Abstract
The hormone prolactin (PRL) contributes to breast cancer pathogenesis through various signaling pathways, one of the most notable being the JAK2/signal transducer and activator of transcription 5 (STAT5) pathway. PRL-induced activation of the transcription factor STAT5 results in the up-regulation of numerous genes implicated in breast cancer pathogenesis. However, the molecular mechanisms that enable STAT5 to access the promoters of these genes are not well understood. Here, we show that PRL signaling induces chromatin decompaction at promoter DNA, corresponding with STAT5 binding. The chromatin-modifying protein high mobility group nucleosomal binding domain 2 (HMGN2) specifically promotes STAT5 accessibility at promoter DNA by facilitating the dissociation of the linker histone H1 in response to PRL. Knockdown of H1 rescues the decrease in PRL-induced transcription following HMGN2 knockdown, and it does so by allowing increased STAT5 recruitment. Moreover, H1 and STAT5 are shown to function antagonistically in regulating PRL-induced transcription as well as breast cancer cell biology. While reduced STAT5 activation results in decreased PRL-induced transcription and cell proliferation, knockdown of H1 rescues both of these effects. Taken together, we elucidate a novel mechanism whereby the linker histone H1 prevents STAT5 binding at promoter DNA, and the PRL-induced dissociation of H1 mediated by HMGN2 is necessary to allow full STAT5 recruitment and promote the biological effects of PRL signaling.
Highlights
The hormone prolactin (PRL) contributes to breast cancer pathogenesis through various signaling pathways, one of the most notable being the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway
Chromatin was digested with micrococcal nuclease (MNase), mononucleosomal DNA was isolated, and protected regions were amplified by quantitative real-time PCR using overlapping amplicons spanning across the CISH promoter
These results suggest that PRLinduced chromatin remodeling at the CISH promoter results in increased accessibility at the STAT5 consensus elements, presumably allowing increased STAT5 binding following activation
Summary
STAT5 Inhibition Results in Decreased Proliferation of Breast Cancer Cells—Because the transcription factor STAT5 is a critical mediator of PRL-induced signaling, we first assessed the role of STAT5 in mediating the biological effects of PRL in breast cancer cells. Because PRL treatment resulted in chromatin remodeling upstream of the STAT5 binding sites as well, we sought to determine whether there are other important regulatory elements in this region that enhance or repress PRL-induced CISH transcription To this end, a Dual-Luciferase reporter assay was utilized. HMGN2 knockdown resulted in decreased expression of CISH, IER3, and PHLDA2 in response to PRL treatment (Fig. 4E) These data indicate that HMGN2 plays a key role in facilitating PRLinduced, STAT5-mediated gene expression. To address the possibility that HMGN2 may affect nucleosome positioning without affecting total levels of H3, the MNase protection assay was carried out following HMGN2 knockdown, covering the region upstream of the CISH TSS, which had previously exhibited PRL-induced chromatin remodeling (Fig. 3B). HMGN2 Promotes Histone H1 Loss to Regulate STAT5 Binding and CISH Transcription—Given these results, we hypothesized that the decrease in PRL-induced gene expression follow-. These findings further validate our model (Fig. 11) and suggest that linker histone H1 occupancy may serve as a general mechanism regulating STAT transcription factor binding
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