The High Mobility Group proteins HMGA1 are nuclear architectural factors that play a critical role in a wide range of biological processes. Since recent studies have identified the microRNAs (miRNAs) as important regulators of gene expression, modulating critical cellular functions such as proliferation, apoptosis and differentiation, the aim of our work was to identify the miRNAs that are physiologically regulated by HMGA1 proteins. To this purpose, we have analysed the miRNA expression profile of mouse embryonic fibroblasts (MEFs) carrying two, one or no Hmga1 functional alleles using a microarray (miRNA microarray). By this approach, we found a miRNA expression profile that differentiates Hmga1-null MEFs from the wild-type ones. In particular, a significant decrease in miR-196a-2, miR-101b, miR-331 and miR-29a was detected in homozygous Hmga1-knockout MEFs in comparison with wild-type cells. Consistently, these miRNAs are downregulated in most of the analysed tissues of Hmga1-null mice in comparison with the wild-type mice. ChIP assay shows that HMGA1 is able to bind regions upstream of these miRNAs. Moreover, we identified the HMGA2 gene product as a putative target of miR-196a-2, suggesting that HMGA1 proteins are able to downregulate the expression of the other member of the HMGA family through the regulation of the miR-196a-2 expression. Finally, ATXN1 and STC1 gene products have been identified as targets of miR-101b. Therefore, it is reasonable to hypothesize that HMGA1 proteins are involved in several functions by regulating miRNA expression.

High mobility group A (HMGA) proteins are chromatinic proteins that do not have transcriptional activity per se, however, by interacting with the transcription machinery, they regulate, negatively or positively, the expression of several genes. We searched for genes regulated by HMGA1 proteins using microarray analysis in embryonic stem (ES) cells bearing one or two disrupted hmga1 alleles. We identified 87 transcripts increased and 163 transcripts decreased of at least 4-fold in hmga1؊/؊ ES cells. For some of them, a HMGA1-dose dependency was observed, because an intermediate level was observed in the heterozygous ES cells. When the expression analysis of these genes was extended to embryonic fibroblasts and adult tissues such as heart, spleen, and liver from hmga1-knockout mice, contrasting results were obtained. In fact, aside some genes showing the same HMGA1 regulation observed in ES cells, there were some genes that did not modify their expression, and others showing a HMGA1-mediated regulation but in an opposite direction. These results clearly indicate that HMGA1-mediated gene regulation depends on the cellular context. Finally for a couple of analyzed HMGA1-regulated genes, electrophoretic mobility shift assay and chromatin immunoprecipitation revealed a direct binding of HMGA1 proteins to their promoters, suggesting a HMGA1-direct regulation of their expression.

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