element, which inserted after the duplications that created the three class I ADH genes, plays an important role in regulating tissue-specificity of ADH1C. The ubiquitous Nuclear factor-Y (NF-Y) and an H4IIE-C3/liver-specific factor bound to the subrepeat sequence. This result suggested that tissue specificity might result from combinatorial regulation by these two transcription factors.

Coordinate repression of regulators of embryonic identity by PICKLE during germination in Arabidopsis.

In angiosperms, germination represents an important developmental transition during which embryonic identity is repressed and vegetative identity emerges. PICKLE (PKL) encodes a CHD3-chromatin-remodeling factor necessary for the repression of expression of LEAFY COTYLEDON1 (LEC1), a central regulator of embryogenesis. A candidate gene approach and microarray analysis identified nine additional genes that exhibit PKL-dependent repression of expression during germination. Transcripts for all three LEAFY COTYLEDON genes, LEC1, LEC2, and FUS3, exhibit PKL-dependent repression, and all three transcripts are elevated more than 100-fold in pkl primary roots that inappropriately express embryonic traits (pickle roots). Three other genes that exhibit PKL-dependent regulation have expression patterns correlated with zygotic or somatic embryogenesis, and one gene encodes a putative Lin-11, Isl-1, MEC-3 (LIM) domain transcriptional regulator that is preferentially expressed in siliques. Genes that exhibit PKL-dependent repression during germination are not necessarily regulated by PKL at other points in development. Our data suggest that PKL selectively regulates a suite of genes during germination to repress embryonic identity. In particular, we propose that PKL acts as a master regulator of the LEAFY COTYLEDON genes, and that joint derepression of these genes is likely to contribute substantially to expression of embryonic identity in pkl seedlings.