Identification Of An Intronic Enhancer Element

Diverse pro-inflammatory mediators (LPS, TNFa, IL-1b, IFN-g) regulate expression of MnSOD.  To comprehend the role of this anti-oxidant enzyme in cytoprotection, we sought to further identify the critical DNA sequences which mediate transcriptional regulation of this gene.  In transfected lung epithelial cells, a human growth hormone reporter gene system was utilized to identify regulatory elements in the promoter and within a 6.1 kb internal genomic fragment containing multiple DNase I hypersensitive sites.  Northern analysis revealed a 10-20 fold response to pro-inflammatory mediators and inclusion of the internal fragment was
necessary to mimic these stimulus-dependent endogenous levels.  The inducible element was localized to a 260 bp fragment in intron 2 coinciding with a previously defined DNase I hypersensitive site. 

A comparison of the sequence in intron 2 from the rat and human genes as shown below demonstrates a high degree of identity.  The overall homology between the two species being 60-65% within this intron.

rat    (1) TTCTGGGAATAATTGTTTTAATGGCTAA-ACAAAATAGTATGCTGTCTTT

human  (1) ---------TTAT-GTTTTAACGTTTTTTACAAAATAGTAT--TGTCTTT

Con    (1)          T AT GTTTTAA G  T   ACAAAATAGTAT  TGTCTTT

rat   (50) TTAATCTATTAGTT-----TTT--ACAAGGAGAATAATTGTTAGCCGTGT

human (39) TTAATTTATTCGTTAGTGGTTTGCACAAGGAAGATAATCGATAGTCATGT

Con   (51) TTAAT TATT GTT     TTT  ACAAGGA  ATAAT G TAG C TGT

rat   (93) CTCTGGGT-TAGCTGTATTGCTTGAGAAAACCAGACAGTAAAA----TTC

human (89) TTTTTAGACTCTCTGTATTGCTTG-GTAAGCTACGTAGTAAAAAATGTTT

Cons  (101)  T T  G  T  CTGTATTGCTTG G AA C A   AGTAAAA    TT

rat  (138) ACTTTTTCTTAAATACACTCTTATTGCCTCTCAGGTCTGGGAAACGGGTT

human(138) ACTTTTCCTTAAAT------GTTTTGAATTTCGGGGTTATGAAATTTGTT

Con  (151) ACTTTT CTTAAAT       T TTG  T TC GG  T  GAAA   GTT

rat  (188) GAGTAATTG-----------------GTTCACTGGGGGCATCTAGTGGAG

human(182) GAGTAATTTTTAGACAGTCACATCTTGTTGACTGGAGGCATCTAGTGGAA

Con  (201) GAGTAATT                  GTT ACTGG GGCATCTAGTGGA

rat  (221) AAGTGTGGTATTTTAGCATAGTTGTGT---AAGTGGCCCAACCAA-GAGA

human(232) AAATGCAGTATTTCAGCCTGATTGTGTTTGAAGTAAATGATTAAAAGAGG

Con  (251) AA TG  GTATTT AGC T  TTGTGT   AAGT     A   AA GAG

rat  (267) AGGAAATTACCACATTCTGGAAATTTTACTTGCAATAAGCAAATCACATA

human(282) AGGAAGTTACCACATTCTGGAAGATTTACTTGAGACAGAC--------GA

Con  (301) AGGAA TTACCACATTCTGGAA  TTTACTTG  A A  C         A

rat  (317) ATCGTGAAT-ACGGGAAGAGACTC--TGATTTAGGAAATGACAGATTTGG

human(324) ACCTTGAATTACGGGAAAAGGCCCCGTGATTTAGGAAATAACAAATTTGG

Con  (351) A C TGAAT ACGGGAA AG C C  TGATTTAGGAAAT ACA ATTTGG

rat  (364) GAAGGCTGTGGTAAT-AGTGAGTAGGGGAAAAGCCCAGTTGGGAAATCGT

human(374) GAAACATGTAATGGGGAGAGACT-GGGGAATACCCCAGTTGTGAAAGTAC

Con  (401) GAA   TGT  T    AG GA T GGGGAA A CCCAGTTG GAAA   

rat  (413) TTCCTCTAAGGTGACATCTGACA------ACTTTCCTCTTAA-TGTTGTA

human(423) TTCCTGTAAGGCAACATCTGACACCAGGAACCTTTCTCTTCAGTATTTTA

Con  (451) TTCCT TAAGG  ACATCTGACA      AC TT CTCTT A T TT TA

rat  (456) AAAACATGGTGATTTCAACCCTTCC---GTGGAGACAGAGCTGTATTTGT

human(473) AAAACAACTTAATTTCAGTCCTTTACTTGTGGAATCAGAGCCTTACTTAT

Con  (501) AAAACA   T ATTTCA  CCTT     GTGGA  CAGAGC  TA TT T

rat  (503) TTAGTGAATGCTGCTGGGAATAAGAAAGCCGTGGTTTTATTG-ACCTGGC

human(523) GTAATACAACCCACTGGAAAAAAGCTTTTTATTGTATTGTACTATATTGT

Con  (551)  TA T  A  C  CTGG AA AAG       T GT TT T   A  T G

rat  (552) TGAGGATGGATTTTGAAAAGGTGTTTACGTTTTATATTTCAG--------

human(573) TTATAAGTGATTGAGTACCTGCAGAGCTTTCTTTTACTTAAACATATTTT

Con  (601) T A  A  GATT  G A   G        T TT TA TT A        

rat  (594) --------------------------

human(623) AAAAATTATTAAAAAGATTTTCATGT

Con  (651)                          

This element functions in an orientation- and position-independent manner as well as with a heterologous promoter, as shown below (left) utilizing the minimal viral thymidine kinase promoter.  The ability to function with a heterologous promoter also satisfies the definition of a true enhancer.  This figure also demonstrates that the homologous sequence within the human MnSOD exhibits identical enhancer activity.  We have also placed the MnSOD enhancer/TK promoter in front of the DsRED reporter gene and as shown can generate red fluorescent cells in a stimulus specific manner. Another novel characteristic of this enhancer element involves its ability to support cytokine-inducible transcription in the absence of a classical promoter.  Thus, we have identified a complex highly conserved, cytokine-responsive, intronic element within rat and human MnSOD which we describe as a prohancer.

Our current regulation efforts are focused on the cloning of a number of these cognate trans-acting factors along with dominant negative experiments to demonstrate physiological relevance for these protein-DNA contacts. We are also currently using in vivo footprinting to detect the stimulus-specific proteins associated with the intronic enhancer element. A model illustrating a potential mechanism for the action of an internal enhancer element is shown below.