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Department of Molecular and Cellular Biology (E.B.K., M.H., W.X., J.M.R.), and Children’s Nutrition Research Center (M.R.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Jeffrey M. Rosen, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: jrosen{at}bcm.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Stat5 was originally identified in the lactating mammary gland (3, 4, 5) and is the primary transcription factor responsible for signaling by Prl (5, 6). After stimulation by Prl, the principal Stat5 isoform expressed in the mammary gland, Stat5a, is rapidly tyrosine phosphorylated by the JAK2 kinase, dimerizes, and translocates into the nucleus, where it interacts as a tetramer with clustered Stat5 binding sites in both the ß-casein promoter and enhancer. However, optimal ß-casein gene expression is only achieved after stimulation by both Prl and glucocorticoids, indicating the requirement for synergy between GR and Stat5 (7, 8, 9, 10, 11, 12).
Closely spaced DNA binding sites for Stat5 and other transcription factors are required for maximal transcriptional activation of many genes. Accordingly, two functional Stat5 binding sites and seven half-palindromic GR elements (GREs) are present in the proximal promoter region of ß-casein gene, as revealed by comparative analysis of ß-casein gene sequences from different species (6, 13, 14). Interestingly, the mutation of Stat5 binding site in the ß-casein promoter abolishes both Prl and GR responsiveness (3). Mutation of upstream half-GREs severely diminishes, whereas deletion of this region or mutation of the three downstream half-GREs completely abolishes the cooperative effects of hydrocortisone (HC) and Prl at the ß-casein promoter in HC11 mammary epithelial cells (12).
C/EBPß is a member of the CCAAT/enhancer binding protein family of bZIP transcription factors, which bind to specific DNA sequences as homo- and heterodimers, and plays critical role in mammary gland development and milk protein gene expression (15, 16). C/EBPß exists in three distinct isoforms, termed liver-enriched transcriptional activator protein (LAP) (full-length protein, 39 kDa), LAP2 (a 21-amino acid truncation at the N terminus, 36 kDa) and liver-enriched transcriptional inhibitory protein (LIP) (a large N-terminal truncation, 20 kDa). LAP and LAP2 are activators of gene transcription. The LIP isoform lacks an activation domain and functions as a dominant-negative version to suppress gene activation (17). Maximal cooperative ß-casein transcription has been observed when all three transcription factors (Stat5, GR, and C/EBPß) are expressed in heterologous cells, and this cooperativity was not seen between Stat5 and C/EBPß in the absence of full-length, transcriptionally active GR (18). The analysis of different GR variants has established several unique roles for GR at the ß-casein proximal promoter including enhanced transactivation by C/EBPß, possibly by promoting a nonrepressive conformation, and increased Stat5 activation in part by prolonging tyrosine phosphorylation. All three transcription factors, Stat5, GR, and C/EBPß, interact with the nuclear transcriptional coactivator p300 (19, 20), which possesses histone acetyltransferase activity involved in chromatin remodeling to facilitate gene transcription.
On the other hand, Ying Yang-1 (YY-1) has been demonstrated to play a functional role in ß-casein gene repression (21). Mutation of the YY-1 site has been shown to lead to a stronger lactation-associated activation complex in extracts from the lactating mammary gland, and the mutation of the Stat5 binding site, which was crucial for the formation of the lactation-associated complex, caused an increase in YY-1 DNA binding (22). The YY-1 site in the ß-casein promoter is a low-affinity site, which is presumably influenced by protein:protein interactions, including interactions with C/EBPß (23).
Lactogenic hormone regulation of ß-casein gene expression in mammary epithelial cells, therefore, provides an excellent system to investigate kinetic chromatin remodeling to understand better the complex transcriptional control of this gene. Using HC11 cells as a model, we have tested the effects of Prl and glucocorticoids both singly and in combination at different time points after hormone treatment. Using chromatin immunoprecipitation (ChIP) analysis, we have determined the dynamics of assembly and disassembly of Stat5, GR, C/EBPß, and YY-1 at the hormonally activated ß-casein proximal promoter as well as the distal mouse ß-casein enhancer located approximately –6 kb upstream of the transcription start site (Fig. 1A
) (2, 14). Prl alone resulted in a rapid recruitment of both Stat5 and histone deacetylase 1 (HDAC1) to the ß-casein promoter and enhancer, and reciprocally the dissociation of YY1 from the proximal promoter. In addition, we have examined the recruitment of coactivator p300 and determined chromatin acetylation status as a function of hormonal treatment. Finally, we have established the time course of RNA polymerase II (Pol II) and phospho-RNA polymerase II (p-Pol II) accumulation at the ß-casein promoter and enhancer after stimulation with HC and Prl. Although glucocorticoids alone led to a rapid increase in histone H3 acetylation, treatment with both hormones was required for stable association of p300 and phospho-Pol II at both the promoter and enhancer. Collectively, these data suggest a model for the assembly of a multiprotein complex that helps to define how the signaling pathways controlled by these lactogenic hormones are integrated to regulate ß-casein gene expression.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), and ß-casein mRNA levels were quantitated by quantitative PCR (qPCR) as described in Materials and Methods. GAPDH was used as a control for mRNA integrity.
Treatment with HC alone produced no detectable increase in ß-casein mRNA. A small (
3-fold) increase in ß-casein RNA accumulation at 24 h was detected in cells treated with Prl alone. However, more than a 500-fold induction of ß-casein mRNA was observed 24 h after treatment with both hormones with a significant induction (7-fold) of mRNA accumulation observed as early as 1 h (Fig. 1C).
Histone Acetylation and HDAC1 Recruitment Are Involved in Activation of ß-Casein Transcription
To explore the possibility that ß-casein expression is regulated by changes in chromatin structure, we examined histone acetylation and HDAC1 recruitment using ChIP assays and antibodies against acetylated histone H3 (Fig. 2, A and B) and HDAC1 (Fig. 3, A and B). A summary of the histone acetylation status of chromatin and HDAC1 recruitment after hormone stimulation for different periods of time (0–24 h) assayed using qPCR is shown in Figs. 2 and 3. In cells treated with HC only and in combination with Prl, a rapid increase in acetylation was detected within 5 min at both the ß-casein promoter and enhancer with maximal histone acetylation observed by 15 min, which declined thereafter. However, the maximal fold change observed with HC only was twice (12- to 15-fold) that seen in cells stimulated with both hormones (6- to 7-fold). In cells stimulated with Prl alone, no change was detected in histone H3 acetylation compared with nontreated cells. Interestingly, as discussed previously, several half-palindromic DNA binding sites (1/2GREs) are present within the ß-casein proximal promoter, but no known GREs are found within the enhancer (Fig. 1A). The observed hyperacetylation at both regions of the ß-casein gene supports the hypothesis that GR may exert multiple functions: it may initiate chromatin remodeling required for transcription initiation, and at the same time it may play a bridging role in ß-casein activation through interactions with Stat5 and C/EBPß as well as binding to different coactivators, comodulators, and/or corepressors.
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, A and B). However, in cells treated with HC alone, we did not detect any changes in HDAC1 occupancy at any time after HC addition. Treatment with Prl alone showed a rapid accumulation of HDAC1 at both the promoter and enhancer within 5 min, which peaked by 15 min, and then declined with longer stimulation (Fig. 3B). In cells treated with both hormones, the peak of HDAC1 accumulation was shifted to 5 min followed by decrease at 15 min, and essentially a complete disappearance occurred by 30 min. These results suggest that Stat5, but not GR, is responsible for the transient increase observed in HDAC1 recruitment and associated deacetylase activity. It has been reported previously that Stat5-induced transcription at some promoters may involve recruitment of HDAC1 and deacetylation of C/EBPß (25, 26, 27).
Recruitment of Transcription Factors Stat5, GR, C/EBPß, and YY-1 in Chromatin Remodeling
To better understand how lactogenic hormones facilitate the recruitment of specific transcription factors to the ß-casein promoter and enhancer, we used antibodies to GR, Stat5, YY-1, and C/EBPß in modified ChIP assays and employed real-time qPCR for quantitative analysis after treatment with Prl alone, HC alone, or both hormones.
Because antibodies were not available for ChIP assays to identify the specific C/EBPß isoforms associated with the ß-casein promoter and enhancer, we instead analyzed their association, as well as that of GR and Stat5, with total chromatin after hormonal treatment. This was accomplished by performing ChIP/Western experiments as shown in Fig. 4, A and B, either at 30 min or 24 h after the addition of Prl and HC. We were able to demonstrate the recruitment to chromatin of Stat5, GR, and C/EBPß after stimulation of HC11 cells with both hormones (Fig. 4, A and B). By using conventional ChIP assays, accumulation of all three transcription factors was detected within 15 min at the proximal promoter after stimulating cells with HC and Prl (Fig. 4C).
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) in cells treated with HC alone or in cells treated with both hormones, showed a transient increase in chromatin association (2-fold) at the proximal promoter region between 5 and 30 min, followed by decreased association at 4–24 h after hormone addition. About the same (2-fold) increase in GR accumulation was found at the distal enhancer in 5–15 min after hormone exposure. In cells treated with Prl alone, as expected, we did not observe any changes in GR binding at both the promoter and enhancer compared with untreated cells.
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). In cells treated with HC alone, we observed only slight increase in C/EBPß accumulation 15 min after treatment compared with nontreated cells. An increase in C/EBPß binding to the promoter (1.5-fold) and enhancer (2-fold) was detected after 15 min of treatment with Prl, and declined thereafter. In cells treated with both hormones, the same increased level (1.5- to 2-fold) of C/EBPß accumulation was found at the promoter in 15–30 min, but in this case C/EBPß appears to stay associated for up to 24 h. However, no significant changes were detected at the enhancer region under the same conditions. The regulation of ß-casein gene expression has been shown previously to be influenced by the LAP:LIP isoform ratio rather than the absolute level of C/EBPß (28). HC11 cells treated with insulin and Prl showed an increased level of both LAP and LIP, whereas the addition of glucocorticoids inhibited preferentially the expression of the LIP isoform (28). Interestingly, by performing ChIP/Western experiments, we were able to detect an increase in the LAP isoform bound to chromatin with no change detected in the amount of LIP in cells treated for 24 h with both HC and Prl (Fig. 4B). However, a lack of C/EBPß isoform-specific antibodies prevents the direct assessment of LIP/LAP association at the ß-casein promoter and enhancer using ChIP assays.
The time course of assembly and disassembly of Stat5 at the ß-casein promoter and enhancer in cells treated with HC, or Prl, or the combination with both hormones is shown in Fig. 6. Treatment with Prl alone resulted in an elevated (2- to 2.5-fold) accumulation of Stat5 at 15 min that was diminished to basal levels by 24 h. However, a much greater accumulation of Stat5 at the promoter (9.5-fold) and enhancer (11.5-fold) was observed at 15 min in cells treated with both Prl and HC. No Stat5 accumulation was detected in cells stimulated with HC alone, as expected.
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). In contrast, a significant increase was observed using an antibody directed at the N terminus of Stat5a (Fig. 6B). In cells treated with both hormones, we consistently observed Stat5 accumulation using both C-terminal and N-terminal antibodies, although with slightly different kinetics (Fig. 6B), suggesting that GR interaction with Stat5a might influence the accessibility of the C terminus of Stat5a. These results support previous observations concerning the differential accessibility of the amphipathic 
-helical region at the C-terminal end of Stat5 under different activation conditions (29). Thus, N- and C-terminal anti-Stat5a antibodies give comparable immunoprecipitation of Stat5 when activated by Prl, but when activated by Src conformational differences due to tyrosine phosphorylation of the carboxyl-terminal activation domain result in decreased recognition and immunoprecipitation by the C-terminal antibody (29).
ChIP assays performed using YY-1 antibody in HC11 cells after Prl treatment showed a rapid disassociation of YY-1 from the ß-casein promoter (Fig. 7A). Maximal disassociation of YY-1 in cells treated with both hormones was observed by 15–30 min. No changes in YY-1 association were observed in cells treated with HC alone.
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, in HC11 cells treated with Prl, Stat5 and YY-1 display a reciprocal relationship in their association with the ß-casein promoter; when Stat5 binding increases, YY-1 decreases. These data are consistent with earlier studies (21, 22), which suggested that YY-1 represses ß-casein gene expression in the absence of Prl and that lactogenic hormones act by relieving this repression. No dynamic changes in YY-1 binding were detected at a distal enhancer in cells exposed to HC alone, Prl alone, or both hormones (data not shown). These results were as expected because the enhancer region of ß-casein does not contain any known DNA binding sites for YY-1.
Recruitment of p300 and Pol II
ChIP assays were performed using antibodies to p300 (Fig. 8A). In cells treated with HC alone, the recruitment of this coactivator was observed at both the proximal promoter and distal enhancer regions by 5 min, with a peak of accumulation seen between 5 and 15 min followed by a return to basal levels at the enhancer, but a biphasic pattern of p300 association was seen at the promoter with ongoing HC stimulation. Treatment of HC11 cells with Prl alone resulted in a maximal accumulation of p300 both at the promoter and enhancer at 15 min followed by a decrease to essentially control levels at both the promoter and enhancer by 24 h. Interestingly, in cells treated with both hormones, an increased level of p300 association was observed with both the promoter and enhancer at 5 min after hormone addition compared with the hormones added singly. Furthermore, in contrast to Prl and HC added alone, the level of p300 binding remained significantly elevated over the controls at 24 h when both hormones were present. These results suggest that coactivator/comodifier p300 is involved in the assembly of the multiprotein complex on the activated ß-casein gene through the protein:protein interactions with GR, C/EBPß, and Stat5 (Fig. 8B).
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. An increased level of Pol II associated both with the proximal promoter and somewhat surprisingly with the distal ß-casein enhancer was observed within 5 min after stimulation cells with both hormones with no significant changes detected up to 24 h. The dynamics of p-Pol II accumulation, however, were quite different; it is present at the promoter by 30 min and at the enhancer by 1 h with maximum levels detected after 1 h at the promoter and later (4 h) at the enhancer, which correlates with the detection of mRNA transcripts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). We also determined the association of the cofactors (YY-1, HDAC1, and p300), which are involved in ß-casein gene expression and possess posttranslational modification activities. Finally, we investigated the dynamics of histone acetylation and established the time course of Pol II and p-Pol II accumulation at the hormonally activated ß-casein promoter and enhancer. We have attempted to integrate these experimental results into a working model for the assembly of a multiprotein complex at the ß-casein regulatory regions, which can be summarized as follows.
The Initial Binding of the Specific Transcription Factors within the ß-Casein Gene Regulatory Regions
In cells treated with HC alone, we observed a statistically significant increase in GR binding to promoter and enhancer within 15 min (Fig. 5). Seven half-palindromic DNA binding sites for GR (1/2GREs) are present at ß-casein proximal promoter, but none has been identified at the enhancer region (Fig. 1A). The increased level of GR association with enhancer in cells treated with HC or in combination with Prl may, therefore, be explained by either of two not necessarily mutually exclusive models: 1) indirect binding through the association with other transcription factors and/or coactivators; 2) a looping model between promoter and enhancer regions at ß-casein gene via protein:protein interactions. The participation of GR in transcriptional activation of the 2-macroglobulin gene (which does not contain GR-binding sites in the enhanceosomal DNA) has been proposed previously to occur through binding to either Stat3 or c-Jun (30), one of many such examples of indirect interactions of nuclear receptors with promoters and enhancers (31). There is, however, also a possibility that GR can be recruited to the enhancer by a noncanonical GRE (32).
Comparative sequence analysis of different mammalian species has revealed two conservative DNA-binding sites for Stat5 at the proximal promoter and two sites at the distal enhancer regions (Fig. 1A) (2, 14). In cells treated with Prl alone, we observed a rapid (15-min) 3-fold increase in Stat5 accumulation at the proximal promoter, which returned to the control levels within 1 h of treatment (Fig. 6A). Combined HC and Prl treatment resulted in a significantly greater accumulation of Stat5 (9-fold), consistent with the known synergism between Prl and glucocorticoids (12). Interestingly, in cells stimulated with both hormones, the level of Stat5 accumulation at the promoter remained increased (3-fold) at 24 h after hormone addition with similar kinetics observed in Prl-induced Stat5 binding at the ß-casein distal enhancer (Fig. 6A).
It has been established previously that the ß-casein promoter contains a low affinity DNA binding site for YY-1 (Fig. 1A) (2, 14). Furthermore, using reporter gene constructs in stably transfected HC11 cells, YY-1 was demonstrated to repress ß-casein expression in the absence of Prl, and mutation of the YY1 DNA binding site resulted in hormone-independent induction (21, 22). Consistent with these data, we were able to demonstrate using quantitative ChIP assays that YY-1 is bound to the ß-casein promoter and represses ß-casein gene expression in the absence of lactogenic hormones, and the binding is relieved after Prl treatment enabling formation of a transcription complex (Fig. 7).
Because C/EBPß (LIP) interacts directly with YY-1 (21) and YY-1 interacts with several HDACs (33, 34), it is possible that interactions between YY-1 and C/EBPß isoform LIP are responsible for the repression of ß-casein promoter in the absence of lactogenic hormones through the recruitment of histone deacetylases. We performed ChIP assays using antibodies to HDAC1 (Fig. 3) and did not observe a correlation of HDAC1 association with the dynamics of YY-1 disassociation at the proximal promoter (Fig. 7). These data are consistent with the results (35), demonstrating that YY-1:HDAC1 complex was not supershifted by HDAC1 antibodies, suggesting that a histone deacetylase other than HDAC1 is most likely associated with YY-1 resulting in transcriptional repression.
The Recruitment of HDAC1
It has been shown recently that histone deacetylase activity is required for transcriptional activation by Stat5 (26, 27) and that Stat5 can activate gene expression by recruiting HDAC1 leading to the deacetylation of C/EBPß (25). Consistent with these results, it has been reported previously that a pharmacological inhibitor of histone deacetylase, trichostatin A, can inhibit endogenous ß-casein gene expression in mammary epithelial cells (36). Furthermore, it has been proposed that C/EBPß proteins when acetylated may be incapable of binding DNA and activating transcription (25). After hormone stimulation, activated Stat5 would then bind to sites adjacent to the C/EBPß binding sites at gene regulatory regions and recruit HDAC1. Consequently, HDAC1 may deacetylate C/EBPß LAP, and once deacetylated, LAP-LAP homodimers and/or LAP-LIP heterodimers perhaps can bind with a high affinity to its DNA binding sites, interact with other transcription factors such as GR, and recruit the necessary coactivators required to initiate transcription. In support of this proposed model, the current experiments have demonstrated HDAC1 recruitment in the first 5 min after treatment of HC11 cells with Prl at both the ß-casein promoter and enhancer. This increase in HDAC1 recruitment correlates well with the dynamics of Stat5 and C/EBPß binding (Fig. 8B). However, because there appears to be multiple acetylation sites perhaps with different functional consequences present in C/EBPß (Johnson, P., personal communication), this model is probably overly simplistic.
Recruitment of Coactivator p300 to ß-Casein Gene Regulatory Regions
Many studies have implicated CBP and the related p300 protein (37) as transcriptional coregulators that participate in the activities of many different transcription factors (38, 39, 40). These large transcriptional coactivators possess intrinsic histone acetyltransferase activities capable of modifying the chromatin organization and facilitating the binding of Pol II transcription complex to the core promoter (41). Using quantitative ChIP assays in cells stimulated with Prl, similar kinetics of Stat5, C/EBPß binding, and the recruitment of p300 were observed at the ß-casein promoter and enhancer (Fig. 8B). Treatment of cells with HC and Prl resulted in a cumulative increase in p300 accumulation compared with either hormone alone (Fig. 8A). These results suggest that coactivator p300 is rapidly recruited at the gene regulatory regions through the protein:protein interactions with the primary transcription factors after their DNA binding.
Histone Acetylation at the ß-Casein Gene Regulatory Regions
Histone acetylation is a key step in transcriptional control. Acetylation of the N-terminal tails of the histone proteins, particularly H3 and H4, is thought to lead to relaxed chromatin structure and, therefore, enhance the rate of transcription. Using antibodies to acetylated H3 in ChIP assays (Fig. 2), we were able to detect a rapid (in 5 min) increase in histone acetylation at both the ß-casein gene promoter and enhancer regions after stimulation with HC and Prl, which peaked at 15 min. In cells treated for 15 min with HC alone, an even greater peak in histone acetylation was observed. The strict dependence of histone acetylation upon HC treatment strongly suggests that acetylation was directly initiated by GR recruitment to the ß-casein promoter and enhancer.
Recruitment of Pol II
We observed an increased level of Pol II accumulation at the ß-casein promoter (3-fold) and enhancer (2.5-fold) after 5 min of stimulation with HC and Prl that remained elevated after 24 h of hormonal treatment (Fig. 9). In parallel, the pattern of p300 recruitment in cells treated with both hormones (Fig. 8): the association of p300 with both promoter and enhancer was increased (6- to 7-fold) within 5 min and then gradually decreased (3-fold) after 24 h of treatment, but remarkably, never reached the basal level (as seen in nonstimulated cells). The correlation between p300 and Pol II recruitment suggests that the p300:Pol II complex might be recruited as a unit, consistent with a previous study (42). It is also believed that the C-terminal domain of nonphosphorylated Pol II is responsible for mediating multiple protein:protein interactions involved in the assembly of the preinitiation complex, whereas the subsequent phosphorylation of the C-terminal domain contributes to the initiation of transcription and elongation of the primary transcript (42).
Binding of the Basal Transcriptional Machinery to the DNA Template and the Initiation of the Transcription
The analysis of the time course of Pol II phosphorylation at the ß-casein promoter (Fig. 9) indicated that Pol II was not activated during the first 15 min of hormonal stimulation, despite the presence of GR, Stat5, C/EBPß, p300, and acetylated histone H3. The increase in p-Pol II at the promoter was first observed at 30 min and peaked at 1–4 h, which correlates well with the time of ß-casein mRNA appearance (Fig. 1, B and C). These data are consistent with the observation that p-Pol II represents a final step in the transcriptional initiation process. It has been proposed that SWI/SNF mediates this final step by remodeling the proximal promoter region, thus stimulating an effective binding of the basal transcriptional machinery to the DNA template (43).
The mechanism by which enhancers participate in the recruitment of chromatin remodeling complexes and Pol II to mediate gene transcription is not yet completely understood, and several alternative, but not necessarily mutually exclusive, models have been proposed. These include the following: 1) tracking along the chromatin between the enhancer and promoter (tracking model), 2) direct contact between enhancer and promoter with the intervening DNA looped out (looping model), and 3) linking of the intervening DNA (linking model) (44). Combined linking and looping (44), or looping and tracking (45) models also have been described. In our experiments, we were able to detect an increased level of p-Pol II with a peak at 4 h within the distal enhancer region approximately 6 kb upstream of the start site of transcription (Fig. 9). The association of p-Pol II at both regulatory regions helps to support a looping model, which posits that the proximal promoter and distal enhancer communicate with each other through the protein:protein interactions. It is possible that a looping structure between enhancer and promoter provides binding surfaces that increase preinitiation complex recruitment and enhance the transition from transcription initiation to elongation (46). The similar dynamics of assembly of transcription factors, the coactivator p300 and RNA Pol II, as well as histone acetylation at both regions provides support for this model, but the future studies will be required to directly test this hypothesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Antibodies
Anti-Stat5 (N-20) (sc-836), anti-YY-1 (c-20) (sc-281), anti-Pol II (N-20) (sc-899), and anti-C/EBPß (c-19) (sc-150) were purchased from Santa Cruz. Antibodies to C-terminal Stat5a were purchased from Zymed Laboratories (San Francisco, CA) (13-3600). Anti-acetyl-histone H3 (06-599), anti-HDAC1 (05-614), and anti-phospho-Pol II (05-623) were purchased from Upstate Biotechnology. Anti-GR (PA1-512) antibodies were purchased from Affinity Bioreagents (Golden, CO).
Tissue Culture
HC11 cells were grown at 37°C and 5% CO2 in RPMI medium supplemented with 10% bovine calf serum (JRH Biosciences, Lenexa, KS; 12-13378), 2 mM glutamine (JRH Biosciences; 59-20277), 50 µg/ml gentamycin (Sigma-Aldrich; G-1272), 5 µg/ml bovine insulin (Sigma-Aldrich; I-6634), and 10 ng/ml murine epidermal growth factor (JRH Biosciences; 85-510-601). After cells reached confluency (2 x 107), they were grown for an additional 3 d, and then primed for 48 h in priming medium (0.5 M glutamine/5 µg/ml insulin/10% stripped donor horse serum/RPMI 1640 medium). Cells were treated with HC alone (1 µg/ml), Prl alone (1 µg/ml), or both hormones at various periods of time (5 min to 24 h).
RNA Isolation and Analysis
Total RNA was isolated from untreated and hormone-treated cells using Trizol (Invitrogen, San Diego, CA). After DNase I treatment, total RNA was reverse-transcribed (SuperScript; Invitrogen) and amplified by PCR using primers specific to exon VII of ß-casein gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers for exon VII were as follows: exonVII-forward (5'-CATATGCTCAGGCTCAAACCATCTCT-3') and exonVII-reverse (5'-GTACTGCAGAAGGTCTTGGACAGAC-3'). PCR products were separated on agarose gels and visualized by ethidium bromide staining.
ChIP Assay
ChIP was performed as described previously (47) with a few modifications. Cells were cross-linked with 1% formaldehyde added directly to cell culture medium for 10 min at room temperature. The cell monolayers were washed twice with ice-cold 1x PBS and collected into dithiothreitol solution [100 mM Tris-HCl (pH 9.4)/10 mM dithiothreitol] followed by incubation at 30 C for 10 min. Cells were washed sequentially with ice-cold PBS, buffer I [0.25 M Triton X-100, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES (pH 6.5)] and buffer II [200 mM NaCl, 1 mM EDTA, 10 mM HEPES (pH 6.5)], containing protease and phosphatase inhibitors, and then lysed in lysis buffer [1% sodium dodecyl sulfate (SDS), 10 mM EDTA, 50 mM Tris-HCl (pH 8.1) plus protease inhibitors and 1 mM phenylmethylsulfonylfluoride] for 10 min on ice. Sonication was performed using a Branson-450 Sonifier with microtip in 7-sec bursts followed by 1 min of cooling on ice for a total sonication time of 21 sec per sample resulted in DNA fragment sizes of 0.3–1.5 kb. Then samples were centrifuged at 14,000 for 10 min at 4 C. Supernatants were diluted 5-fold in ChIP dilution buffer [1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8.1) plus protease and phosphatase inhibitors] and precleared for 30 min at 4 C with 20 µl preimmune serum and 80 µl salmon sperm DNA/protein A/G agarose slurry. Ten percent of total supernatant was saved as a total input control and processed with the eluted immunoprecipitates (IPs) beginning with the cross-linking reversal step. Then, 5 µg of specific antibodies were added to the chromatin solutions (with no antibody control included), and samples were incubated at 4 C for overnight with rotation. Immunocomplexes were collected with 60 µl of the salmon sperm DNA/protein A/G agarose beads for 1 h at 4 C with rotation. Beads were then washed consecutively for 10 min each with low salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8.1)], high salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl (pH 8.1)], and LiCl wash buffer [0.25 mM LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)] and twice in 1x TE buffer. Complexes were then eluted twice in 100 µl freshly made elution buffer (1% SDS/0.1 M NaHCO3). To reverse formaldehyde cross-links, 1 µl 10 mg/ml RNase and 5 M NaCl to a final concentration of 0.3 M was added to the eluates and they were incubated at 68 C for overnight. To 200 µl of eluent, 4 µl of 0.5 M EDTA, 8 µl of 1 M Tris (pH 6.5), and 1 µl of 20 mg/ml proteinase K was added, and samples were incubated for 2 h at 45 C. DNA was recovered using the QiaQuick spin columns (Qiagen, Valencia, CA) and eluted in 80 µl of 10 mM Tris (pH 8.0). Recovered DNA was then quantified by real-time PCR.
Quantitative Real-Time PCR
qPCR was performed on ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) using SYBR Green (Molecular Probes, Eugene, OR) as a marker for DNA amplification. The following primers were used: promoter-forward, 5'-CACTTGGCTGGAGGAACATGTAGTT-3'; promoter-reverse, 5'-ACATCTGAAGTTCTTACCTTTAGTGGAGG-3'; enhancer-forward, 5'-AGTCTCAAGGAAATACTGGATCTATTG-3'; enhancer-reverse, 5-GAGTTTGTGAACCATCTTTTACTAACC-3'. Real-time PCR was performed with 5 µl DNA (input DNA was diluted 1:10) using 40 cycles of three-step (94 C for 30 sec, 55 C for 30 sec, 72 C for 30 sec) amplification. IP data were normalized to input DNA, and the amounts of DNA recovered in the IPs were expressed as percentages of input DNA. Each PCR generated only the expected specific amplicon that was proved by running the melting temperature profiles of the final products (dissociation curve). No PCR products were observed in the absence of template. The relative amounts of IP and input DNA were determined by comparison to a standard curve generated by serial dilutions (1:100, 1:200, 1:500, 1:1000, and 1:2000) of input DNA. Both experimental IPs and input DNA were run in triplicate. The average of the experimental IP DNA triplicate was divided by the average of input triplicate, and the resulting normalized values were used for statistical analysis. All differences reported were statistically significant (P < 0.05). PCRs for each antibody were performed at least three times. All ChIPs were performed on chromatin from at least two different cell culture experiments with essentially similar results.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The authors have nothing to disclose.
First Published Online June 13, 2006
Abbreviations: C/EBPß, CCAAT enhancer binding protein ß; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GR, glucocorticoid receptor; GRE, GR element; HC, hydrocortisone; HDAC1, histone deacetylase 1; IP, immunoprecipitate; LAP, liver-enriched transcriptional activator protein; LIP, liver-enriched transcriptional inhibitory protein; Pol II, RNA polymerase II; p-Pol II, phosphorylated RNA polymerase II; Prl, prolactin; qPCR, quantitative PCR; SDS, sodium dodecyl sulfate; Stat5, signal transducer and activator of transcription 5; YY-1, Ying Yang-1.
Received for publication April 11, 2006. Accepted for publication June 5, 2006.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, is essential for ductal morphogenesis, lobuloalveolar proliferation, and functional differentiation in the mouse mammary gland. Genes Dev 12:1917–19282-macroglobulin gene. Genes Dev 17:2564–2577NURSA Molecule Pages Link:
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