Patent Application: US-366901-A

Abstract:
a method is described for regulating gene expression related to iron metabolism to ameliorate diseases that include sickle cell disease , cancers , neurodegenerative diseases , friedreich &# 39 ; s ataxia and other neuromuscular disorders , and atherosclerosis . this approach is illustrated by recent findings that show that ferritin - h , an iron - binding protein that is present in cell nuclei , can repress the human β - globin gene , the gene that is mutated in sickle cell disease . increased expression of ferritin - h or a related ferritin - family peptide , given to effected cells either as the peptide itself , as an expression clone of the ferritin - h - subfamily gene , or via a gene regulator that increases expression of the ferritin - h - subfamily gene itself , prevents or ameliorates expression of the disease state in disorders where increased availability of iron is implicated in the etiology of the disease , including those named above .

Description:
it has been found that ferritin is a repressor of the human β - globin gene , the same gene that is mutated in sickle cell disease and in some forms of β - thalassemia . the repressor is a nuclear form of ferritin ( broyles et al ., “ a ferritin - like protein binds to a highly conserved cagtgc sequence in the β - globin promoter , in sickle cell disease and thalassaemias : new trends in therapy , of the ferritin h subfamily of ferritin peptides . briefly , the inventors have found the following : 1 ) a ferritin - family protein from human k562 erythroleukemia cell nuclear extracts ( as well as pure human ferritin - h ) binds to the promoter of the human β - globin gene ( the promoter that drives the mutated form of the gene in sickle cell ) at − 150 bp from the transcription start site , in vitro . the binding is very specific to that dna sequence . 2 ). an expression clone of ferritin - h represses this β - globin promoter in transient co - transfection experiments . this is very reproducible in multiple experiments with two different reporter genes , with no repression seen by control / null plasmids . 3 ) ferritin - h no longer represses if the promoter contains a mutated binding site . the inventors have a perfect control plasmid — a β - globin promoter mutated only in the ferritin - h binding site and hooked to the same reporter gene ( cat , in this case ). this is not only the perfect control for the transfections , but it also connects the in vitro dna binding with in vivo function quite nicely . since a decrease in β - globin expression is compensated by an increase in gamma ( fetal )- globin expression in human erythroids cells , and since a modest amount of this switching is known to totally ameliorate sickle cell and wholly or partially ameliorate β - thalassemias , this new finding makes ferritin useful for curing the phenotype of these classic genetic diseases . reports in the scientific literature indicate that h ferritin ( heavy chain ferritin ) is decreased by 50 % in aged rat brains and in other neurodegenerative diseases such as alzheimer &# 39 ; s and show that ferritin - h is found in the neurodegenerative diseases where iron - mediated oxidative damage has been demonstrated , as in parkinson &# 39 ; s disease and possibly huntington &# 39 ; s disease . there are also studies that indicate a protective role of ferritin against cancers , such as liver and skin cancers . it has been reported that uv light induces ferritin production in skin cells and that ferritin is protective against uv damage . indeed , ferritin h can be used to treat any diseases in which cellular injury is caused by iron - mediated oxidative damage . delivering the ferritin - h peptide or a truncated form of it to erythroid precursor cells is a more effective , more natural form of therapy than the partial measures currently in use to treat sickle cell disease and β - thalassemias . similar delivery of ferritin - derived peptides provides effective treatments and protection in alzheimer &# 39 ; s and other neurodegenerative diseases and cancers . the peptide can also be delivered as a fusion protein , with parts or all of the ferritin - h peptide fused to another protein such as transferrin or other ligand for which specific receptors exist on the surface of erythroid precursor cells , neurons , or other cell types for which protection is desired . the making of fusion proteins targeted to specific tissues is well know to those skilled in the art . alternatively , an expression clone that encodes ferritin - h or a part of it , delivered to erythroid precursor cells , to hematopoietic stem cells , to neurons or to other tissue cells in an appropriate vector , either ex vivo or in vivo ; and the protein expressed from such a vector also cures and protects against disease . the ferritin - h described here is distinct from other known trans - acting proteins in its physical properties and its proposed function as a repressor that binds primarily to the β - globin promoter . the h - ferritin subfamily is represented by a larger number of genes than the l - ferritin subfamily and includes a cluster of genes / pseudogenes on the x chromosome . one of these , ferritin - x , appears to encode a peptide identical in size and very similar in predicted three - dimensional structure to ferritin - h . the possibility remains that the actual dna - binding of the b - globin promoter − 150 cagtgc motif is mediated in vivo by a ferritin - associated protein that would be protected from proteinase k and heat treatments and react with anti - ferritin antisera because of its strong association with ferritin . however , if this is the case , it is a protein that is ubiquitous in human nuclear extract and there would be no need to upregulate it and it is ineffective in the absence of ferritin - h . upregulation of ferritin - h is enough . from the inventors &# 39 ; transient expression assays , it is clear that ferritin - h can repress the human b - globin gene and that this repression is mediated by binding of ferritin - h and / or a co - repressor to the − 150 region of the promoter containing a highly conserved cagtgc motif ( fig1 and 6 ). the binding site of this ferritin - h is within an important are required for activation of transcription of the β - globin gene . thus , the binding of this protein and displacement of other factors could be important in the repression of the human β - globin gene as apparently the mouse bb1 protein ( which recognizes the same sequence ) is in the repression of the mouse β - major globin gene in uninduced mel cells . subsequent interaction of this binding site with upstream negative regulatory regions creates a tightly - bound complex that prevents binding of other positive factors such as gata - 1 as well as sterically hinder the formation of an active transcription complex on the proximal promoter , by dna looping . fig7 shows a schematic representation of the ferritin - h protein having a bound iron ion . the active site responsible for ferroxidase activity has been elucidated . however , the active site or sites the protein responsible for transcription repression has not been identified . as with all other gene regulation proteins , derivatives of ferritin - h will repress dna transcription as well as or better than ferritin - h itself . these derivatives include fragments of ferritin proteins and any fusion proteins into which the active site or sites of ferritin h responsible for transcription repression has been spliced . ferritin h derivatives may also include larger transcription or translation products of a ferritin family protein . ferritin - h derivatives further include any mimetic proteins that represses dna transcription by means of an active site that is substantially the same as the ferritin - h active site responsible for dna binding and transcription repression . those skilled in the art will appreciate that the ferritin active site or sites responsible for repression of dna transcription may include both dna binding and protein binding sites . ferritin - h derivatives may be found in fragments of any of the ferritin family proteins . ferritin h is only one member of the family of ferritin proteins . ferritin h and ferritin l are the most studied . there are likely to be ferritin family proteins that have not yet been identified . ferritin family proteins are generally involved in iron metabolism . now that the inventors have elucidated the gene regulatory activity of ferritin h and its derivatives , it is likely that other ferritin family proteins will also have gene regulatory functions . the ability of ferritin family proteins to bind to the 5 ′ promoter region of the beta globin gene was ascertained only after lengthy and rigorous experimentation as described below . the first example shows that ferritin - h binds to the cagtgc ferritin binding site , seq id no : 1 , found at bases − 148 to − 153 of the 5 ′ promoter region of the human beta - globin gene . example 2 shows that in addition to binding to the ferritin binding site , ferritin - h binds to another nuclear protein that binds to the beta - globin 5 ′ promoter region further upstream of the ferritin binding site . fig8 through 12 show the experiments directed toward elucidating the mechanism by which ferritin - h represses the human beta [ greek symbol ]- gene . these results represent work - in - progress and show that human k562 cell nuclear ferritin interacts with other dna - binding proteins to repress this promoter , especially upstream silencer - binding proteins via dna - looping .” cell lines . k562 ( human erythroleukemia ) cells were grown in suspension in rpmi 1640 medium with 10 % or 15 % fetal bovine serum ( fbs ) and antibiotics as described ( berg , p . e ., williams , d . m ., qian , r . l ., cohen , r . b ., cao , s . x ., mittelman , m . & amp ; schechter , a . n . ( 1989 ) nucleic acids res 17 , 8833 - 52 ) and harvested at a density of 10 6 cells / ml for making nuclear extracts . cv - 1 ( african green monkey kidney epithelial ) cells ( adherent cells used for transfections / transient gene expression assays ) were grown in dmem with l - glutamine , 10 % fbs and antibiotics ( miller , i . j . & amp ; bieker , j . j . ( 1993 ) mol cell biol 13 , 2776 - 86 ). clones , transfections , and gene expression assays . the upstream region (− 610 /+ 20 ) of the human b - globin gene , previously cloned in psv2cat ( berg , p . e ., williams , d . m ., qian , r . l ., cohen , r . b ., cao , s . x ., mittelman , m . & amp ; schechter , a . n . ( 1989 ) nucleic acids res 17 , 8833 - 52 ), was subcloned through pgem and pselect ( now called palter ) and recloned into pcat - basic ( all vectors from promega ). mutants of the − 153 /− 148 site of the b - globin promoter were generated by transcription from mutant oligonucleotides corresponding to the − 164 /− 128 region using the pselect system . transfections of cv - 1 cells were carried out with dmrie - c transfection reagent ( gibco / brl ) in optimem serum - free medium and were optimized using the green fluorescent protein plasmid pegfp - c1 ( clontech ), fluorescence microscopy and quantitative fluorescence of cell lysates with a microtiter plate reader . the reporter gene chloramphenicol acetyl transferase ( cat ) was quantified in lysates of transfected cells using an elisa ( promega ) standardized with purified cat . the eklf ( erythroid kruppel - like factor ) expression plasmid has the eklf gene cloned into psg - 5 ( stratagene ;( miller , i . j . & amp ; bieker , j . j . ( 1993 ) mol cell biol 13 , 2776 - 86 ) and the ferritin - h expression clone is in the eucaryotic expression vector pcexv - 1 ( wu , y . j . & amp ; noguchi , c . t . ( 1991 ) j biol chem 266 , 17566 - 72 ). total cellular protein was determined with the bca microtiter plate assay ( pierce ) using bovine serum albumin as a standard . proteins and antibodies . ferritins from human liver ( enriched in l chains ) and from human heart ( enriched in h chains ), human transferrin ( iron saturated ) and apotransferrin , polyclonal ( rabbit ) antiserum to human spleen ferritin and nonimmune rabbit serum were obtained from sigma chemical company . restriction fragments and oligonucleotides . the 5 ′ region of the human b - globin gene ( from − 610 to + 20 ), previously cloned in psv2cat , was cut into three fragments by sequential digestions with hind iii and rsa i . the three fragments , electroeluted from agarose ( maniatis , t ., fritsch , e . f . & amp ; sambrook , j . ( 1989 ) molecular cloning : a laboratory manual ( cold spring harbor laboratory , cold spring harbor , n . y . ), were 416 bp (− 638 /− 223 ), 147 bp (− 127 /+ 20 , containing the proximal promoter region ), and 95 bp ( the rsa fragment , − 222 /− 128 , containing mainly distal promoter sequences ). the three fragments were phenol / chloroform treated , dephosphorylated , and end - labelled with 32 - p as described ( maniatis , t ., fritsch , e . f . & amp ; sambrook , j . ( 1989 ) molecular cloning : a laboratory manual ( cold spring harbor laboratory , cold spring harbor , n . y . ; kurien , b . t ., scofield , r . h ., & amp ; broyles , r . h . ( 1997 ) anal biochem 245 , 123 - 126 ). synthetic oligonucleotides corresponding to − 232 /− 188 and − 164 /− 128 were purified and annealed as previously described ( berg , p . e ., williams , d . m ., qian , r . l ., cohen , r . b ., cao , s . x ., mittelman , m . & amp ; schechter , a . n . ( 1989 ) nucleic acids res 17 , 8833 - 52 ), and the double - stranded oligos were end - labeled as above and / or used as unlabeled competitors in gel mobility shift assays . preparation of nuclear extracts . each nuclear extract preparation was made from two liters of k562 cells ( 1 × 10 6 cells / ml ) using the procedure of dignam , lebovitz , and roeder ( dignam , j . d ., lebovitz , r . m . & amp ; roeder , r . g . ( 1983 ) nucleic acids res 11 , 1475 - 89 ). protein content of the extracts ranged from 3 to 6 mg / ml . extracts enriched 80 - 90 % in ferritin - like protein ( s ) were prepared by treating the crude extracts with proteinase k and / or heat at 75 ° c . ( atkinson , b . g ., dean , r . l ., tomlinson , j . & amp ; blaker , t . w . ( 1989 ) biochem cell biol 67 , 52 - 7 ). gel mobility shift assays . gel retardation assays ( i . e ., gel shifts ) were used to determine dna binding of the extract proteins to the rsa i ( 95 bp ) fragment and synthetic oligonucleotides ( berg , p . e ., williams , d . m ., qian , r . l ., cohen , r . b ., cao , s . x ., mittelman , m . & amp ; schechter , a . n . ( 1989 ) nucleic acids res 17 , 8833 - 52 ; fried , m . & amp ; crothers , d . m . ( 1981 ) nucleic acids res 9 , 6505 - 25 ). each reaction contained 0 . 5 - 2 ng of dna , 1 . 0 - 5 . 0 ug of extract protein , 1 . 0 - 5 . 0 ug of poly di : poly dc , 100 mm kcl , and binding buffer ( berg , p . e ., williams , d . m ., qian , r . l ., cohen , r . b ., cao , s . x ., mittelman , m . & amp ; schechter , a . n . ( 1989 ) nucleic acids res 17 , 8833 - 52 ). unlabeled competitor oligonucleotides ranged from 15 - to 2000 - fold molar excess and were included in the reaction mixture with the probe before adding protein . gels used for retardation assays were 4 %, 5 %, or 6 % acrylamide and the running buffer was low ionic strength tae ( berg , p . e ., williams , d . m ., qian , r . l ., cohen , r . b ., cao , s . x ., mittelman , m . & amp ; schechter , a . n . ( 1989 ) nucleic acids res 17 , 8833 - 52 ). sequence alignments and homology searches . all mammalian β - globin promoter sequences (− 200 /+ 1 ) were obtained directly from genbank and manipulated using the pileup program followed by the lineup program of the university of wisconsin gcg package . ferritin - h represses expression driven by the b - globin promoter in transient co - transfection assays . a transient expression assay was set up with cv - 1 cells in which b - globin promoter - driven expression of a reporter gene is low unless the cells are co - transfected with an expression clone of eklf , a developmentally - specific activator of transcription . results with a b - cat reporter plasmid are shown in fig1 . the expression level of b - cat stimulated by eklf was repressed by over 60 percent by co - transfection of an expression clone of human h - chain ferritin ( i . e . ferritin - h ). controls included a positive cat - control plasmid ( expresses cat constituitively ), a negative cat - basic plasmid ( contains no promoter ), and b - cat without eklf stimulation . the experiment shown in fig1 has been repeated five more times with b - cat and three times with a b - luc ( b - promoter - luciferase ) construct with very similar results . the repression is also evident ( although reporter activity is lower ) when eklf is omitted ( data not shown ). other controls have included co - transfection of the “ empty ” carrier plasmids for all the expression clones ( no effect on reporter gene expression ) as well as keeping the total amount micrograms of transfected dna constant ( e . g ., fig6 ), to rule out the possibility of non - specific inhibition of gene expression due to excess dna or to some aspect of the structure of a carrier plasmid . co - transfection of an expression clone for ferritin - l sometimes resulted in some repression of reporter gene expression ; but the effect was less dramatic and inconsistently observed . binding of ferritin to b - globin promoter dna . a restriction fragment containing part of the distal promoter of the human β - globin gene , from − 222 /− 128 , is bound by human liver - or human heart - derived ferritin , as shown in gel retardation assays ( fig2 a , left side , lanes 3 and 4 ). in the inventors &# 39 ; experiments , ferritin from human heart ( which is enriched in h - type ( heavy ) subunits ) showed a higher degree of binding than liver ferritin ( which is relatively enriched in l - type subunits ), as indicated by the darker band in lane 3 ( left ). a restriction fragment containing the proximal promoter (− 127 /+ 20 ) does not show this binding ( fig2 a , right side , lanes 2 - 5 ); and another iron - binding protein , transferrin ( known to be primarily extracellular except when bound to its receptor ) does not bind the distal fragment bound by ferritin ( fig2 a , left side , lanes 5 and 6 ). the shift bands produced by the binding of human liver ferritin and by human heart ferritin usually correspond to the lower and upper shift bands produced by k562 cell crude nuclear extracts , respectively ( fig2 a , left side , lanes 2 - 4 ). the inventors have also found that nuclear extract ferritin can produce either shift band and that the higher molecular weight band will yield the lower band when eluted and reshifted . multiple shift bands with crude extracts ( e . g ., fig3 ) are the result of different sized aggregates of ferritin subunits or oligomers of the dna - protein complex and / or complexes with other proteins in crude extracts , since the inventors have found that at least one of the multiple bands contains gata - 1 . enrichment of a ferritin - like protein from k562 cell nuclear extracts . a polyclonal antiserum to human spleen ferritin ( which is composed of a mixture of heavy and light chains of ferritin ) was found to cause a supershift of part of the dna - protein complexes formed from crude k562 nuclear extract and the − 222 /− 128 restriction fragment , and the intensity of the super - shift band was proportional to the amount of antiserum added ( fig2 b ). the supershift with anti - ferritin antiserum was found to be specific for this dna - protein complex , very little - to - no dna was shifted in the absence of nuclear extract , neither anti - transferrin antiserum nor nonimmune rabbit serum ( not shown ) shifted the complex , anti - rabbit igg inhibited the supershift , and protein complexes with other dnas such as β - ivs2 were not shifted by the anti - ferritin antiserum ( fig2 c ). ferritin , unlike most proteins , is resistant to proteinase k digestion and heat at 75 ° c ., and can be obtained ninety percent pure from extracts of embryonic / larval erythroid cells using these two treatments . when this procedure was applied to k562 nuclear extracts , the remaining protein gave a single shift band with the − 222 /− 128 restriction fragment ( fig2 b , third lane from the left ). furthermore , when the anti - ferritin antiserum was added to the reaction mixture after incubation of the dna and binding protein , a larger complex was formed , resulting in a supershift band ( fig2 b , fourth lane ). it should be noted that the primary shift band with nuclear extract treated with proteinase k did not shift as far as the gel bands obtained with untreated extract . the inventors have investigated this in a series of timed digests and have found that ferritin is subject to partial digestion by proteinase k ; what remains after a 10 - 15 min digestion ( e . g ., fig2 c , third lane ) appears to be a proteinase k - resistant core that still binds dna . furthermore , increasing the amount of the enriched peptide preparation gives an increasing intensity to the shift band , and the band gradually moves up the gel as the complex builds in amount . as shown in fig3 b , the left series of lanes , a single supershift band was obtained ( arrow ) with the 95 - bp distal promoter that increased in intensity with increasing antiserum . to further localize the binding of the anti - ferritin reactive protein , the supershift was also performed with 32 p - labeled double - stranded oligonucleotides of the − 232 /− 188 and − 164 /− 128 sequences . the more 39 oligonucleotide gave a supershift band , whereas the more 59 oligonucleotide did not ( fig3 b ), indicating that the protein recognized by the antiserum binds to a 37 - bp sequence between − 164 and − 128 . the lack of a supershift with the − 232 /− 188 oligonucleotide also serves as a control for the specificity of the antibody . localization of the binding region with the antibody super - shift assay . the antibody gel shift was also used with 32 p - labeled double stranded oligonucleotides corresponding to the 3 ′ and 5 ′ ends of the 95 bp restriction fragment ( fig3 a ) and with crude k562 nuclear extracts to further localize the binding of ferritin . thus only the 3 ′ oligo gave the supershift band ( fig3 b ), indicating that the protein recognized by the antiserum binds to a 37 base pair ( bp ) sequence between − 164 and − 128 . definition of the binding site with competition gel shifts . to further localize the binding of ferritin , the inventors mutated the 37 bp oligonucleotide in different places , replacing six nucleotides at a time with all a &# 39 ; s , all c &# 39 ; s , or all g &# 39 ; s , with complementary nucleotide replacements in the opposing strand ( fig4 a ). a competition gel shift assay was done with the partially purified protein from heated k562 nuclear extract , in which each of the unlabeled mutant oligos as well as the native sequence was competed against the 32 - p - labeled native sequence for binding . all mutants competed for binding as well as the native sequence except those mutated in the − 153 /− 148 region , i . e ., mutated in the sequence cagtgc ( e . g ., fig4 d ). the inventors conclude that these six base pairs comprise the binding site of ferritin - h . in fig4 c , the specificity of this binding is titrated and quantified in oligo competitions with the unlabeled native sequence compared with the sequence mutated in all of the six nucleotides found to be important for binding , i . e ., the sequence cagtgc ( native ) compared with mutant # 4 ( fig4 a ). whereas the binding to the labeled native sequence is significantly competed with 50 - fold excess of unlabeled self , it took 1 , 000 - fold excess of the unlabeled mutant oligo to begin to compete with the binding to the native sequence , a twenty - fold difference . sequence alignments of the promoters (− 162 /+ 1 ) from twelve mammalian adult β - globin genes show that the − 150 cagtgc of the human β - promoter is very highly conserved . in a phylogenetic comparison of twelve mammalian adult β - globin promoters from the cap site to − 162 ( fig5 ), the inventors have found that the cagtgc sequence in the − 150 region is among the most conserved of the cis - acting elements , second only to the tata and ccaat boxes in its high degree of conservation , as highly conserved as the proximal cacc motif and more highly conserved than the distal cacc . the inventors &# 39 ; results show that in cv - 1 cells , an expression clone of human h - ferritin down - regulates expression of an eklf - stimulated b - globin promoter - driven cat reporter gene ( fig1 ). the inventors have identified a protein in k562 cell nuclear extracts that has unique properties , i . e ., stability to proteinase k and heat ( 75 ° c .) and reactivity with anti - ferritin antisera . ferritin - h binds to a 5 ′ region of the β - globin gene that is required for activation of the β - globin gene in k562 and normal erythroid cells , i . e ., the region between − 128 and − 222 from the cap site ( fig3 ). this dna region has been shown to bind native human ferritin in gel shift experiments ( fig2 ). the specificity of the binding of the ferritin - like protein has been confirmed using different dna segments and oligonucleotides in an antibody gel shift assay , and the oligos and antiferritin antiserum have been used to show that the binding site is between − 128 and − 165 ( fig3 ). competition gel shift assays with mutated oligonucleotides have shown that the binding of ferritin requires the nucleotides cagtgc , at − 153 /− 148 of the human β - globin gene ; and when this cagtgc motif is mutated , in vitro binding is reduced approximately twenty fold ( fig4 ). the ability of ferritin - h to repress in this system is abolished , but eklf - stimulation is retained , when the − 153 /− 148 ferritin binding site is mutated in the co - transfected b - globin - reporter plasmid ( fig6 ). these results show that ferritin h can repress the human adult b - globin gene by binding the promoter in a sequence - specific manner . the biology of ferritin - h and its highly conserved binding site ( fig5 ), as well as its demonstrated function in transient assays , mean that in k562 cells it is indeed functioning as a b - globin repressor . such a repressor is useful in ameliorating sickle cell and other genetic diseases . it is noteworthy that an rna sequence cagugn has previously been found to function in the regulation of translation and stability of mrnas coding for proteins involved in iron metabolism , e . g ., mrnas for ferritin subunits and for the transferrin receptor . in this quite different context , the hexanucleotide is at the apex of a stem - loop structure referred to as an ire ( iron - responsive element ), a stable secondary structure formed in the 5 ′ or 3 ′ untranslated regions of the single - stranded mrnas . the regulatory protein which binds to the ire ( the ire - bp ) has been identified as the cytosolic form of aconitase , a cubane iron - sulfur cluster protein with a molecular mass close to 97 kda . in contrast , the heat - stable , ferritin - h recognizes the cagtgc sequence in dna and apparently has a molecular mass of about 20 kda , or less if partially proteolyzed . globin gene regions are enriched in cagtgc / cagtgn sequences relative to the frequency one would expect for the sequence to occur at random . the human genome , as well as the 73 , 326 bp sequence of the β - like globin gene cluster on chromosome 11 , is approximately forty percent g + c . therefore , the frequency of occurrence of g and c nucleotides will be 0 . 2 each , and the frequency of a and t will be 0 . 3 each . the random frequency of occurrence of the sequence cagtgc will be ( 0 . 2 )( 0 . 3 )( 0 . 2 )( 0 . 3 )( 0 . 2 )( 0 . 2 )= 0 . 000144 . therefore , the sequence would be expected to occur by chance ten - to - eleven times in the 73 , 326 bp of the β - like cluster . the actual occurrence is thirty - six times , three - to - four times the number expected by chance . similarly , the pentamer cagtg ( in the sequence cagtgn ) occurs 205 times , again about four times the fifty - two / fifty - three occurrences expected by chance . the function of this sequence , like other cis - regulatory elements , is context - dependent . the sequence occurs in the 5 ′ and 3 ′ regions of the epsilon - and gamma - globin genes , but these locations and their surrounding sequences are markedly different from the − 153 location for the β - globin gene . binding of ferritin - h to sites 5 ′ and / or 3 ′ to the epsilon - and gamma - globin genes will have a stimulatory rather than an inhibitory effect on transcription . phylogenetic footprinting is useful for identifying important binding sites for regulatory proteins . in this regard , it is interesting that the cagtgc / cagtgn sequence is very highly conserved in sequence and location within mammalian β - globin gene promoters ( fig5 ), and is found in the β - promoters of chickens and frogs as well . the high conservation of this sequence means that this binding site has an important function . the xenopus adult major β - globin gene has the cagtgc sequence at − 45 from the cap site , and an oligonucleotide containing this sequence binds the human ferritin - h from k562 nuclear extracts more strongly than the corresponding region of the human β - globin promoter . consistent with the inventors &# 39 ; discovery that ferritin - h acts as a repressor of adult β - globin in human k562 cells is the inventors &# 39 ; finding that the − 150 binding site for this protein competes with the mouse β - major − 160 site known to bind the repressor protein bb1 . materials : calf intestine alkaline phosphatase , t4 polynucleotide kinase , and sau 96 i were obtained from promega / fisher . 32p - γ - atp was from dupont / nen . polyclonal ( rabbit ) antiserum to human spleen ferritin was obtained from sigma chemical company . all other reagents were molecular biology grade . restriction fragments and oligonucleotides : the 5 ′ region of the human beta globin gene ( from − 610 to + 20 ), previously cloned in psvocat , was cut from the purified plasmid by digestions with hind iii and bam hi . the 630 bp fragment was phenol / chloroform treated , dephosphorylated , and end - labelled with 32 - p . synthetic oligonucleotides corresponding to the core / bp - 1 binding site of ncr1 (− 584 /− 527 ), the more distal of the two 5 ′- β - globin silencers , and − 164 /− 128 region of the promoter were purified and annealed , and the double - stranded oligos were end - labeled as above and / or used as unlabeled competitors in gel mobility shift assays . preparation of nuclear extracts : nonadherent k562 cells were grown in suspension in a medium composed of rpmi 1640 and 15 % fetal bovine serum as described and harvested at a density of 106 cells per ml . for each preparation , nuclear extract was prepared from two liters of cells . protein content of the extracts ranged from 3 to 6 mg / ml . extracts enriched approximately 80 % in proteins which specifically bind the − 150 promoter region and the − 550 silencer region were prepared by treating the crude extracts with heat at 80 ° c . gel mobility shift assays : gel retardation assays ( i . e ., gel shifts ) were used to determine dna binding of the partially purified extract proteins , first to the synthetic oligonucleotides corresponding to − 550 silencer and to the − 150 region of the promoter , and subsequently to the 630 bp fragment of the human β - globin gene containing both the promoter and upstream regulatory sequences with the modifications described in the legend to fig1 . gels used for retardation assays were 4 % acrylamide and the running buffer was low ionic strength tae . experimental design : the dna looping assay is performed by mixing an extract containing proteins specific for regulatory sites that are proposed to interact , with dna containing the contiguous sites separated by intervening dna ; and the binding of the proteins is detected with a standard emsa . if proteins bound to separate sites interact with each other in a stable way , the intervening dna will form a loop which can be cut at a unique restriction site in the loop . the test for looping is that the dna - protein complex retains its emsa migration as a single band after the cut . controls include lanes with deproteinized aliquots of the reaction before and after the restriction digest , to prove that the loop was indeed cut . the conditions used for cutting the looped complex with sau 96 i are given in the legend to fig1 . we have reported in a preliminary paper that a restriction fragment containing part of the distal promoter of the human β - globin gene , from − 222 /− 128 bp , is bound by ferritin - h protein in k562 cell nuclear extracts , and is specific for the − 150 region . at least two proteins specific for the functionally defined silencers that map upstream of the proximal and distal promoter of the human β globin gene , in the regions of − 300 (− 338 /− 233 ) and − 530 (− 610 /− 490 ) from the cap site . with the ultimate aim of exploring interactions between these silencers and the β - promoter , we designed an experimental approach for detecting dna looping stabilized by interactions between proteins bound to sites separated by moderate lengths of intervening dna . we have used a partially purified k562 cell nuclear extract that contains proteins that bind these separate regions . fig8 is a diagram of the β - globin gene 5 ′ region used as a probe in these experiments . fig1 and 10 show emsas using this 630 bp probe (− 610 /+ 20 ) combined with restriction digests to demonstrate looping , and fig9 gives a diagrammatic interpretation of the results . the partially purified protein extract used for these experiments was found to contain both the − 150 promoter - binding protein and silencer (− 530 )- binding activity by separate gel shift assays with their respective oligonucleotides ( data not shown ). fig1 , lane 1 , and fig1 , lane 1 , show the migration of the dna alone , which gives a single band . in lane 2 of fig1 and 11 , all of the dna is retarded in its migration , due to the binding of proteins from the partially purified k562 nuclear extract . in lane 3 of fig1 and 11 , the material was reacted with the restriction enzyme sau 96 i , after the dna and proteins had formed a complex ; the large majority of this material was retarded in its migration similar to that in lane 2 . ( as shown in fig8 , there is a single sau 96 i site in the 5 ′ β - globin sequence , at − 210 , which cuts the dna between the promoter and the upstream regions .) in lanes 4 and 5 of fig1 and 11 , the complexes in lanes 2 and 3 , respectively , were deproteinized and run as the pure dna , showing that one large piece of dna was recovered from the complex in lane 2 , whereas all the dna from the complex in lane 3 was cut , giving two clean bands ( lanes 5 ) identical in their migration to bands obtained when pure dna was reacted with the restriction enzyme ( lane 6 , fig1 ). the lengths of these fragments are 229 bp (+ 20 /− 209 , containing the promoter ) and 401 bp ( containing the upstream sequences , including the silencers ). when the mixture containing these fragments of pre - cut dna was reacted with the partially purified proteins , the two fragments were shifted independently , but the large ( looped ) complex was not formed ( lane 7 , fig1 ). the observed fact that the complex detected in lane 2 of both figures holds together after the dna has been cut completely with sau 96 i indicates that a loop was initially formed between a site or sites downstream from − 209 and a site or sites upstream from − 210 . an interpretive drawing of these results is shown in fig9 , and the legend indicates which parts of the drawing correspond to which gel lanes in fig1 . fig9 shows a schematic diagram of the β - globin gene 5 ′ region 30 and the experiments used to elucidate protein binding to it . ferritin 34 binds to promoter region 30 at ferritin binding site 44 . several dna binding proteins 50 also bind to the β - globin promoter region . binding proteins 50 all bind upstream of the ferritin binding site 44 . repression of the β - globin gene by ferritin is enhanced by a protein - protein interaction between ferritin and at least one of the promoter binding proteins 50 . fig9 illustrates the protein - protein - dna complex ferritin 34 forms with at least one of binding proteins 32 . promoter region 30 has a ferritin binding site 44 and upstream of that a protein binding site 46 . binding protein 32 attaches to binding site 46 , and ferritin 34 binds to ferritin binding site 44 . ferritin 34 and binding protein 32 then bind to one another , thereby creating a loop in the dna . promoter region 30 may be cut into two smaller fragments 40 and 42 at restriction enzyme site 36 by restriction enzyme sau96i . because of the protein - protein interaction between binding protein 32 and ferritin 34 , the complex remains intact . thus , application of a restriction enzyme does not result in a mobility shift on a gel assay . this can be seen in lanes 2 and 3 of both fig1 and fig1 . removing proteins from the uncut dna loop results in an intact promoter region 30 illustrated in lane 4 of fig1 and land 4 of fig1 . removing proteins from the dna loop after being cut by a restriction enzyme results in two dna fragments 40 and 42 . these fragments may be seen in lane 5 of fig1 and lane 5 of fig1 . lane 7 of fig1 shows the result of adding nuclear extract to fragments 40 and 42 . the same complex found in lane 3 may be formed by adding nuclear extract to dna fragments 40 and 42 . when promoter fragments 40 and 42 are combined with a nuclear extract having ferritin 34 and binding protein 32 , a gel shift results . this is shown in lane 7 of fig1 . this shows that the dna loop is caused by ferritin binding to the promoter region . controls : controls incorporated into the experiments described above , as depicted in fig1 and 11 , include deproteinizing the complexes to show that the loop was cut by the restriction enzyme , and showing that unrelated dna sequences ( e . g ., p . putida dna ) do not form a complex with this extract . as a further control , the single - band complex in lane 3 from gels identical to that in fig1 was isolated , deproteinized , and also shown to contain equal amounts of the two restriction fragments resulting from sau 96 i digestion . as shown in fig1 , lanes 6 and 7 , the two sau 96 i fragments of the beta globin 5 ′ region shift independently with this extract and do not form the large complex unless they are linked ; furthermore , an approximately eight - fold greater protein concentration is required to begin to shift the separated restriction fragments than is required to initiate formation of the looped complex . interpretations : the reported experiments have shown that dna looping can be detected in vitro with an emsa assay combined with digestion with a specific restriction enzyme . in these dna looping experiments , the results show that sequences between − 209 and + 20 bp of the human β - globin gene interact with upstream sequences between − 210 and − 610 bp . the looping is mediated by a partially purified extract containing a − 150 promoter - binding protein and β - globin silencer - binding protein , confirmed by binding experiments with the extract and the separate binding sequences . when the single , large dna - protein complex detected by our emsas was cut with sau 96 i , the complex still migrated as a single , large complex high on the gels . ( there was a small increase in migration of the cut complex which is to be expected since a single , double - stranded restriction cut will change the dna conformation slightly .) it should also be noted that the binding of the proteins in this looped complex appears to be very tight ; it takes a high excess of unlabeled − 164 /− 128 and − 584 /− 527 oligonucleotides to break up the complex ( not shown ). furthermore , a comparison of the binding affinity of the full 630 bp dna with the binding affinities of a mixture of the fragments generated by sau 96 i , showed that it takes approximately eight - fold less protein to form a shifted complex with the large , intact ( 630 bp ) dna than with a mixture of the separate fragments , showing that the binding to the larger 630 bp dna is cooperative and that looping is occurring . all of these results are consistent with known parameters and forces controlling dna looping , which is mediated by two or more proteins showing cooperative ( and , usually , tight ) binding . the results of these experiments also show that repression of the β - globin gene by upstream silencers can be mediated by dna looping . this approach does not allow one to determine the identity of the proteins involved and may not work in cases where there is dna supercoiling , as with certain plasmid contructs in vitro , or weak protein - protein interaction . the loop in the promoter region formed by the interaction between ferritin and one or more upstream binding proteins enhances repression of the β - globin gene . human cells generally have sufficient amounts of upstream binding proteins such that addition of ferritin alone to a human cell by the methods described herein is generally sufficient to cause repression of the β - globin gene and other genes regulated by this activity . in addition , binding of ferritin to the cagtgc ferritin binding site is generally sufficient to repress transcription of the β - globin gene . one method of increasing ferritin - h expression is to repress expression of ferritin - l or other ferritin family proteins . this may be accomplished by using antisense dna oligonucleotides specific for the genes that encode ferritin family proteins other than ferritin - h . reduction and expression of these ferritin proteins leads to a higher concentration and heightened expression of ferritin - h . by shifting the ratios between ferritin - h and other ferritin family proteins , β - globin is repressed and the deleterious effects of sickle cell anemia are reduced to acceptable levels . heightened expression of ferritin - h also cures intracellular iron mismanagement , resulting in lower levels of harmful ferrous ions . while ferritin - h ferroxidase activity may play a role in proper management of intracellular iron , higher concentrations of ferritin - h affect expression of a number of genes involved in iron metabolism . this genetic regulatory function of ferritin - h facilitates proper iron management in cells that have been adversely affected by a wide variety of diseases . as described in the background , cancer , neurodegenerative diseases , neuromuscular disorders and atherosclerosis all lead to improper iron management within the body &# 39 ; s cells . increasing the concentration of ferritin - h and the resulting genetic regulatory effects alleviate the deleterious effects of improper iron management . studies have shown that ferritin - h exhibits the most efficient ferroxidase activity when it is expressed at roughly the same levels as ferritin - l . equal expression levels result in the highest number of ferritin - h / ferritin - l heteropolymers . the heteropolymeric form of the 24 - mer ferritin complex is the most efficient at converting the ferrous ion to the ferric ion and at sequestering iron ions . this suggests that maintaining equal concentrations of ferritin - h and ferritin - l is most likely to result in proper iron management . increasing levels of ferritin - h would result in the formation of ferritin - h homopolymers . ferritin - h homopolymers exhibit low ferroxidase activity . it would be expected that this would lead to higher levels of the more harmful ferrous ion and have adverse affects on the cells . however , the inventors have discovered that the gene regulatory functions of ferritin - h causes just the opposite to occur . those skilled in the art will realize that there are a number of ways in which to elevate levels of ferritin - h within a cell . it may involve introduction of the ferritin - h protein itself by any number of pharmaceutically acceptable means well known to those skilled in the art . this may include using liposomal constructs containing ferritin - h protein . these constructs may or may not have ligands or antibodies . an alternative method for increasing intracellular levels of ferritin - h is to regulate expression of ferritin family molecules . this may be done in a number of ways . antisense dna oligonucleotides that target ferritin family genes other than ferritin - h will result in decreased expression of the targeted gene and cause greater concentrations of ferritin - h within the cell . it is also possible to introduce proteins or other compounds that increase transcription or translation of an endogenous ferritin - h gene or a related ferritin family gene . these activating compounds may be introduced to cells in methods similar to the introduction of the ferritin - h protein itself as discussed above . yet another method of increasing intracellular levels of ferritin - h is to introduce a ferritin - h expressing vector into the cells . those skilled in the art will appreciate that there are a number of methods to transfect cells with a number of different vectors , including plasmids , phagemids , and cosmids . the type of vector used , the promoter region within the vector and any control sequences used with the vector will vary depending on a variety of factors known to those skilled in the art . these factors include but are not limited to the cell tissue targeted , the level of desired expression and the level of ferritin family protein expression within the targeted cells . yet another method of increasing intracellular levels of ferritin - h is to increase levels of proteins or compounds that elevate transcription or translation of ferritin - h promoters . methods of increasing intracellular methods may be considered either intracellular induction methods , where the cell creates its own ferritin , or extracellular introduction methods , where ferritin is added to the cell as when liposomal constructs are used . transfection of cells with vectors coding for a ferritin family protein may be performed either ex vivo or in vivo . when performed in vivo , the vectors are introduced into the patient &# 39 ; s body . when performed ex vivo , cells are transfected with a vector and then implanted into the patient &# 39 ; s body tissue . stem cells are especially well suited for this , however other cells may also be used . whereas , the present invention has been described in relation to the drawings attached hereto , it should be understood that other and further modifications , apart from those shown or suggested herein , may be made within the spirit and scope of this invention . citations in the following list of references are incorporated in pertinent part by reference . 1 . blau , c . a . & amp ; stamatoyannopoulos , g . 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