Precise alignment of sites required for mu enhancer activation in B cells.
The lymphocyte-specific immunoglobulin mu heavy-chain gene intronic enhancer is regulated by multiple nuclear factors. The previously defined minimal enhancer containing the muA, muE3, and muB sites is transactivated by a combination of the ETS-domain proteins PU.1 and Ets-1 in nonlymphoid cells. The core GGAAs of the muA and muB sites are separated by 30 nucleotides, suggesting that ETS proteins bind to these sites from these same side of the DNA helix. We tested the necessity for appropriate spatial alignment of these elements by using mutated enhancers with altered spacings. A 4- or 10-bp insertion between muE3 and muB inactivated the mu enhancer in S194 plasma cells but did not affect in vitro binding of Ets-1, PU.1, or the muE3-binding protein TFE3, alone or in pairwise combinations. Circular permutation and phasing analyses demonstrated that PU.1 binding but not TFE3 or Ets-1 bends mu enhancer DNA toward the major groove. We propose that the requirement for precise spacing of the muA and muB elements is due in part to a directed DNA bend induced by PU.1.
Nikolajczyk
B S
BS
Rosenstiel Basic Medical Sciences Research Center, Department of Biology, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Nelsen
B
B
Sen
R
R
D000818:Animals; D001402:B-Lymphocytes / Q000502:physiology*; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002474:Cell-Free System; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D011817:Rabbits; D051381:Rats; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D015534:Trans-Activators; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D000099232:Proto-Oncogene Protein Spi-1
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D017931:DNA Primers; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D015534:Trans-Activators; D014157:Transcription Factors; D000099232:Proto-Oncogene Protein Spi-1; C494041:ETS1 protein, human; C494042:Ets1 protein, mouse; C494043:Ets1 protein, rat; C069203:TFE3 protein, human