In order to analyze the events occurring at the promoter of a gene upon mitogenic stimulation, we investigated the mitogen-inducible collagenase promoter. Using ChIPs, we established that first the transcription factors c-Jun and c-Fos, the basal transcription factor TBP, RNA polymerase II, and the methyltransferase SET9 are recruited to the collagenase promoter (Fig. ). Simultaneous with the appearance of SET9 at the collagenase promoter, first dimethylation and later trimethylation of lysine 4 of histone H3 are observed. Next, the HAT p300 and the kinase RSK2 are recruited. The recruitment of p300 might occur via its interaction with c-Jun (23
), while RSK2 might be recruited via its interaction with p300 (Fig. ). The presence of p300 and RSK2 at the collagenase promoter correlates with the appearance of acetylated and phosphorylated histones. Finally, the cascade of recruitment events culminates with the association of the remodeling complex component Brg-1, which coincides with initiation of transcription. The timing of recruitment suggests that recruitment of the nucleosome remodeling machinery requires prior histone methylation, acetylation, and phosphorylation of the nucleosome.
FIG.8. Model depicting the ordered recruitment of chromatin-modifying and basal factors to the collagenase promoter. Shortly after TPA and 20% serum treatment, c-Jun, c-Fos, TBP, RNA polymerase II, and SET9 assemble on the collagenase promoter and histones are (more ...)
Thus far, the in vivo sequence of nucleosomal modifying and remodeling events leading to transcriptional activation has been studied for four types of promoters: the cell cycle-regulated yeast HO promoter, the virus-induced beta interferon promoter, the differentiation-induced α1
-antitrypsin promoter, and the hormone-induced cathepsin D promoter (see the introduction). The recruitment results described here predominantly resemble the situation described for the α1
-antitrypsin gene (40
), in which TBP and RNA polymerase II appear at the promoter before transcriptional activation. Transcription is started only after the recruitment of HAT and remodeling complexes, suggesting that at these promoters chromatin reconfiguration is the defining step of the initiation process. Interestingly, although the recruitment of factors is an ordered event for all promoters examined so far, the order and timing of recruitment are different in all cases (HO, beta interferon, α1
-antitrypsin, cathepsin D, and collagenase), suggesting that each gene has its own order and timing of events leading to transcription.
The chromatin reconfigurations that are required for collagenase expression to be induced are most likely brought about by nucleosomal modifications. Two observations are relevant in this respect. First, the modifications at the collagenase gene are controlled at the local level and do not reflect the average level of these modifications in the cell (for example, compare in Fig. the total level of phosphorylated histone H3 with phosphorylated histone H3 levels at the collagenase promoter). Second, it seems that different histone modifications follow each other during gene activation and there is no single modification event associated with gene activation. While acetylation and phosphorylation represent late modifications, methylation is an early event. As methylation of lysine 4 has been suggested not only to preclude repression complex formation but also to be involved in the recruitment of other chromatin-modifying enzymes (30
), it might be the earliest step in collagenase gene activation. This is supported by the observation that lysine-4-methylated histone H3 is a preferred target for subsequent acetylation over unmodified histone H3 (53
). SET9 recruitment prior to association of p300 as observed at the collagenase promoter (Fig. and ) supports this model.
The nucleosomal changes observed at the collagenase promoter are brought about by specific histone-modifying factors. Apparently, in each case recruitment is sufficient to get enzymatic activity, since the presence of the kinase, acetyltransferase, and methyltransferase activity coincided with the appearance of the associated histone modification. However, RSK2 and SET9 appear to be still present at the collagenase promoter when the modification of histones by these enzymes is already decreasing. This might suggest the involvement of specific demethylase and phosphatase activities, but it could also be due to the fact that more modifications occur at the single histone tail (2
). Hence, the presence of RSK2 and SET9 at the promoter could still reflect enzymatically active molecules but proof of functionality would require the use of antibodies that are not affected by additional modifications. Our results also indicate that p300 and RSK2 are both enzymatically active at the collagenase promoter, possibly as a complex. This is in apparent contrast to a study by Merienne et al. (27
) that showed the preferential interaction of the p300 homologue CBP with unphosphorylated RSK2, forming a complex in which both kinase activity and acetyltransferase activity are inhibited. This apparent contradiction might be explained by the differences between p300 and CBP as well as by cell type-specific effects, affecting both binding and activity. For example, under conditions where we demonstrate binding of p300 to RSK2 (Fig. ) we could not detect an interaction between CBP and RSK2 (data not shown).
One of the questions remaining is how SET9 is recruited to the collagenase promoter. As our ChIP results indicate that SET9 is not present before stimulation (T
= 0), it should be targeted to the collagenase promoter. One possibility is that SET9 associates with a putative repression complex present at the collagenase promoter before stimulation. In this complex it could methylate lysine 4 of histone H3 and thereby prevent the repression complex from binding to the collagenase promoter. Alternatively, SET9 could be recruited by another complex containing specific DNA binding factors. Finally, SET9 might also be a component of the PIC, thereby precluding formation of a repression complex and stimulating PIC formation at the collagenase promoter. As our ChIP assays indicate that SET9 is present at the collagenase promoter during the entire PIC formation, we favor the last explanation, although our attempts to identify the specific recruitment factor have thus far failed. Consistent with the idea of SET9 being a component of the PIC is the observation that SET9 can methylate only core histones and not nucleosomes (30
), implying that SET9 functions in concert with other chromatin-reconfiguring enzymes, as was recently shown for the yeast homologue SET1 (46
). Together with histone acetylation and phosphorylation, SET9-mediated histone methylation would then create the conditions which preclude formation of repression complexes and stimulate stable PIC assembly and subsequent transcription activation.
In conclusion, we show for the first time that in addition to the well-established requirement of acetylation, induction of collagenase expression also involves di- and trimethylation of lysine 4 of histone H3 and phosphorylation of serine 10 of histone H3. These modifications are brought about by different histone-modifying enzymes like SET9, p300, and RSK2, which are recruited sequentially to the collagenase promoter culminating in the initiation of transcription.