Role of the Intronic Enhancer in Tumor Necrosis Factor-mediated Induction of Manganous Superoxide Dismutase

Abstract

Manganous superoxide dismutase (Mn-SOD), a tumor necrosis factor (TNF)-inducible gene product, plays an important role in removing superoxide anions produced inside mitochondria. Two regulatory regions, the proximal promoter region (PPR), which is upstream from the transcription initiation site, and the TNF-responsive element (TNFRE), which is inside intron 2, are responsible for Mn-SOD expression. To understand how each of these regions contributes to the transcription of Mn-SOD, quantitative reverse transcription-PCR, chromatin immunoprecipitations, and in vivo nuclease sensitivity assays were performed on the murine Mn-SOD gene. These assays demonstrate that Sp1 and nuclear factor (NF)-κB p65 are required for Mn-SOD induction by TNF. Sp1 bound the PPR constitutively, whereas NF-κB p65 and C/EBP-β bound the TNFRE only after TNF treatment. Binding of C/EBP-β to the TNFRE was dependent on the presence of NF-κB p65. The chromatin structure within the TNFRE became more accessible to nuclease digestion after TNF treatment. This accessibility required Sp1 and NF-κB p65. Treatment of cells with an inhibitor of histone deacetylation, or transient transfection with coactivator-expressing plasmids, enhanced the expression of Mn-SOD. NF-κB p65 binding was required for acetylation of histones H3 and H4 at the PPR and the TNFRE. Together, these data suggest communication between the PPR and the TNFRE which involves chromatin remodeling and histone acetylation during the induction process of Mn-SOD in response to TNF. Manganous superoxide dismutase (Mn-SOD), a tumor necrosis factor (TNF)-inducible gene product, plays an important role in removing superoxide anions produced inside mitochondria. Two regulatory regions, the proximal promoter region (PPR), which is upstream from the transcription initiation site, and the TNF-responsive element (TNFRE), which is inside intron 2, are responsible for Mn-SOD expression. To understand how each of these regions contributes to the transcription of Mn-SOD, quantitative reverse transcription-PCR, chromatin immunoprecipitations, and in vivo nuclease sensitivity assays were performed on the murine Mn-SOD gene. These assays demonstrate that Sp1 and nuclear factor (NF)-κB p65 are required for Mn-SOD induction by TNF. Sp1 bound the PPR constitutively, whereas NF-κB p65 and C/EBP-β bound the TNFRE only after TNF treatment. Binding of C/EBP-β to the TNFRE was dependent on the presence of NF-κB p65. The chromatin structure within the TNFRE became more accessible to nuclease digestion after TNF treatment. This accessibility required Sp1 and NF-κB p65. Treatment of cells with an inhibitor of histone deacetylation, or transient transfection with coactivator-expressing plasmids, enhanced the expression of Mn-SOD. NF-κB p65 binding was required for acetylation of histones H3 and H4 at the PPR and the TNFRE. Together, these data suggest communication between the PPR and the TNFRE which involves chromatin remodeling and histone acetylation during the induction process of Mn-SOD in response to TNF. Manganous superoxide dismutase (Mn-SOD), 1The abbreviations used are: Mn-SOD, manganous superoxide dismutase; C/EBP, CCAAT/enhancer-binding protein; CBP, cAMP-responsive element-binding protein-binding protein; ChIP, chromatin immunoprecipitation; MCP, monocyte chemoattractant protein; MEF, murine embryonic fibroblast; MNase, micrococcal nuclease; NF-1, nuclear factor-1; NF-κB, nuclear factor-κB; PPR, proximal promoter region; RT-PCR, reverse transcription-PCR; TNF, tumor necrosis factor; TNFRE, tumor necrosis factor-responsive element; TSA, trichostatin A. 1The abbreviations used are: Mn-SOD, manganous superoxide dismutase; C/EBP, CCAAT/enhancer-binding protein; CBP, cAMP-responsive element-binding protein-binding protein; ChIP, chromatin immunoprecipitation; MCP, monocyte chemoattractant protein; MEF, murine embryonic fibroblast; MNase, micrococcal nuclease; NF-1, nuclear factor-1; NF-κB, nuclear factor-κB; PPR, proximal promoter region; RT-PCR, reverse transcription-PCR; TNF, tumor necrosis factor; TNFRE, tumor necrosis factor-responsive element; TSA, trichostatin A. a reactive oxygen scavenging metalloenzyme, catalyzes the conversion of superoxide radicals to hydrogen peroxide and oxygen inside mitochondria (1McCord J.M. Fridovich I. J. Biol. Chem. 1969; 244: 6049-6055Google Scholar). The expression of Mn-SOD is important in protecting mitochondria from oxidative damage mediated by superoxide radicals generated during normal cellular metabolism or under conditions of inflammation and oxidative stress, such as tumor necrosis factor (TNF)-mediated apoptosis (2Aggarwal B.B. Natarajan K. Eur. Cytokine Netw. 1996; 7: 93-124Google Scholar, 3Weisiger R.A. Fridovich I. J. Biol. Chem. 1973; 248: 3582-3592Google Scholar). The importance of Mn-SOD function is illustrated by the neonatal death of mice containing targeted disruptions of their Sod2 genes (4Lebovitz R.M. Zhang H. Vogel H. Cartwright J. Dionne L. Lu N. Huang S. Matzuk M.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9782-9787Google Scholar, 5Li Y. Huang T. Carlson E.J. Melov S. Ursell P.C. Olson J.L. Noble L.J. Yoshimura M.P. Berger C. Chan P.H. Wallace D.C. Epstein C.J. Nat. Genet. 1995; 11: 376-381Google Scholar). A variety of factors, which include TNF, interleukin-1, lipopolysaccharide, interferon-γ, and x-ray irradiation, induce Mn-SOD transcription (6Visner G.A. Dougall W.C. Wilson J.M. Burr I.A. Nick H.S. J. Biol. Chem. 1990; 265: 2856-2864Google Scholar, 7Hirose K. Longo D.L. Oppenheim J.J. Matsushima K. FASEB J. 1993; 7: 361-368Google Scholar, 8Akashi M. Hachiya M. Paquette R.L. Osawa Y. Shimizu S. Suzuki G. J. Biol. Chem. 1995; 270: 15864-15869Google Scholar). The important role of Mn-SOD in protection against inflammation and oxidative stress emphasizes the need to regulate Mn-SOD expression precisely. Sequence analysis, promoter mutagenesis, and in vivo genomic footprinting identified two regulatory regions separated by 2.2 kbp of DNA (Fig. 1) (9Jones P.L. Kucera G. Gordon H. Boss J.M. Gene (Amst.). 1995; 153: 155-161Google Scholar, 10Jones P.L. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Google Scholar). The 5′-proximal promoter region (PPR) contains numerous putative regulatory motifs that can be recognized by Sp1, AP-1, C/EBP, and NF-κB family members. Results of promoter mutagenesis experiments demonstrated that the PPR contributed only a 1.7-fold increase to the total induced expression in response to TNF, indicating that TNF responsiveness was not within this region. However, in vivo genomic footprinting suggested a role for the Sp1 binding sequences in constitutive expression of Mn-SOD. A TNF-responsive element (TNFRE) was identified within the second intron of the Mn-SOD gene. Sequence analysis of this 238-bp element revealed numerous potential transcription factor binding sites. TNF induced the occupancy of the binding sites for NF-κB, C/EBP, and NF-1 within the TNFRE. In vitro DNA binding assays showed that the 5′-region of the TNFRE bound C/EBP-β, and the 3′-region bound C/EBP-β and NF-κB. One report suggested an NF-κB binding site (originally named as C/EBPX because of its homology to a C/EBP binding site) within the 5′-region of the TNFRE which is indispensable for the responsiveness to TNF (11Maehara K. Hasegawa T. Isobe K. J. Cell. Biochem. 2002; 77: 474-486Google Scholar). The 3′-NF-κB site, however, was not independently responsive to TNF and was suggested to provide position and orientation independence to the TNFRE in a heterologous setting (10Jones P.L. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Google Scholar). The intronic location of the TNFRE leads to an intriguing question about how the PPR and the TNFRE communicate over 2.2 kbp of DNA. The packaging of eukaryotic DNA into nucleosome and higher order structures of chromatin allows for the possibility that these two regulatory regions are physically close enough for direct interaction or communication through bridging molecules. The role of chromatin structure in Mn-SOD expression was suggested by the observation that the promoter of the rat Mn-SOD gene became more sensitive to DNase I after TNF treatment, indicating that some form of nucleosome remodeling occurred (12Kuo S. Chesrown S.E. Mellott J.K. Rogers R.J. Hsu J.L. Nick H.S. J. Biol. Chem. 1999; 274: 3345-3354Google Scholar). Whether the remodeling involves acetylation of specific lysine residues on the N-terminal tails of core histones or nucleosome repositioning is not known. To determine the role of chromatin in controlling the accessibility of transcription factors to the Mn-SOD gene, we examined changes in nuclease-hypersensitive sites, the requirement for coactivators that have the potential to modify chromatin, and examined changes in histone acetylation at the PPR and the TNFRE of the Mn-SOD gene in response to TNF. transcription Sp1 and NF-κB p65 were to be required for the induction of Mn-SOD by TNF. The TNFRE not the PPR became to nuclease after TNF treatment, and Sp1 and NF-κB p65 were for these assays showed that Sp1 bound PPR constitutively, whereas C/EBP-β and NF-κB p65 bound the TNFRE only after TNF and the binding of C/EBP-β to DNA required NF-κB p65. In a NF-κB TNF induced changes in histone acetylation at the PPR and the TNFRE. of the coactivators element-binding protein-binding and and an inhibitor of the these suggest that induction of Mn-SOD by TNF communication between the PPR and the TNFRE, a process chromatin remodeling and histone acetylation that leads to the of the transcription factor at the PPR and the TNFRE, and in gene and murine were from the The embryonic containing targeted disruptions of the NF-κB p65 was by D. of were in with and and and trichostatin A were used at and To an embryonic that have more to the murine embryonic were from mice and The Sp1 was from embryonic cells containing a in Sp1 gene as D. Zhang A. S. Boss J.M. J. Biol. Chem. Scholar). The of NF-κB p65 by TNF is normal in this as by analysis of the gene D. Zhang A. S. Boss J.M. J. Biol. Chem. Scholar). This analysis and in vivo genomic in a to the is cells were in with and and and CBP, and were by L. and Y. transfection of cells was performed by the were with TNF at and for Mn-SOD by as was from cells the each of was reverse to the reverse were as of each reverse transcription was in a containing and of each was a with at for at for and at for for a total of Two of assays were performed for each the specific for of Mn-SOD and and and The for Mn-SOD was to that of and the was to a assays were performed at from was from L. cells were with the J. Scholar). cells were with TNF for was and for Mn-SOD by cells were and to about The cells were with containing and The cells were for at with in the the cells were with containing and by with a of for at The digestion was by the of containing and K. genomic DNA was and the of DNA were with was as (10Jones P.L. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Google specific for the regulatory region of Mn-SOD, which are in specific for PPR and TNFRE of Mn-SOD in a was performed as Boss J.M. Nat. Scholar). from cells was and with of to and at for The were and the was from the digestion and the DNA was in and used as in and were from histone H3 and histone H4 were from was performed in a The were used for the and were performed for a total of with at for and and at for the TNFRE, the and were used in a for with at for at for and at for was performed as The of DNA was by of the with a generated a of genomic DNA as To for in chromatin the of DNA were to the of chromatin DNA to each Sp1 and NF-κB p65 for the TNF of of Mn-SOD expression by TNF is at the in murine (10Jones P.L. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Google Scholar, J.M. Nick H.S. J. Biol. Chem. Scholar). In vitro DNA binding assays and in vivo genomic footprinting analysis of the regulatory region of the Mn-SOD gene suggest for Sp1 in binding to the PPR and for NF-κB binding to the TNFRE in response to TNF (10Jones P.L. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Google Scholar, K. Hasegawa T. Isobe K. J. Cell. Biochem. 2002; 77: 474-486Google Scholar, K. T. H. A. Isobe K. 1999; Scholar). To the of Sp1 and NF-κB in TNF induction of Mn-SOD, the of Mn-SOD in and and murine embryonic was by (Fig. Mn-SOD expression was the The of TNF induced the expression of Mn-SOD in the not in the and indicating that Sp1 and p65 are required for TNF induction of Mn-SOD in The transcription of gene, which is not by TNF, was not induced not A murine embryonic was for its to induce Mn-SOD in response to TNF. that Mn-SOD was induced in this with at (Fig. at of TNF treatment, Mn-SOD was induced in the or not TNF at the structure and its remodeling in response to have to a role in of interferon-γ, the and the murine tumor S.E. 1999; 11: Scholar, S. A. Scholar, S. A. Scholar, S. A. Scholar, C. J. 1993; Scholar, P.C. J. Mol. Cell. Biol. Scholar, Mol. Cell. Biol. 11: Scholar). To determine changes in chromatin structure to the of Mn-SOD sensitivity assays were DNA in regions as as in regions between with two of was performed on and cells after with potential during C. J. 1993; Scholar, Scholar). The genomic DNA was and by that the PPR and the TNFRE of the Mn-SOD gene. were on the PPR (Fig. to and to indicating that the region was accessible to the nuclease and is in an region to and to on the and to and to on the was to the sites of the of this region was not a of for as DNA was by at this region. the data into a (Fig. that the region upstream from the transcription initiation of sensitivity A of the PPR and the TNFRE regions by digestion in is the PPR, and and regions, the TNFRE, the regions that after TNF treatment, and regions that after TNF treatment. The the changes that in the digestion at the TNFRE (Fig. revealed regions on the to to and to and two on the to and to These regions the binding sites of NF-κB, and In a region and C/EBPX was to and to on the and to on the of TNF treatment, a increase in was within the potential region on the of the PPR, whereas the to on the and their (Fig. A and In changes were inside the TNFRE after of TNF of the TNFRE, were within the regions, whereas the and C/EBPX became (Fig. The in the are in the (Fig. The induced by TNF or of at of a transcription factor at and C/EBPX the accessibility to that to is important to that the produced to of the that of these a NF-κB p65 and Sp1 for experiments in demonstrate that Sp1 and NF-κB p65 were required for the induction of Mn-SOD by TNF. To determine the changes in chromatin structure are dependent Sp1 or NF-κB assays were performed on and NF-κB In the of TNF treatment, the at the PPR and the TNFRE in cells were from in TNF treatment, the to changes in the sensitivity (Fig. whereas the showed only in within the PPR (Fig. and region of the TNFRE to and to on the and to on the In the region to was upstream from the and C/EBPX in cells (Fig. These suggest that Sp1 and p65 are required for the chromatin structure changes in and These changes are to Mn-SOD induction by TNF and are through interaction between the PPR and the TNFRE. To the of Sp1, and C/EBP in the analysis was performed (Fig. The showed that and the of p65. and the of Sp1, whereas cells about of the The showed the for the of C/EBP-β expression were in and on in to of Sp1, NF-κB, and C/EBP have to regulatory regions of Mn-SOD through in vitro analysis (10Jones P.L. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Google Scholar, K. Hasegawa T. Isobe K. J. Cell. Biochem. 2002; 77: 474-486Google Scholar, K. T. H. A. Isobe K. 1999; Scholar). In vivo genomic footprinting the occupancy of binding sequences for Sp1 at the PPR and for NF-κB and C/EBP at the TNFRE (10Jones P.L. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Google Scholar). To determine these transcription factors to the Mn-SOD gene under in vivo assays with quantitative analysis of the DNA were against Sp1, NF-κB and C/EBP-β were The showed that p65 and C/EBP-β were induced to the TNFRE in response to TNF with and increase in the of DNA in cells with (Fig. In the binding of the two factors occurred as as treatment. A increase in p65 binding to the PPR was in the of Sp1 PPR DNA was after TNF treatment. with the only a of Sp1 DNA containing the TNFRE was that Sp1 not to this region. These are with the that TNF p65 and C/EBP-β to the TNFRE, whereas Sp1 the C/EBP-β the TNFRE in a factor at the Mn-SOD gene, assays against Sp1, and C/EBP-β were performed on and The showed that p65 to the Mn-SOD regulatory regions in whereas Sp1 only bound the PPR in In C/EBP-β was induced by TNF to the TNFRE in not cells (Fig. However, the of C/EBP-β binding in cells was about that in The of C/EBP-β binding to the TNFRE in cells was not the of the of C/EBP-β because of C/EBP-β were in and (Fig. These suggest 1) the presence of p65 is required for the binding of C/EBP-β to the TNFRE of and of transcription factors on the TNFRE Sp1 binding to the PPR 2.2 kbp Mn-SOD by to potential direct between the regions, is that coactivators to the of the TNFRE on the binding of Sp1 to the the of gene transcription can be by transcription of coactivators to J. Sci. Scholar). The is to transcription of some cellular genes by the function of A. J. 1997; Scholar, D. 1995; Scholar). To the requirement of coactivators for Mn-SOD cells were or not with of cells were with TNF for was and to quantitative specific to Mn-SOD and The showed that TNF induction of Mn-SOD by (Fig. The of was not by not To determine this observation is by and to of the of in cells was The showed that was to TNF induction of Mn-SOD by (Fig. is intriguing that a was not transfection is a factor in the experiments and some for the of the data suggest that an in to can function in this and TNF of the potential of and in Mn-SOD the of their was or were into cells by with TNF for was and by (Fig. of each of the two coactivators to a increase in Mn-SOD and the increase was in a of was not by not These suggest that function as for Mn-SOD induction by TNF. of to Mn-SOD changes in nuclease sensitivity and the potential for as by the experiments suggest the possibility that acetylation of the histone N-terminal which is to be with the of gene a role in this TSA, a histone was to induce the expression of the gene, D. G. Boss J.M. J. Biol. Chem. 1999; 274: Scholar). To determine of histone can Mn-SOD cells were with was and in quantitative analysis specific for Mn-SOD and (Fig. The showed that of to in a induction of Mn-SOD after a with These suggest that of histone is to induce the expression of Mn-SOD gene. TNF H3 and H4 at the PPR and the determine TNF induction leads to acetylation at the Mn-SOD gene, assays were performed on cells specific for H3 or H4 histone (Fig. The showed that TNF induced a of increase in acetylation of H3 and increase in acetylation of H4 at the A increase of in acetylation of H3 and H4 was at the TNFRE after TNF treatment. These changes occurred in a with the factor binding after TNF treatment. be the histone H3 acetylation in the PPR is in the constitutive and is the of the at the TNFRE not H3 and H4 acetylation of the gene were during the TNF treatment, indicating that the changes in histone acetylation were specific for the Mn-SOD gene. These demonstrate that TNF induction in acetylation of H3 and H4 at the PPR and the TNFRE. NF-κB p65 for at the PPR and the understand the role of Sp1 and NF-κB p65 in histone acetylation of the Mn-SOD gene, assays were performed on and (Fig. The showed increase in histone acetylation at the PPR and the TNFRE in cells after TNF treatment. This that changes in histone acetylation are dependent NF-κB p65 In a increase in histone H3 and H4 acetylation was at the TNFRE in which was to that in the The of these data is that Sp1 plays direct role in histone acetylation at the TNFRE in response to TNF. These are with the that in response to TNF the PPR and the TNFRE communicate to factor on the Mn-SOD gene through of the chromatin The data demonstrate that changes in chromatin structure are in the induction process of Mn-SOD transcription in response to TNF. and NF-κB the of Sp1, NF-κB and Mn-SOD gene by TNF were The binding of these factors to their regions within the Mn-SOD gene was demonstrated by a role in Mn-SOD expression. The of the nuclease digestion and assays suggest that TNF an chromatin structure within the TNFRE and histone acetylation at the PPR and the TNFRE. These changes were dependent on NF-κB p65. NF-κB p65 in the TNFRE Sp1 binding to the PPR, the suggest communication between the two regions during TNF on the and (9Jones P.L. Kucera G. Gordon H. Boss J.M. Gene (Amst.). 1995; 153: 155-161Google Scholar, 10Jones P.L. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Google the for of the Mn-SOD gene is constitutive or Mn-SOD histone H3 acetylation at the PPR is that the region is Binding of Sp1 to the in the PPR expression of Mn-SOD. In the the TNFRE is to be accessible as suggested by the of the at and at TNF treatment, NF-κB is into the and can the TNFRE. NF-κB to two sites within the TNFRE, C/EBPX and the NF-κB site, of which were to NF-κB in vitro (10Jones P.L. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Google Scholar, K. Hasegawa T. Isobe K. J. Cell. Biochem. 2002; 77: 474-486Google Scholar). on the observation that NF-κB p65 to the TNFRE in the binding of Sp1 to the PPR NF-κB binding to the TNFRE. NF-κB a or with histone such as between NF-κB and have in S. Y. T. Proc. Natl. Acad. Sci. U. S. A. 1997; Scholar, Genet. Scholar). The histone H3 and H4 at the TNFRE and the NF-κB the binding of C/EBP-β to the region. This is by data that in not in C/EBP-β bound the TNFRE after TNF In vitro assays demonstrated that NF-κB p65 the DNA binding of C/EBP-β P.C. Mol. Cell. Biol. 1993; Scholar). chromatin changes C/EBP-β to the remodeling A. Mol. Cell. 1999; Scholar). in this chromatin structure within the TNFRE, in the of as as its A of this is the increase in factor The of factors within the are to in the of the transcription to the The role of Sp1 in Mn-SOD by TNF was because the constitutive expression of Mn-SOD in the was to that in the not factors, such as for the of Sp1 during Mn-SOD transcription in Binding of to the PPR by in vitro analysis (11Maehara K. Hasegawa T. Isobe K. J. Cell. Biochem. 2002; 77: 474-486Google Scholar). However, Sp1 is required for expression of Mn-SOD, indicating specific between Sp1 and assays have that NF-κB p65 not the TNFRE in the of Sp1, that Sp1 the binding of NF-κB to DNA. have for the TNF induction of the gene D. Zhang A. S. Boss J.M. J. Biol. Chem. Scholar, D. Rogers Boss J.M. J. 1999; Scholar). The DNA binding regions of Sp1 and NF-κB are separated by 2.2 kbp of DNA which the question of how Sp1 and NF-κB physically The between Sp1 and NF-κB binding sites be 2.2 kbp of the DNA between Sp1 and NF-κB is into This Sp1 and NF-κB to each or communicate through bridging such as The binding of C/EBP-β to the TNFRE in that NF-κB p65 is for C/EBP binding the possibility that C/EBP-β in with the have the importance of NF-κB in gene T. D. T. FASEB J. 1995; Scholar). TNF induction of Mn-SOD in a NF-κB we that Mn-SOD was not indicating that NF-κB p65 is required for Mn-SOD and the sites in the TNFRE are specific for A report suggested that of Mn-SOD not NF-κB R.J. J.M. Nick H.S. J. Biol. Chem. a to In that an inhibitor to Mn-SOD induction by TNF, to the that NF-κB is not for Mn-SOD expression. The be the of used in the two is that are during TNF and of cells with inhibitor not of and the of NF-κB. in assays the chromatin DNA is into with an of about is for such assays to the of the two sites in the TNFRE because of the between the of the two C/EBP sites in the TNFRE be The data the role of the TNFRE in response to TNF and NF-κB as as C/EBP-β only the TNFRE. in the PPR DNA by the p65 be by the of a bridging the PPR and the TNFRE during Mn-SOD of the of the PPR DNA with the TNFRE DNA by the was that the region of Mn-SOD sites that NF-κB in vitro (10Jones P.L. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Google Scholar). these sites are of expression of Mn-SOD, in NF-κB p65 and in expression. The data be with binding of NF-κB to these sites. of histones with A. Biol. Scholar). In the of with gene whereas is with gene and of suggest a role for histone acetylation in Mn-SOD a requirement for coactivators with histone was suggested by of Mn-SOD and the that the Mn-SOD of histone with was to Mn-SOD assays showed that histone acetylation at the PPR and the TNFRE Mn-SOD induction by TNF. assays of histone acetylation in and suggest that of coactivators with histone the Mn-SOD gene is through NF-κB p65. and remodeling are important of of genes which are induced in response to J. J.L. Mol. Cell. Biol. 1999; Scholar, J. D. A. D. J. 17: Scholar). structures are to transcription their binding sites of transcription factors or transcription sites S.E. 1999; 11: Scholar). of leads to chromatin structures that of transcription factors or transcription on sensitivity analysis demonstrated nucleosome repositioning within the TNFRE after TNF of This is with the role of the TNFRE in response to TNF. Sp1 and NF-κB p65 are for the of Mn-SOD, their in the changes in chromatin nuclease sensitivity was in the experiments with NF-κB cells not nucleosome indicating that NF-κB p65 is for chromatin changes in the TNFRE in response to TNF. nucleosome repositioning occurred in the This that the region is accessible to some This accessibility be by the presence of within the which can to to of The to this region an important role for Sp1 in this which by at the TNFRE through direct between the PPR and the TNFRE, the of coactivators or chromatin or through its role in which to the region as G. and for for the and of this