Mediator and SWI/SNF are required for chromatin remodeling of RNR3

RNA polymerase II is reported to copurify with two chromatin modifying activities. One is the mediator that contains a subunit, Nut1p, with intrinsic histone acetyltransferase activity (Lorch et al. 2000), and the other is the SWI/SNF complex that disrupts nucleosomes in an ATP-dependent manner (Wilson et al. 1996). Thus, we explored the requirement for the mediator as a whole, and NUT1 specifically, by analyzing the expression and chromatin remodeling of RNR3 in the srb4-138 temperature-sensitive mutant and in a strain containing a deletion of NUT1. Inactivation of the srb4-138 mutant abolished the derepression of RNR3 (Fig. ​(Fig.3A),3A), and nucleosome mapping reveals that the loss of transcription is accompanied by reduced chromatin remodeling in this strain (Fig. ​(Fig.3B).3B). Deletion of NUT1 reduced the DNA damage-dependent expression of RNR3 (Fig. ​(Fig.3C)3C) to ~50% of the level observed in wild-type cells, but expression in the Δnut1 strain was significantly higher than in the srb4-138 mutant (Fig. ​(Fig.3,3, cf. A and C). Also, nucleosome mapping of the RNR3 promoter revealed that deleting NUT1 had no detectable effect on chromatin remodeling (Fig. ​(Fig.3D).3D). Similar results, in regards to the transcript level and the degree of chromatin remodeling, were also observed in a Δsrb2 and Δsrb5 mutant, two other nonessential subunits of the mediator complex (data not shown). Whereas the mediator as a whole is necessary for high levels of transcription and chromatin remodeling of RNR3, these two functions are not strictly dependent upon NUT1.

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Figure 3

Mediator (SRB4), but not NUT1 specifically, is required for the DNA damage-induced chromatin remodeling of RNR3. Wild type (Z579) and temperature-sensitive srb4-138 (Z628) mutant were grown and processed as in Figure ​Figure1.1. (A) Northern blot analysis of RNR3 mRNA. (B) MNase mapping. Analysis of expression and chromatin structure of RNR3 in the srb4-138 mutant. (C). Northern blot analysis of RNR3 mRNA levels in wild type and Δnut1 mutant. Cells were treated with (+) or without (−) MMS for 2.5 h at 30°C. Quantification of RNR3 transcripts was performed using the ImageQuant software program. The expression level in wild-type cells without MMS treatment is arbitrarily defined as a unit of 1. scR1 is a loading control. (D) MNase chromatin mapping was performed using wild type (PH499) and the Δnut1 (YJR709) mutant treated with or without MMS for 2.5 h at 30°C. ND, digested naked DNA; M, a DNA size marker.

We next examined the requirement for the SWI/SNF complex in the derepression of RNR3 by using a mutant deleted of the gene encoding the catalytic subunit of the complex, SNF2. Deletion of SNF2 reduced the derepression of RNR3 to ~20% of the level of wild-type cells (Fig. ​(Fig.4A).4A). The formation of the preinitiation complex at the promoter was examined using the chromatin immunoprecipitation assay (ChIP) with antibodies to TBP and RNA polymerase II. MMS treatment increased the cross-linking of both factors approximately fivefold in wild-type cells, but was strongly reduced in the Δsnf2 mutant (Fig. ​(Fig.4B).4B). Analysis of the nucleosomal organization of RNR3 revealed that the extent of chromatin remodeling was likewise greatly reduced in the Δsnf2 strain, suggesting that the reduced transcription and preinitiation complex (PIC) formation is largely attributed to reduced nucleosome remodeling (Fig. ​(Fig.4C).4C). Similar results were obtained using a strain containing a point mutation in the ATPase motif (K798A) of SNF2 (data not shown), suggesting that the ATP-dependent nucleosome remodeling activity is required for transcription and remodeling. Next, we sought to verify that SWI/SNF is directly regulating RNR3 by using the ChIP assay. We analyzed the association of Snf2p with RNR3 using primer pairs directed to regions upstream and downstream of the promoter, and found that the highest level of cross-linking was observed over the promoter (Fig. ​(Fig.4D).4D). The cross-linking to the promoter in untreated cells was approximately twofold above background levels (anti-HA), and MMS treatment increased its cross-linking an additional 3.2-fold (Fig. ​(Fig.4D).4D). The cross-linking of Snf2 diminished further away from the promoter, and only background levels were detected +1500 base pairs away from the promoter within the open reading frame (ORF). Moreover, no specific cross-linking was observed at the promoter of the SWI/SNF independent ADH1 (data not shown). Thus, Snf2 is predominantly localized over the promoter, and is increased by DNA damage.

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Figure 4

SWI/SNF regulates RNR3 expression and nucleosome remodeling. (A). Northern blot analysis of RNR3 mRNA levels. (B). Chromatin immunoprecipitation assay (ChIP) for TBP and RNA polymerase II cross-linking. SNF2 and Δsnf2 strains were grown at 30°C and treated with MMS for 2 h (gray bars) or left untreated (white bars). Immunoprecipitations (IPs) were carried out using a polyclonal antibody against TBP, a monoclonal antibody against RNA polymerase II (8WG16), and preimmune serum (PI) or anti-HA (HA) as controls. Immunoprecipitated and input DNA were amplified by PCR using primers specific for the core promoter regions of RNR3. Data are expressed as the means and standard deviations of the cross-linking from three independent experiments. The amount of cross-linking in untreated wild-type cells was arbitrarily set at a value of 1. (C) MNase mapping. The lane on the right (ND), indicates digestion of naked DNA by MNase. (D) ChIP assay for Snf2-myc13 binding. Strain YJR589 (SNF2-MYC13) was treated with (2 h) and without MMS prior to cross-linking as indicated. ChIPs were carried out using monoclonal antibodies to the MYC tag and HA tag (as a negative control for background). Similar background was observed in IPs using untagged strains. Immunoprecipitated and input DNA were amplified by PCR using primers directed to the regions of RNR3 indicated in the panel. For reference, +1 is the major transcription start site; the TATA box is located at −75 and the URS between −330 and −200.

TFIID and RNA polymerase II are required for SWI/SNF and mediator recruitment, but not histone acetylation

To increase our understanding of the TAF_II- and GTF-dependent nucleosome remodeling, we analyzed the cross-linking of transcription factors to the RNR3 promoter in wild-type and mutant cells. First, we examined the cross-linking of TBP, TAF_IIs, and Rpb1 to the promoter in wild-type cells grown at 30°C. The results in Figure ​Figure5A5A show that even in the absence of DNA damage, a significant level of TBP and TAF_II cross-linking was detected at the promoter. The amounts of RNR3 promoter precipitated using antiserum against five different TAF_IIs and TBP were approximately two- to three- and sevenfold above that observed using preimmune serum, respectively. Furthermore, the amount of promoter DNA brought down in untreated cells was above that of ORF DNA (Fig. ​(Fig.5A)5A) or telomeric DNA (data not shown). The addition of MMS resulted in an additional approximately threefold, approximately fivefold, and approximately sevenfold increase in the cross-linking of TAF_IIs, TBP, and RNA polymerase II, respectively. The MMS-induced cross-linking of TBP was greater than that of each of the TAF_IIs examined. What accounts for this difference is not known, but it may result from either the presence of other TBP-containing complexes at the promoter or more efficient cross-linking of TBP to DNA.

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Figure 5

Cross-linking of transcription factors to the RNR3 promoter in the presence and absence of DNA damage. (A) YJR589 was grown at 30°C and treated with or without MMS where indicated. IPs were carried out using polyclonal antibodies against TBP, TAF_IIs, and a monoclonal antibody against RNA polymerase II (8WG16). (Upper panel) Results from one experiment. The IPs using preimmune sera shown correspond to the rabbits used to raise antiserum against TAF1p and TBP. Similar levels of background were observed using three other preimmune sera or HA monoclonal antibody. IP and input DNA were amplified by PCR using primers specific for the promoter of RNR3 or +1500 bp from the start site of transcription (ORF). Lanes 1–6 show PCR reactions using 3.3-fold dilutions of input DNA. The DNA from the RNA polymerase II IP was diluted fivefold prior to amplification to assure linearity. The ChIP results are expressed as signal above preimmune sera, which was arbitrarily set at 1.0. (Lower panel) Graphical representation of the MMS-induced increase in cross-linking from three to five experiments, using at least three independently prepared chromatin preparations. (B) Chromatin immunoprecipitation analysis of transcription factor occupancy at the RNR3 promoter in temperature-sensitive mutants. Wild-type (YJR589) and taf1-2 (YJR595), taf12-9 (YJR592), and rpb1-1 (YJR598) strains, all containing SNF2-13MYC, were grown at 25°C and then shifted to 37°C for 45 min to inactivate the transcription factors. Cells were then treated with 0.02% MMS for 2 h (gray bars) or left untreated (white bars). IPs were carried out with antibodies against TBP, TAF1p, TAF12p, Pol II (8WG16), Ac-H3 (Di-acetyl Lys 9 and Lys 14), and (9E10) c-myc. Cross-linking of Srb4-myc was performed the same as the others except strains contained SRB4-13MYC (YJR588, YJR594, YJR591, and YJR597). The data are presented as a ratio of the amount of PCR product in wild-type cells, −MMS. Data are from at least three independent chromatin preparations and experiments. The increase in TAF1p cross-linking in the taf1-2 mutant (−MMS) is the result of a single aberrant value, and is not truly indicative of increased cross-linking in all repetitions.

Next, we examined the cross-linking of these factors in TAF_II and RNA polymerase II mutants at the restrictive temperature. The results in Figure ​Figure5B5B indicate that inactivating taf1-2, taf12-9, or rpb1-1 abolished the MMS-induced increases in TBP, TAF_II, and RNA polymerase II cross-linking to the promoter, indicating that their association with the promoter is mutually dependent. Also, inactivation of srb4-138 abolished the cross-linking of RNA polymerase II, and reduced the cross-linking of TAF1p and TAF12p (see Supplemental Material). Surprisingly, we found that the increased cross-linking of TFIID subunits to RNR3 requires RNA polymerase II. This differs from a previous analysis showing that TBP is recruited to a number of other yeast genes in the rpb1-1 allele (Kuras and Struhl 1999), suggesting that TBP and TFIID recruitment can occur by multiple mechanisms and display distinct transcription factor dependencies. In addition, since TBP cross-linking was eliminated upon the loss of TAF1 function, it is likely that the increase in TBP cross-linking is dependent on its association with TAF_IIs within the TFIID complex.

The requirement for TAF_IIs and RNA polymerase II for nucleosome remodeling of RNR3 suggests that they participate in the recruitment of SWI/SNF. This was examined by comparing the cross-linking of Snf2-myc to the RNR3 promoter in mutants before and after MMS treatment. The cross-linking of Srb4-myc was also examined. The results displayed in Figure ​Figure5B5B reveal that treating wild-type cells with MMS at 37°C resulted in an increase in the cross-linking of both Srb4-myc and Snf2-myc to the promoter by ~1.8-fold and 2.5-fold, respectively. More importantly, we found that inactivation of taf1-2, taf12-9, or rpb1-1 eliminated the DNA damage-induced enhancement of Snf2-myc and Srb4-myc cross-linking to the RNR3 promoter (Fig. ​(Fig.5B),5B), thus indicating that SWI/SNF and mediator recruitment is dependent on RNA polymerase II and TAF_IIs. Further, inactivation of srb4-138 reduced SWI/SNF recruitment (see Supplemental Material), which is consistent with the extent of nucleosome remodeling observed in this strain (Fig. ​(Fig.33A).

Derepression of RNR3 transcription is accompanied by an increase in histone H3 acetylation caused by the release of Ssn6/Tup1 and HDACs from the promoter (Davie et al. 2002; V. Sharma and J. Reese, unpubl.). Given the interdependence of nucleosome remodeling and histone acetylation (Belotserkovskaya and Berger 1999; Hassan et al. 2001a,b; Narlikar et al. 2002), we examined the acetylation state of histone H3 at the promoter in the TAF_II and RNA polymerase II mutants. Consistent with our previous observations, treating cells with MMS resulted in an ~2.7-fold increase in histone H3 acetylation in wild-type cells (Fig. ​(Fig.5B).5B). Interestingly, the level of acetylation of histone H3 in both the taf1-2 and rpb1-1 mutants was very similar to that observed in wild-type cells, increasing by 2.4- and 2.2-fold (89% and 80% of wild type), respectively. Thus, whereas both nucleosome remodeling and SWI/SNF recruitment are strictly dependent on TAF1 and RNA polymerase II, histone H3 acetylation occurs independently of these factors. This observation strongly suggests that these mutants are specifically blocked at the nucleosome remodeling step, and that the phenotypes we observe are unlikely to result from trivial defects in the DNA-damage sensing and response pathways. In contrast to what was observed in the taf1-2 mutant, inactivation of taf12-9 eliminated the MMS-induced histone H3 acetylation (Fig. ​(Fig.5B).5B). TAF12p is a component of TFIID and SAGA, and is essential for SAGA's nucleosomal HAT activity in vitro (Grant et al. 1998). Thus, SAGA may be the HAT complex responsible for the MMS-induced histone H3 acetylation. To test this, we examined the derepression of RNR3 and its histone acetylation levels in Δgcn5 and Δada2 mutants. Gcn5 is the catalytic subunit of SAGA, and Ada2 is required for the acetylation of nucleosomal substrates (Balasubramanian et al. 2002). Deletion of either GCN5 or ADA2 reduced the level of derepression of RNR3 to ~50%–60% of that observed in wild-type cells (Fig. ​(Fig.6A),6A), and abolished the DNA damage-induced increase in histone H3 acetylation (Fig. ​(Fig.6B).6B). Since Gcn5 is an integral subunit of both the SAGA and ADA complexes (Grant et al. 1997), we confirmed the SAGA dependence by examining the histone acetylation in a SAGA-specific (Δspt7) and ADA-specific (Δahc1) mutant. Consistent with our results using a TAF12 ts mutant, disrupting SAGA by deleting SPT7 eliminated histone acetylation, whereas histone acetylation occurred normally in the Δahc1 strain (Fig. ​(Fig.6B).6B).

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Figure 6

SAGA facilitates SWI/SNF remodeling at RNR3. (A) Northern blot for RNR3 in Δgcn5 and Δada2 mutants. PSY316, PSY316Δgcn5, and PSY316Δada2 were grown in YPAD at 30°C and treated with MMS for 2 h. scR1 is a loading control. (B) Analysis of histone H3 acetylation in HAT complex mutants. A single experiment is shown. Results from three independent chromatin preparations and experiments yielded similar results with errors of 10%. (C) Chromatin mapping in wild-type and Δgcn5 cells. (D) SWI/SNF and polymerase II recruitment. As in Figure ​Figure5,5, except strains YJR589 (SNF2-MYC13) and YJR715 (Δgcn5; SNF2-MYC13) were used. The data are presented relative to the amount of PCR product in IPs from untreated wild-type cells (−MMS), and is the result of at least three independent chromatin preparations and experiments. White bars, −MMS; gray bars, +MMS.

The SAGA dependence was unexpected given that we failed to observe increases in TAF12p cross-linking in the taf1-2 or rpb1-1 mutant even though acetylation increased upon MMS treatment (Fig. ​(Fig.5B).5B). Furthermore, we failed to detect specific cross-linking of multiple SAGA-specific subunits to RNR3 in the absence or presence of MMS anywhere around the promoter (data not shown). Precipitations were carried out using polyclonal antibodies to ADA1p, ADA3p, and Spt7p, and strains containing Gcn5-myc, Spt3-myc, or Ada2-myc were examined; all produced negative results (data not shown). All of the subunits indicated above cross-linked to the GAL1-GAL10 promoter by two groups (Bhaumik and Green 2001; Larschan and Winston 2001) and thus are capable of being cross-linked at that locus. Either SAGA is associated with the RNR3 promoter in a form that is refractory to cross-linking, or perhaps more likely, it may not be directly targeted to the promoter by an activator and acetylation occurs via a global, nontargeted mechanism (Vogelauer et al. 2000; Katan-Khaykovich and Struhl 2002).

Histone acetylation and nucleosome remodeling cooperate to regulate the expression of many genes; thus, we examined whether deletion of GCN5 affects the remodeling of nucleosomes at RNR3. The results in Figure ​Figure6C6C show that the extent of nucleosome remodeling in the Δgcn5 mutant was less than that observed in wild type. Digestion within the TATA-box region and the diminished intensity of the bands marking the internucleosomal hypersensitive sites were observed, but to a lesser degree than the wild-type strain. Thus, acetylation by Gcn5 is not essential for nucleosome remodeling, but rather increases the extent of remodeling or the fraction of promoters (cells) remodeled in the steady state.

Next, we investigated whether the reduced remodeling results from impaired SWI/SNF recruitment, and whether acetylation affects a postrecruitment step. Deleting GCN5, in fact, slightly increased the cross-linking to RNR3 in the presence and absence of MMS (Fig. ​(Fig.6D),6D), 3.8-fold versus 2.7-fold and 1.7-fold versus 1-fold, respectively. The cause of this is unknown. Nonetheless, Snf2-myc was cross-linked at least as well as in a wild-type strain. Thus, the recruitment of SWI/SNF appears not to require GCN5-dependent acetylation. Deleting GCN5 did, however, reduce the MMS-induced cross-linking of RNA polymerase II to RNR3 (Fig. ​(Fig.6D),6D), to a level ~70% of that observed in a wild-type strain. Taken together, these results suggest that acetylation facilitates, but is not essential for, SWI/SNF-induced nucleosome remodeling and is not required for SWI/SNF recruitment.

Why does nucleosome remodeling at RNR3 require the PIC?

Models of the mechanisms of chromatin remodeling have largely focused on the role of sequence-specific DNA binding proteins in recruiting nucleosome-remodeling machines and histone-modifying activities (Peterson and Workman 2000; Hassan et al. 2001b; Narlikar et al. 2002). Clearly, this mechanism is likely to be essential for the remodeling of chromatin at the promoters of a large number of genes in vivo. Our present results provide some convincing evidence that chromatin remodeling of at least some promoters requires components of the general transcription machinery, defining another path towards chromatin remodeling. The results described here are in contrast to what has been described for a number of other yeast genes, including SWI/SNF-dependent SUC2 (Hirschhorn et al. 1992; Gavin and Simpson 1997). We even verified the RNA polymerase II independence of the remodeling at SUC2 in our assays, which argues against differences in methodologies. This leads to the question of why RNR3 requires contributions from GTFs, whereas genes such as CHA1, PHO5, and SUC2 do not? One possibility may be the types or strengths of the regulatory proteins that bind to the promoters of these genes. It is known that certain types of activation domains, for example, glutamine- or proline-rich, cannot recruit chromatin remodeling activities to promoters on their own, yet they can direct high levels of transcription (Utley et al. 1998; Neely et al. 1999; Yudkovsky et al. 1999; Hassan et al. 2001a). It is clear from our data in Figures ​Figures5B5B and ​and7A7A that the activator(s), if any, that bind to the RNR3 promoter are not sufficient to recruit or retain SWI/SNF without contributions from PIC components. Given that strong activators can recruit chromatin remodeling activities directly (for review, see Peterson and Workman 2000; Hassan et al. 2001b; Narlikar et al. 2002), and can even do so at sites lacking core promoter sequences (Kuo et al. 2000; Park et al. 2001), it is likely that certain activators can bypass the contributions from PIC components. Perhaps GTFs play a subtle role in remodeling even at genes such as SUC2, but their contributions are not detectable under the experimental conditions employed. Evidence suggesting that factors other than the activator can affect the recruitment mechanisms of chromatin remodeling activities is available. For example, the DNA-binding protein Pho4 drives the expression and chromatin remodeling of PHO5 and PHO8, but only PHO8 shows dependence on the SWI/SNF complex (Gregory et al. 1999). Furthermore, the codependence and order of recruitment of coactivators by the enhanceosome of the IFN-β promoter are regulated by core promoter architecture (Agalioti et al. 2000; Lomvardas and Thanos 2002). The core promoter dependence suggests that the composition of the PIC may be important.

How do GTFs and RNA polymerase II contribute to remodeling?

The identification of chromatin-modifying activities within yTAF1/dTAF1 (Mizzen et al. 1996; Pham and Sauer 2000) and mediator/RNA polymerase II holoenzyme (Myer and Young 1998; Lorch et al. 2000; Malik and Roeder 2000; Myers and Kornberg 2000) might suggest that remodeling is dependent on these activities. However, we believe this is not the case at RNR3. First, it is unlikely that the putative intrinsic HAT activity of TAF1p or HAT activity associated with RNA polymerase II is involved in nucleosome modification, because inactivating the taf1-2 mutant or the rpb1-1 mutant had a minimal to no effect on histone acetylation. Furthermore, Gcn5 accounts for the entirety of the DNA damage-induced histone H3 acetylation at the RNR3 promoter. Thus, it is unlikely that histone-modifying activities of TFIID (TAF1p), mediator, or RNA polymerase II are directly involved in remodeling nucleosomes at RNR3.

We argue that the function of TFIID and polymerase is to assist in the recruitment of the SWI/SNF complex to the promoter. In other words, the PIC can perform recruitment functions normally attributed to activator proteins. Significantly, a complex composed of the mediator and TFIID undergo reciprocal, cooperative interactions at promoters that can bypass the requirement for activators in PIC assembly by forming a platform on immobilized templates (Johnson et al. 2002). Likewise, TFIID and other PIC components may form a platform and recruit the SWI/SNF complex to RNR3 in vivo. Alternatively, TFIID and PIC components may stabilize SWI/SNF association after it has been directed to the promoter by a gene-specific factor (activator). We found that deletion of CRT1 causes the constitutive remodeling and recruitment of the transcription factors and remodeling machineries in wild-type cells, and disrupting the PIC under this situation causes the loss of SWI/SNF recruitment and maintenance of the remodeled state. This supports two conclusions: (1) It is unlikely that Crt1 itself or Ssn6/Tup1 is required for recruiting the remodeling machinery. This was a possibility, given recent analyses of galactose and stress-regulated genes (Papamichos-Chronakis et al. 2002; Proft and Struhl 2002). (2) Even if an unidentified gene regulatory protein binds to the RNR3 promoter, it is insufficient to maintain the presence of SWI/SNF and PIC components at the promoter. It seems logical that there is some contribution from a gene-specific factor(s) because, otherwise, TFIID could independently recruit chromatin remodeling activities to most promoters. Specifically, the core promoter architecture of the IFN-β promoter specifies SWI/SNF recruitment (Lomvardas and Thanos 2002). However, since the TAF_II dependence of RNR3 is specified by the upstream repression sequences (URSs), and not the core promoter (Li and Reese 2000), contributions from the core promoter alone cannot explain our results, but they cannot be ruled out as a contributing factor. Studies identifying this factor(s) are beyond the scope of this study.

Compared to other genes that have been characterized to date, the recruitment of transcription factors and coactivators to RNR3 appears to display a higher degree of cooperativity. For example, the recruitment (as measured by increased cross-linking) of TFIID, mediator, and SWI/SNF is dependent on RNA polymerase II. In contrast, recruitment of TBP to many yeast genes does not require Pol II (Kuras and Struhl 1999; Li et al. 1999), nor does the recruitment of mediator or SWI/SNF (Bhoite et al. 2001; Cosma et al. 2001; Park et al. 2001). Our data could be interpreted as if the recruitment of transcription factors occurs as part of a holoenzyme or larger complex, but such conclusions are premature. Our results do indicate that RNA polymerase II is required for the association of mediator and SWI/SNF with the RNR3 promoter; however, our results cannot distinguish whether or not these two complexes are corecruited with RNA polymerase as a holoenzyme, or recruited separately, or whether polymerase is required to stabilize their association after they have been recruited. Regardless of whether or not SWI/SNF and mediator are stable or stoichiometric components of an RNA polymerase II holoenzyme, the copurification of chromatin-remodeling activities with certain PIC components (Wilson et al. 1996; Grant et al. 1997; Nakajima et al. 1997; Cho et al. 1998; Neish et al. 1998), the genetic interactions between subunits of these complexes (Carlson 1997; Roberts and Winston 1997; Myer and Young 1998; Hampsey and Reinberg 1999), and the results described here indicate that an interaction among mediator, SWI/SNF, and RNA polymerase II may be essential for the regulation of gene expression.

MNase mapping

Temperature-shift experiments were typically performed as follows: 0.5 L of cell culture was grown to log-phase (OD₆₀₀=1.0 to 1.2) at 23°C, and then an equal volume of prewarmed YPD medium (50°C) was added. Following a 45-min preincubation, MMS was added to the final concentration of 0.015%, and the culture was maintained at 37°C for 2–2.5 h. Nuclei preparation was carried out essentially as described (Li and Reese 2001). For low-resolution mapping of nucleosome by indirect end labeling, the specific DNA sequences were detected by hybridization with a probe directed towards the end of the PstI site +468 to +725 relative to the major start site of RNR3, and to the HinfI site of SUC2 (Hirschhorn et al. 1992)

We thank Mai Xu and Robert Simpson for their assistance in establishing the chromatin mapping methods in our laboratory. Drs. Michael Green, Richard Young, Mark Soloman, Fred Winston, and Shelley Berger are recognized for strains used in these experiments, and those not shown. We thank Drs. Simpson, Jerry Workman, and members of the Reese lab and the Penn State gene regulation group for advice and comments on this work. This research was supported by funds provided by the NIH (GM58672) and by an Established Investigator Grant from the American Heart Association to J.C.R.

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