Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Feb 10;292(6):2237-2254.
doi: 10.1074/jbc.M116.764233. Epub 2016 Dec 29.

Histone H1 and Chromosomal Protein HMGN2 Regulate Prolactin-induced STAT5 Transcription Factor Recruitment and Function in Breast Cancer Cells

Affiliations
Free PMC article

Histone H1 and Chromosomal Protein HMGN2 Regulate Prolactin-induced STAT5 Transcription Factor Recruitment and Function in Breast Cancer Cells

Suzanne M Schauwecker et al. J Biol Chem. .
Free PMC article

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.

Keywords: STAT transcription factor; breast cancer; chromatin remodeling; linker histone H1; non-histone chromosomal protein HMG-17 (HMGN2); prolactin; prolactin receptor; signal transducers and activators of transcription 5 (STAT5); transcription regulation.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

FIGURE 1.
FIGURE 1.
STAT5 inhibition results in decreased proliferation of breast cancer cells. A, effectiveness of chemical inhibition of STAT5 in preventing PRL-induced STAT5 phosphorylation and nuclear translocation. T47D cells were pretreated with the STAT5 inhibitor CAS 285986-31-4 or DMSO as the vehicle control (VC) for 1 h before stimulation with PRL. Nuclear and cytoplasmic lysates were isolated, and activation of STAT5 was analyzed by Western blotting using an antibody against phosphorylated (p-) STAT5. The membranes were then stripped and reprobed with an antibody against total STAT5. Histone H4 and tubulin were used as loading controls for the nuclear and cytoplasmic fractions, respectively. B, STAT5 inhibition reduces cell proliferation and prevents the PRL-induced increase in proliferation. T47D cells were treated with the indicated concentrations of STAT5 inhibitor or vehicle control, with or without PRL, for 3 days. BrdU incorporation was measured by absorbance as an indication of cell proliferation. Results are presented as the mean ± S.E. (error bars) of three independent experiments. Within each individual experiment, each set of treatment conditions was carried out in triplicate. Statistical significance was determined by two-sided t test assuming equal sample variance, comparing without versus with PRL (No PRL versus +PRL) at each concentration of STAT5 inhibitor. *, p ≤ 0.05; **, p ≤ 0.01.
FIGURE 2.
FIGURE 2.
PRL treatment induces chromatin decompaction and promotes binding of the transcriptional machinery. A–F, ChIP-qPCR analysis of the CISH promoter following a time course of PRL treatment. Nuclear lysates were precipitated with antibodies against H3 (A), H4 (B), H1 (C), RNAPII (D), phosphorylated (p-) RNAPII (E), or STAT5 (F). Normal IgG served as a control for nonspecific binding. For all histones and STAT5, primers amplify the region of the CISH promoter from −81 to −9 bp relative to the TSS, which includes STAT5 consensus elements. For RNAPII and phospho-RNAPII, primers amplify the region from +46 to +132 bp of the CISH TSS. See Fig. 3B for a map of the CISH promoter. The amount of DNA recovered was calculated relative to the input control and is graphed as a percentage of input. Results are presented as the mean ± S.E. (error bars), n ≥ 3 independent experiments. Statistical significance was determined by a two-sided ratio paired t test, comparing PRL treatment time points with the untreated sample. STAT5 ChIP was analyzed by two-sided paired differences t test. p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
FIGURE 3.
FIGURE 3.
PRL treatment results in increased chromatin accessibility at STAT5-binding sites. A, schematic of the qPCR amplicons used for the MNase protection assay at the CISH promoter. Amplification indicates that the DNA region was protected from MNase digestion, suggesting that it was nucleosome-bound. B, PRL treatment results in nucleosome remodeling at the CISH promoter. Top, T47D cells were treated with or without PRL for 1 h. Nuclei were permeabilized, and the chromatin was digested with MNase. Mononucleosomal DNA was purified, and MNase protection was determined by qPCR using amplicons tiling across the CISH promoter (A). Enrichment of mononucleosomal DNA was calculated relative to amplification of undigested genomic DNA. Values are plotted at the midpoint of each amplicon. Results are presented as the mean ± S.E. (error bars) of three independent experiments. Statistical significance was determined by two-way repeated measures ANOVA. The shaded region indicates statistical significance between the PRL-treated and untreated conditions, p ≤ 0.05. Bottom, diagram of the presumed nucleosome core particle positions at the CISH promoter indicated by the MNase protection assay above, assuming ∼150 bp of protection per nucleosome core particle. STAT5 consensus elements within the CISH promoter are indicated, corresponding to the axis positions shown above. C, diagram of CISH luciferase reporter constructs with sequential truncation of the distal promoter. Distances are relative to the CISH TSS. D, T47D cells were transfected with the CISH luciferase reporter constructs in C along with a Renilla luciferase control vector. Luciferase readings were normalized to the Renilla luciferase internal control, and -fold change was calculated relative to the −975 bp construct without PRL treatment. Results are presented as the mean ± S.E. of three independent experiments. Within each individual experiment, each transfection was carried out in triplicate, and each sample was read in duplicate. Statistical significance was determined by two-way repeated measures ANOVA. RLU, relative luciferase units. **, p ≤ 0.01 for −975 bp versus −84 bp, with PRL treatment. n.s., p > 0.05 for −975 bp versus all other constructs, with PRL treatment.
FIGURE 4.
FIGURE 4.
HMGN2 promotes the transcription of STAT5 target genes. A, HMGN2 knockdown in T47D cells. Shown is Western blotting analysis of cells stably infected with shRNA vectors targeting HMGN2 (shHMGN2) or a nonspecific control (shCTL). Whole cell lysates were probed with an antibody against HMGN2, and tubulin was used as a loading control. B, HMGN2 knockdown results in decreased binding at CISH. T47D shCTL and shHMGN2 cells from A were analyzed by ChIP-qPCR. Nuclear lysates were precipitated with an antibody against HMGN2, and normal IgG served as a control for nonspecific binding. Primers amplify the region of the CISH promoter from −81 to −9 bp. Recovered DNA is graphed as a percentage of input. Results are presented as the mean ± S.E. (error bars), n ≥ 3 independent experiments. Statistical significance was determined by a two-sided ratio paired t test. C, T47D shCTL and shHMGN2 cells were transfected with the CISH luciferase reporter constructs in Fig. 3C along with a Renilla luciferase control vector. Luciferase readings were normalized to the Renilla luciferase internal control, and -fold change was calculated relative to the −975 bp construct with PRL treatment. Results are presented as the mean ± S.E. of three independent experiments. Within each individual experiment, each transfection was carried out in triplicate, and each sample was read in duplicate. Statistical significance was determined by two-way repeated measures ANOVA. RLU, relative luciferase units. D, STAT5 mediates the PRL-induced expression of CISH, IER3, and PHLDA2. T47D cells were pretreated with the STAT5 inhibitor CAS 285986-31-4 or DMSO as the VC for 1 h before treatment with PRL for 1 h. RNA was isolated and analyzed by qRT-PCR for the genes indicated. -Fold change was calculated relative to VC with no PRL treatment. Results are presented as the mean ± S.E., n ≥ 3 independent experiments. Statistical significance was determined by a two-sided ratio paired t test, comparing STAT5 inhibition versus VC with PRL treatment. E, HMGN2 promotes the expression of CISH, IER3, and PHLDA2. T47D shCTL and shHMGN2 cells were treated with PRL for the indicated times. RNA was isolated and analyzed by qRT-PCR for the genes indicated. -Fold change was calculated relative to shCTL with no PRL treatment. Results are presented as the mean ± S.E., n ≥ 3 independent experiments. Statistical significance was determined by a two-sided ratio paired t test, comparing shCTL versus shHMGN2 at each time point. *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.
FIGURE 5.
FIGURE 5.
HMGN2 affects STAT5 chromatin accessibility. A, HMGN2 knockdown results in decreased STAT5 binding at CISH. T47D cells expressing shCTL or shHMGN2 were treated with or without PRL for 45 min and analyzed by ChIP-qPCR. Nuclear lysates were precipitated with an antibody against STAT5, and normal IgG served as a control for nonspecific binding. Primers amplify the region of the CISH promoter from −81 to −9 bp, which includes STAT5 consensus elements. Recovered DNA is graphed as a percentage of input. Results are presented as the mean ± S.E. (error bars) of three independent experiments. Statistical significance was determined by two-way repeated measures ANOVA. *, p ≤ 0.05. B, STAT5 is probably not recruited to the promoter region of IER3 or PHLDA2. T47D cells were analyzed by ChIP-qPCR for STAT5 recruitment following 45 min of PRL treatment. The x axis labels indicate the location of each qPCR amplicon relative to the TSS of the indicated gene. All amplicons target STAT5 consensus elements, except IER3 −0.2 kb, IER3 TSS, and PHLDA2 +0.1 kb. CISH primers are described in A. Results are presented as the mean ± S.E. of three independent experiments. Statistical significance was determined by a two-sided t test assuming equal sample variance. ****, p ≤ 0.0001 comparing without and with PRL (No PRL and +PRL) at CISH. No other regions exhibited significant STAT5 enrichment. C, HMGN2 knockdown does not affect STAT5 activation, as indicated by STAT5 phosphorylation and nuclear translocation. T47D cells expressing shCTL or shHMGN2 were treated with or without PRL for 45 min. Nuclear lysates were isolated and analyzed by Western blotting using an antibody against phosphorylated (p-) STAT5. The membrane was stripped and reprobed with an antibody against total STAT5. Knockdown of HMGN2 was verified, and histone H4 was used as a loading control. D, HMGN2 knockdown does not affect global chromatin accessibility. Nuclei from T47D shCTL and shHMGN2 cells were permeabilized, and the chromatin was digested with MNase for the indicated times. Purified DNA was analyzed by agarose gel electrophoresis.
FIGURE 6.
FIGURE 6.
HMGN2 facilitates the loss of histone H1 but does not affect nucleosome core particle remodeling. A–C, HMGN2 knockdown impairs H1 dissociation and RNAPII loading at CISH but does not affect the dissociation of H3. T47D cells expressing shCTL or shHMGN2 were treated with or without PRL for 45 min and were analyzed by ChIP-qPCR at the CISH promoter. Nuclear lysates were precipitated with antibodies against H3 (A), H1 (B), or RNAPII (C). Normal IgG served as a nonspecific control. CISH primers are described in the legends to Figs. 2 and 5A. The amount of DNA recovered was calculated relative to the input control and is graphed as a percentage of input. Results are presented as the mean ± S.E. (error bars), n ≥ 3 independent experiments. Statistical significance was determined by two-way repeated measures ANOVA. D, HMGN2 knockdown does not affect nucleosome core particle positioning by MNase accessibility. T47D cells expressing shCTL or shHMGN2 were treated with or without PRL for 1 h. Nuclei were permeabilized, and the chromatin was digested with MNase. Mononucleosomal DNA was purified, and MNase protection was determined by qPCR using amplicons tiling across the CISH promoter (Fig. 3A). Enrichment of mononucleosomal DNA was calculated relative to amplification of undigested genomic DNA. Values are plotted at the midpoint of each amplicon. Results are presented as the mean ± S.E. of three independent experiments. Statistical significance was determined by two-way repeated measures ANOVA. The shaded region indicates statistical significance between the PRL-treated and untreated conditions, p ≤ 0.05. The shCTL and shHMGN2 conditions were not significantly different. E, HMGN2 overexpression in T47D cells. Shown is Western blotting analysis of cells stably infected to express exogenous HMGN2 with either a C-terminal (HMGN2-C) or N-terminal FLAG tag (HMGN2-N) or an empty vector (EV) control. Nuclear lysates were probed with an antibody against HMGN2, and histone H4 was used as a loading control. FLAG-tagged HMGN2 runs slightly higher than endogenous HMGN2. F and G, HMGN2 overexpression does not further promote STAT5 binding or H1 dissociation at CISH. Cells from E were analyzed by ChIP-qPCR as in A–C, using antibodies against STAT5 (F) or H1 (G). Results are presented as the mean ± S.E. of three independent experiments. Statistical significance was determined by two-way repeated measures ANOVA. n.s., p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.
FIGURE 7.
FIGURE 7.
HMGN2 does not affect post-translational histone modifications. A, HMGN2 does not affect histone acetylation at H3K9 or H3K14. T47D cells expressing shCTL or shHMGN2 were treated with or without PRL for 45 min and were analyzed by ChIP-qPCR at the CISH promoter. Nuclear lysates were precipitated with antibodies against acetylated H3K9 (H3K9Ac), H3K14 (H3K14Ac), or total H3. CISH primers are described in the legends to Figs. 2 and 5A. The percentage of input DNA recovered with the acetyl-specific antibodies was normalized to the percentage of input recovered for total H3 in a parallel sample. Results are presented as the mean ± S.E. (error bars), n ≥ 3 independent experiments. Statistical significance was determined by two-way repeated measures ANOVA. B, the CISH promoter is not enriched for histone trimethylation at H3K9 or H3K27. ChIP-qPCR analysis was carried out as in A using antibodies against H3K9me3 or H3K27me3. Positive (+) controls for enrichment are ZNF554 for H3K9me3 and α-Satellite for H3K27me3 (EMD Millipore). The amount of DNA recovered was calculated relative to the input control and is graphed as a percentage of input. Results are presented as the mean ± S.E. of three independent experiments. Statistical significance was determined by two-way ANOVA. n.s., p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01.
FIGURE 8.
FIGURE 8.
HMGN2 promotes histone H1 loss to regulate STAT5 binding and gene transcription. A, efficiency of H1.2 knockdown. T47D cells were transiently transfected with siRNA targeting H1.2 using two different sequences (siH1.2 #1 and #2) or a nonspecific control (siCTL). Whole cell lysates were analyzed by Western blotting and probed with antibodies against H1.2 or tubulin (loading control). B, H1 knockdown rescues gene expression following HMGN2 knockdown. T47D shCTL and shHMGN2 cells were transiently transfected with the siRNA constructs in A and treated with or without PRL for 1 h. RNA was isolated, and cDNA was synthesized by RT-PCR and analyzed by qPCR. -Fold change was calculated relative to shCTL and siCTL with no PRL treatment. Results are presented as the mean ± S.E. (error bars), n ≥ 3 independent experiments. Statistical significance was determined by a two-sided ratio paired t test. C, H1 knockdown rescues STAT5 binding following HMGN2 knockdown. Cells were treated as in (B) and were analyzed by ChIP-qPCR for STAT5 at CISH following 45 min of PRL treatment. Recovered DNA was normalized to input and calculated as -fold enrichment compared with shHMGN2 and siCTL. Results are presented as the mean ± S.E. of three independent experiments. Statistical significance was determined by a two-sided t test assuming equal sample variance. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.
FIGURE 9.
FIGURE 9.
H1 knockdown enhances gene expression and breast cancer cell proliferation in response to reduced STAT5 activation. A, H1 knockdown rescues gene expression following partial STAT5 inhibition. T47D cells were transfected with siCTL, siH1-1, or siH1-2 (Fig. 8A). Transfectants were pretreated with the STAT5 inhibitor (200 μm) or DMSO VC for 1 h, followed by PRL treatment for 2 h. RNA was isolated, and cDNA was synthesized by RT-PCR and analyzed by qPCR. -Fold change was calculated relative to VC, siCTL with no PRL treatment. Results are presented as the mean ± S.E. (error bars), n ≥ 3 independent experiments. Statistical significance was determined by a two-sided ratio paired t test. B, H1 knockdown rescues cell proliferation in response to intermediate levels of STAT5 inhibition. T47D cells were transfected with siCTL or siH1 (pooled 1 and 2). Transfectants were treated with the indicated concentrations of STAT5 inhibitor, with or without PRL, for 3 days. BrdU incorporation was measured by absorbance as an indication of cell proliferation. Results are presented as the mean ± S.E. of three independent experiments. Within each individual experiment, each set of treatment conditions was carried out in triplicate. Statistical significance was determined by two-sided t test assuming equal sample variance. Statistical significance shown in the figure is comparing siCTL + PRL versus siH1 + PRL at the indicated concentration of STAT5 inhibitor. Other statistically significant comparisons are as follows: siCTL No PRL versus siH1 No PRL (p ≤ 0.01 at 0 inhibitor; p ≤ 0.05 at 25 μm); siH1 No PRL versus siH1 + PRL (p ≤ 0.05 at 25 μm; p = 0.052 at 50 μm). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.
FIGURE 10.
FIGURE 10.
Genes regulated by H1 are enriched for STAT signaling pathways. A and B, genes found to be up-regulated by H1.2 knockdown in MCF7 cells by Kim et al. (40) were analyzed by the Enrichr enrichment analysis tool (50, 51). Significantly enriched gene sets of interest are plotted with their adjusted p values (q values), which were calculated using the Benjamini-Hochberg method for correction for multiple-hypothesis testing. KEGG pathways are shown in A along with the associated KEGG identifiers. ChIP enrichment analysis through ChEA 2015 is shown in B, with numbers indicating the publication PMID for each study.
FIGURE 11.
FIGURE 11.
Diagram of PRL-induced chromatin remodeling and regulation of STAT5 binding. Gray arrow, possible contribution (see “Discussion”).

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback