Histone H1 enhances synergistic activation of the MMTV promoter in chromatin
2003; Springer Nature; Volume: 22; Issue: 3 Linguagem: Inglês
10.1093/emboj/cdg052
ISSN1460-2075
Autores Tópico(s)Immunotherapy and Immune Responses
ResumoArticle3 February 2003free access Histone H1 enhances synergistic activation of the MMTV promoter in chromatin Ronald Koop Ronald Koop Institut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität, E.-Mannkopff-Strasse 2, D-35033 Marburg, Germany Centre de Regulació Genòmica (CRG), Universitat Pompeu Fabra, Passeig Maritim 37–49, E-08003 Barcelona, Spain Search for more papers by this author Luciano Di Croce Luciano Di Croce Institut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität, E.-Mannkopff-Strasse 2, D-35033 Marburg, Germany Present address: Department of Experimental Oncology, Istituto Europeo d'Oncologia, Via Ripamonti 435, 20141 Milano, Italy Search for more papers by this author Miguel Beato Corresponding Author Miguel Beato Institut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität, E.-Mannkopff-Strasse 2, D-35033 Marburg, Germany Centre de Regulació Genòmica (CRG), Universitat Pompeu Fabra, Passeig Maritim 37–49, E-08003 Barcelona, Spain Search for more papers by this author Ronald Koop Ronald Koop Institut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität, E.-Mannkopff-Strasse 2, D-35033 Marburg, Germany Centre de Regulació Genòmica (CRG), Universitat Pompeu Fabra, Passeig Maritim 37–49, E-08003 Barcelona, Spain Search for more papers by this author Luciano Di Croce Luciano Di Croce Institut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität, E.-Mannkopff-Strasse 2, D-35033 Marburg, Germany Present address: Department of Experimental Oncology, Istituto Europeo d'Oncologia, Via Ripamonti 435, 20141 Milano, Italy Search for more papers by this author Miguel Beato Corresponding Author Miguel Beato Institut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität, E.-Mannkopff-Strasse 2, D-35033 Marburg, Germany Centre de Regulació Genòmica (CRG), Universitat Pompeu Fabra, Passeig Maritim 37–49, E-08003 Barcelona, Spain Search for more papers by this author Author Information Ronald Koop1,2, Luciano Di Croce1,3 and Miguel Beato 1,2 1Institut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität, E.-Mannkopff-Strasse 2, D-35033 Marburg, Germany 2Centre de Regulació Genòmica (CRG), Universitat Pompeu Fabra, Passeig Maritim 37–49, E-08003 Barcelona, Spain 3Present address: Department of Experimental Oncology, Istituto Europeo d'Oncologia, Via Ripamonti 435, 20141 Milano, Italy *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:588-599https://doi.org/10.1093/emboj/cdg052 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Minichromosomes assembled on the mouse mammary tumor virus (MMTV) promoter in vitro exhibit positioned nucleosomes, one of which covers the binding sites for progesterone receptor (PR) and nuclear factor 1 (NF1). Incorporation of histone H1 into MMTV minichromosomes improves the stability of this nucleosome and decreases basal transcription from the MMTV promoter, as well as its response to either PR or NF1. However, histone H1-containing minichromosomes display better PR binding and support a more efficient synergism between PR and NF1, leading to enhanced transcription initiation. A mutant MMTV promoter lacking positioned nucleosomes does not display enhanced transcriptional synergism in the presence of H1. Binding of PR leads to phosphorylation of H1, which leaves the promoter upon transcription initiation. Thus, H1 assumes a complex and dynamic role in the regulation of the MMTV promoter. Introduction The fundamental unit of eukaryotic chromatin is the nucleosome, which consists of a core histone octamer with 147 bp of superhelical DNA wrapped around in 1.65 left-handed turns (Richmond et al., 1984; Luger et al., 1997). A chain of nucleosomes connected by linker DNA builds the 10 nm nucleosomal fiber, which is compacted further in interphase nuclei to form a more irregular 30 nm fiber, stabilized by interactions between nucleosomes and by the so-called linker histones. Linker histones exist in a variety of subtypes (e.g. H1, H1o, H5, etc.; reviewed in Khochbin, 2001; Parseghian and Hamkalo, 2001). Binding of histone H1 stabilizes the nucleosome and protects an additional 20–30 bp of linker DNA against micrococcal nuclease (MNase) digestion to form a structure called the chromatosome (Simpson, 1978). Interactions among linker histones further stabilize and compact the higher order structure of chromatin (Ramakrishnan, 1997; Widom, 1998). As transcriptional activation requires decondensation of chromatin structures (Gregory and Hörz, 1998), histone H1 has long been regarded as a general repressor of transcription (Weintraub, 1985). However, there are indications of a more differential role of H1 in transcriptional regulation (Zlatanova, 1990). Several years ago, it was shown that H1 regulates individual genes in two different systems: Xenopus laevis and Tetrahymena thermophyla. Disruption of the H1 gene in macronuclei of Tetrahymena displays a specific rather than a global effect on gene expression (Shen and Gorovsky, 1996). In Xenopus, the oocyte and somatic type 5S rRNA genes are transcribed differentially during early development. Repression of oocyte 5S RNA transcription coincides with the appearance of histone H1A during the early gastrula stage (Kandolf, 1994). The increase in histone H1 content specifically restricts TFIIIA-activated transcription of oocyte 5S rRNA but does not significantly influence somatic 5S rRNA gene transcription. Thus, the regulated expression of histone H1 during Xenopus development has a specific and dominant role in mediating the differential expression of the oocyte and somatic 5S rRNA genes (Bouvet et al., 1994). One model proposes that H1 relocates the multiple octamer positions over the oocyte 5S RNA gene, some of which allow TFIIIA binding, to a unique translational position incompatible with TFIIIA binding (Sera and Wolffe, 1998). An effect of linker histone on the distribution of nucleosome positions has also been shown by others (Meersseman et al., 1991). A second model proposes differential nucleosome positioning on somatic and oocyte 5S rRNA genes per se, which favor either H1 or TFIIIA binding (Panetta et al., 1998; Crane-Robinson, 1999). Both models underline the importance of nucleosome positioning for gene regulation. Although chromatin generally is viewed as an obstacle for transcription that has to be remodeled or modified in the process of transcriptional activation, we have shown previously that a well-organized chromatin template is a necessary prerequisite for the proper activation of the mouse mammary tumor virus (MMTV) promoter (Chávez and Beato, 1997). The MMTV promoter is organized into positioned nucleosomes in vivo (Richard-Foy and Hager, 1987) and in vitro (Perlmann and Wrange, 1988), with a nucleosome covering the five hormone-responsive elements (HREs) and the binding site for nuclear factor 1 (NF1) (Piña et al., 1990; Truss et al., 1995). The binding site for NF1 is not accessible on this positioned nucleosome, and only two of the five HREs can be bound by hormone receptors (Piña et al., 1990; Eisfeld et al., 1997). The MMTV promoter assembled in minichromosomes also exhibits positioned nucleosomes (Venditti et al., 1998) and is activated in a process involving a two-step synergism. First, the hormone receptor binds to the exposed HREs and triggers a chromatin-remodeling event that facilitates access of NF1. Bound NF1 in turn stabilizes an open nucleosomal conformation required for efficient binding of further receptor molecules to the hidden HREs and full transactivation (Di Croce et al., 1999). A few years ago, it was shown that H1 overexpression in 3T3 cells led to an increase in transcriptional activity of integrated, but not transient transfected MMTV promoters (Gunjan and Brown, 1999). These results suggested that histone H1 played a positive role in MMTV induction depending on its chromatin organization, but did not prove that the effect was a direct consequence of H1 incorporation into the MMTV promoter chromatin. Here, we show that a similar H1-dependent increase in transcriptional activation takes place on MMTV minichromosomes assembled in vitro and that this increase is a direct effect of H1 binding to the MMTV promoter. Addition of H1 to an in vitro chromatin reconstitution system based on extracts from pre-blastodermic Drosophila embryos, which do not contain H1, leads to tightening and stabilization of the nucleosome covering the regulatory elements of the MMTV promoter, which reduces accessibility for restriction enzymes and NF1. However, incorporation of histone H1 in minichromosomes leads to a better binding of PR and to improved synergism between PR and NF1, resulting in enhanced transcription initiation. On a truncated MMTV promoter, which displays no proper nucleosome positioning (Prado et al., 2002), H1 has no positive effect on the synergism between PR and NF1, but rather leads to a general reduction in transcriptional activation. Our results suggest that the positive effect of H1 on transcriptional activation of the MMTV promoter is at least partly due to a more homogeneous nucleosomal organization, which favors PR binding to a larger proportion of MMTV promoters and enhances the functional synergism between PR and NF1. Following PR binding, histone H1 is phosphorylated and is displaced from MMTV promoter chromatin upon activation of transcription initiation. Results Incorporation of histone H1 reduces basal transcription and the activation by either PR or NF1 The extract of pre-blastodermic Drosophila embryos (DREX) used for assembly of minichromosomes is known to lack histone H1, but contains HMG-D, a Drosophila member of the HMG 1 protein family. It is only after the mid-blastula transition that HMG-D is replaced progressively by histone H1 (Ner and Travers, 1994). We have confirmed the lack of histone H1 in our extract by western blotting using post-blastoderm extract as a positive control (data not shown). When histone H1 was added at the onset of a chromatin reconstitution on the plasmid pMMTVCAT B-B, an increase of the nucleosome repeat length from 185 bp (without H1) to 210 bp was observed at a calculated ratio of one molecule of H1 per nucleosome (Figure 1). In a chromatin immunoprecipitation (ChIP) experiment with an H1-specific antibody, we confirmed that H1 was incorporated in the reconstituted chromatin over the promoter region (data not shown). Figure 1.Incorporation of histone H1 in reconstituted minichromosomes. Effect of H1 on nucleosome spacing. Chromatin assembled with or without histone H1 on pMMTVCAT B-B DNA was digested with micrococcal nuclease (MNase) for increasing times. The resulting DNA fragments were separated by electrophoresis on a 1.3% agarose gel in 1× TBE buffer and stained with ethidium bromide. The numbers on each site indicate the approximate size of the fragments in base pairs as determined by comparison with appropriate size markers. Download figure Download PowerPoint To study the effect of incorporation of histone H1 on the activity of the MMTV promoter chromatin, we used a HeLa nuclear extract for in vitro transcription (Dignam et al., 1983). Increasing concentrations of histone H1 in the minichromosome assembly reactions consistently reduced the basal transcriptional activity of the promoter (Figure 2, lanes 1, 5, 9, 13 and 17) and decreased its response to the addition of either purified PR (Figure 2, lanes 2, 6, 10, 14 and 18) or purified NF1 (Figure 2, lanes 3, 7, 11, 15 and 19). Maximal inhibition reached 60% in the case of PR (compare lanes 2 and 18, and Figure 2B) and 50% in the case of NF1 (compare lanes 3 and 19, and Figure 2B). These findings are in agreement with the postulated repressive effect of linker histones on chromatin transcription and could reflect a more compact or less dynamic structure of the minichromosomes containing H1. Figure 2.Influence of histone H1 on transcription from the MMTV promoter. (A) In vitro transcription analysis of MMTV minichromosomes with increasing amounts of histone H1. Chromatin reconstituted with an increasing molar ratio of histone H1 was incubated with purified recombinant PR or/and NF1 as indicated and transcribed in vitro with HeLa nuclear extract. For each template, 25 ng of DNA were used in each reaction. Products were visualized by primer extension analysis and sequencing gel electrophoresis. The positions of the products from the wild-type MMTV promoter (wt) and from a control MMTV promoter lacking the HREs (−77) are indexed on the left. The synergism of PR and NF1 was calculated as: (activity PR + NF1)/(activity PR) + (activity NF1), and is indicated at the bottom. (B) Quantification of the wild-type MMTV transcripts. Transcription signals were quantified in a phosphoimager (Fuji) and are shown in relative units, with the transcription in the absence of H1 and activators (lane 1) set to 1. The numbers on the x-axis refer to the lanes in (A). Download figure Download PowerPoint Histone H1 incorporation reduces the accessibility of restriction sites in the MMTV promoter The efficiency of cleavage with restriction enzymes is a measurement of the accessibility of DNA sequences in a chromatin context. In the past, we have used the restriction enzyme HinfI for this kind of analysis, since its recognition sequence overlaps with the NF1-binding site. We have shown that this site is relatively inaccessible in MMTV minichromosomes assembled in DREX compared with naked DNA (Venditti et al., 1998). The incorporation of histone H1 into wild-type MMTV minichromosomes further reduced the accessibility of the HinfI restriction site by a factor of 4 (Figure 3A, compare lanes 1–3 with lanes 4–6). A similar but even more pronounced inhibition of cleavage by histone H1 incorporation was observed with the restriction enzyme SacI, which cleaves near the dyad axis of the promoter nucleosome between HRE2 and HRE3 (Figure 3B). Therefore, the compaction of nucleosome structure by histone H1 affects the complete nucleosomal DNA and is not restricted to a particular region of the DNA superhelix. This increased compaction offers a plausible explanation for the inhibitory effect of histone H1 on basal transcription and on the activation by the individual transcription factors PR or NF1. Figure 3.Effect of histone H1 on accessibility for restriction enzymes of promoter DNA sequences in chromatin. (A) Cleavage by HinfI of wild-type MMTV (wtMMTV) and mutant MMTV (HRE/MMTVΔ) promoters in minichromosomes assembled in the presence or absence of histone H1. MMTV minichromosomes (200 ng of DNA) were assembled as described in Materials and methods. After assembly, the samples were digested at 26°C with 50 U of HinfI for 2, 4 or 8 min, and the DNA was purified and restricted with DraI (wtMMTV) or PvuII (HRE/MMTVΔ). The digestion products were analyzed by linear PCR. The positions of the HinfI uncleaved and cleaved fragments are indicated. The amount of cleavage as a percentage of total radioactivity is indicated below the lanes. (B) Cleavage by SacI of the wild-type MMTV promoter in minichromosomes assembled in the presence or absence of histone H1. MMTV minichromosomes (200 ng of DNA) were assembled in DREX, digested at 26°C with 50 U of SacI for 1, 2, 4 or 8 min, and the DNA was purified and restricted with DraI. The digestion products were analyzed by linear PCR. The positions of the SacI uncleaved and cleaved fragments are indicated. The amount of cleavage as a percentage of total radioactivity is indicated below the lanes. Download figure Download PowerPoint The synergism between PR and NF1 as well as the actual transcription from the activated MMTV promoter are enhanced by histone H1 In contrast to the inhibitory effect of histone H1 on transcriptional activation by isolated PR or NF1, adding both factors together yielded an unexpected result: H1 enhanced the synergism between PR and NF1 as well as the absolute levels of transcription (Figure 2A, lanes 4, 8, 12, 16 and 20). The synergism increased concomitantly with the amount of H1 up to a calculated molar ratio of 1.25 molecules of H1 per nucleosome. Higher molar ratios resulted in aggregation of the templates and a complete loss of transcriptional activity (data not shown). We also observed an increase in overall transcription (compare lane 4 with 8, 12, 16 and 20), though less pronounced than the augmentation of synergism between PR and NF1. This results from the above-mentioned inhibitory effect of H1 on the levels of transcription induced by the single activators, PR and NF1. In 12 independent experiments, the inhibitory effect of H1 on basal transcription was 28% (±16), while the inhibition in the presence of PR alone was 43% (±5) and with NF1 alone 52% (±6). The synergism between PR and NF1 was 3.5-fold (±1.6) in the absence of histone H1 and increased to 8.2-fold (±2.4) in the presence of H1. Most of our studies were performed with commercial histone H1 preparations. However, experiments with recombinant histone H1o and H1.2 isoforms expressed in Escherichia coli (Doenecke et al., 1997) yielded virtually indistinguishable results. In contrast to the results obtained with minichromosomes, transcription activation on ‘naked’ MMTV DNA was not significantly influenced by the addition of H1 with any combination of activators (data not shown). We conclude that, at the concentrations used in these experiments, H1 has no direct, inhibiting or enhancing effect on the transcriptional machinery as such. This excludes a possible interaction between H1 and PR as a mechanism underlying the positive effects observed with minichromosomes. Histone H1 increases the proportion of activated MMTV chromatin templates The enhanced transcription from MMTV minichromosome templates containing histone H1 could result from the utilization of a larger proportion of the available promoters for transcription or, alternatively, from a higher efficiency of reinitiation on the same number of promoters. To distinguish between these two possibilities, we studied the effect of sarcosyl, which prevents transcription reinitiation but does not preclude elongation by already engaged RNA polymerase II complexes (Hawley and Roeder, 1985, 1987). The complete transcription reactions lacking UTP were incubated for 30 min before the addition of sarcosyl (final concentration 0.07%), where indicated, and 5 min later the missing UTP was supplemented. Incubation was continued for 30 min before addition of a defined amount of riboprobe as internal standard, followed by quantitation of transcripts by primer extension (Di Croce et al., 1999). With naked DNA templates, 100% of the MMTV promoters were activated by PR, but the efficiency of reinitiation was low. Each template was used only 1.7-fold, whereas two control promoters showed a much higher reinitiation. A Zta-dependent promoter was reinitiated 7.5-fold in the presence of the Zta transactivator (Lieberman and Berk, 1991), and a Gal4 reporter showed a 12-fold reinitiation in the presence of the Gal4-VP16 transactivator (Croston et al., 1991) (Figure 4A). Figure 4.Influence of histone H1 on the recruitment of promoters to the active state and on the efficiency of reinitiation. (A) In vitro transcription analysis of free DNA in the presence or absence of sarcosyl. Free reporter DNA (50 ng of template/reaction) for PR (pMMTVCAT B-B), Zta (pZ7E4T CAT) or Gal4-VP16 (pG5E4T CAT) was incubated in the absence of UTP for 30 min with HeLa nuclear extract and purified recombinant PR, Zta or Gal4-Vp16 as indicated. Sarcosyl was added to a final concentration of 0.07% and, after 5 min, transcription was started by addition of UTP. After 30 min, transcription was stopped and a riboprobe was added corresponding to 50 or 100% of the template concentration as indicated. Products were visualized by primer extension analysis and sequencing gel electrophoresis. The positions of the products from the different promoters and the riboprobe are indexed on the left. The template usage was calculated by comparing the signal intensities of the riboprobe with those of the transcription reactions in the presence of 0.07% sarcosyl (single round transcriptions). Comparing the single round transcriptions with the corresponding signals in the absence of sarcosyl (multiple rounds of transcription) yielded the values for the reinitiation rates. (B) In vitro transcription analysis of MMTV minichromosomes in the presence or absence of H1 and sarcosyl. Chromatin reconstituted in the presence or absence of histone H1 (50 ng of template/reaction) was purified via Sephadex G-50 spin columns to remove NTPs. This chromatin showed no transcription signal without addition of UTP (lanes 17 and 18), whereas transcription on non-purified minichromosomes proceeded with internal UTP (data not shown). Purified minichromosomes were incubated in the absence of UTP for 30 min with purified recombinant PR and NF1 as indicated. HeLa nuclear extract was then added for an additional 30 min. Reactions were then processed further with or without sarcosyl addition as in (A). (C) In vitro transcription analysis of pZ7E4T CAT or pG5E4T CAT minichromosomes in the presence or absence of H1 and sarcosyl. Except for the utilization of Gal4-VP16 and Zta instead of PR and NF1, reactions were carried out as in (B). Download figure Download PowerPoint On minichromosomes assembled in the absence of histone H1, the proportion of MMTV promoters activated in the absence of added factors was 3% in this particular experiment. In the presence of either PR alone or NF1 alone, the proportion was 6 and 8%, respectively, in this particular experiment, and this value increased to 26% in the presence of both factors (Figure 4B, lanes 1–8). The efficiency of reinitiation was low and comparable with that on free DNA templates. On minichromosome templates containing histone H1, the proportion of templates activated by PR alone or NF1 alone was slightly lower, 5 and 4%, respectively, but in the presence of both factors 86% of the templates were activated (Figure 4B, lanes 9–16). The absolute figures varied in three independent experiments, but in all cases H1 inhibited template usage in the presence of either PR or NF1 alone while it increased template usage in the presence of both factors together. Addition of H1 had little effect on the efficiency of reinitiation. Thus, the positive effect of histone H1 on activated transcription from MMTV chromatin templates is due mainly to a better synergism between PR and NF1 in terms of promoter recruitment to the active state. The complex effect of histone H1 was selective for the MMTV promoter, since the Gal4 reporter promoter assembled in minichromosomes was insensitive to the addition of H1, and the Zta reporter promoter responded to the addition of H1 with a reduction in transcription (Figure 4C). The efficiency of reinitiation on control promoters in chromatin was reduced compared with free DNA (Figure 4, compare C with A) but, as in the case of MMTV promoters, was not signficantly affected by histone H1 (Figure 4C). The population of nucleosomes on the MMTV promoter is more homogeneous in minichromosomes containing histone H1 The positioning of a nucleosome along the DNA is defined by the rotational and translational phasing. The rotational phasing refers to the relationship between the nucleosome and the helical periodicity of the DNA, whereas the translational phasing describes the position of the nucleosome relative to a given point along the DNA molecule. We set out to investigate if the incorporation of H1 had any effect on the positioning of the MMTV promoter nucleosome. This nucleosome covers the HREs and has been shown to exhibit preferential translational and rotational phasing in different systems (Richard-Foy and Hager, 1987; Perlmann and Wrange, 1988; Piña et al., 1990; Chávez et al., 1995; Truss et al., 1995; Flaus and Richmond, 1998), including reconstitution with a Drosophila embryo extract (Venditti et al., 1998). There fore, we performed a high resolution analysis of nucleosome structure using MNase footprinting. Confirming previous results (Chávez et al., 1995; Venditti et al., 1998), a protected region flanked by hypersensitive sites at −40 and −190 was detected. Within this region, the protection was less clear over the proximal and distal parts of the footprint and more evident in the central part (Figure 5A), as had been observed with other positioned nucleosomes (Tanaka et al., 1996). In the presence of histone H1, the limits of the protection, as determined by the flanking hypersensitive sites, were the same, but the degree of protection was strikingly more pronounced (compare lanes 2 and 3, see especially the protected bands around −84 and −100, indicated by circles). Thus, we conclude that the MMTV promoter nucleosome is stabilized by the incorporation of histone H1. Figure 5.Structural analysis of the promoter nucleosome in the presence or absence of histone H1. (A) High resolution mapping of the translational positioning. Reconstituted minichromosomes were mildly digested with MNase (20 s at 26°C) and the resulting fragments were amplified by linear PCR. Lane D: MNase digestion pattern on naked DNA. Lanes M and C: minichromosomes reconstituted in the presence or absence of H1 (as indicated above) treated with MNase. Cleavage sites protected in chromatin are indicated by open circles; hypersensitive sites are marked by black arrows. The numbers refer to the distance from the start of transcription. The diagram on the right shows the approximate position of the promoter nucleosome. (B) Rotational phasing. The rotational setting of the promoter nucleosome was determined by DNase I digestion of the minichromosomes followed by linear PCR amplification. Lane D: DNase I digestion pattern on naked DNA. Lanes Minichrom.: minichromosomes (with or without H1 as indicated above) digested for 2 min (lanes 5 and 7) and 4 min (lanes 6 and 8) with DNase I. The alternate enhancements (arrows) and protections (circles) show a periodicity of ∼10 bp. The numbers refer to the distance from the transcription start. Download figure Download PowerPoint To analyze the effect of H1 on the rotational orientation of the double helix on the nucleosome surface, we used DNase I digestion (Figure 5B). In both the absence and presence of histone H1, we observed a pattern of preferential cleavage sites (marked by arrows) spaced by ∼10 bp alternating with protection of cleavage sites (marked by circles), indicative of a preferential rotational phasing of the DNA double helix, as reported previously (Piña et al., 1990; Truss et al., 1995; Venditti et al., 1998). The strong hypersensitive sites at −162, −100, −82 and −73 indicate deformations of the double helix in these regions, with widening of the minor groove particularly around the nucleosome axis. The pattern was similar in the presence of histone H1, but the protections and enhancements were more prominent (compare lanes 5 and 6 with lanes 7 and 8). At the hypersensitive positions, −162, −100 and −82, there is a clear increase in the intensity of the main band accompanied by a decrease in the intensity of the flanking bands. We conclude that the dominant rotational setting on the promoter nucleosome is preserved and stabilized in the presence of H1. Taken together, the structural data suggest a more homogeneous population of nucleosomes in the presence of H1, with a higher percentage of molecules adopting a dominant position. Histone H1 does not enhance the synergism between PR and NF1 on a truncated MMTV promoter, which does not position nucleosomes To test the relationship between nucleosome positioning and H1 effects on transcription, we utilized a mutant promoter, HRE/MMTVΔ, in which the region containing the five natural HREs of the MMTV promoter has been replaced by a single canonical HRE. This truncated MMTV promoter does not position nucleosomes (Prado et al., 2002). We compared the accessibility of the NF1-binding site for cleavage by HinfI in minichromosomes containing the HRE/MMTVΔ promoter and on wild-type MMTV minichromosomes (Figure 3A). In the absence of histone H1, the accessibility of this site in the HRE/MMTVΔ promoter chromatin was ∼3-fold higher than in the wild-type chromatin, probably due to the lack of nucleosome positioning (compare lanes 1–3 with lanes 7–9). However, as on the wild-type MMTV promoter, H1 incorporation led to a 3-fold reduction in restriction enzyme cleavage on HRE/MMTVΔ minichromosomes (compare lanes 7–9 with lanes 10–12). Therefore, the difference in accessibility between wild-type MMTV and HRE/MMTVΔ minichromosomes was maintained in the presence of histone H1. In both cases, restriction enzyme access was reliant on ATP-dependent chromatin-remodeling complexes, since reactions performed in the presence of apyrase, which degrades any ATP in the extract (Tsukiyama et al., 1994), almost completely eliminated HinfI cleavage (Venditti et al., 1998; data not shown). Thus, histone H1 has a similar compacting effect on wild-type and HRE/MMTVΔ promoter chromatin. However, in contrast to the situation with wild-type MMTV, where histone H1 had an enhancing effect on the synergism between PR and NF1 (see also Figure 2A), the synergism between the two factors was not affected by H1 in the HRE/MMTVΔ promoter (Figure 6). Moreover, a clear reduction in overall activation of the HRE/MMTVΔ promoter could be observed in the presence of histone H1,
Referência(s)