Molecular analysis of the interaction between an enhancer binding factor and its DNA target

Abstract

ABSTRATTIei fine contacts of a mouse nuclear factor, called PEB1, with the B enhancer of polyoma virus were analyzed. It protects against DNaseI attack a region of about 50 base pairs that can be divided in two domains. The first contains a GC-rich palindrome and the homology to the SV40 enhancer. The second is homologous to a sequence in the immunoglobulin (Ig) heavy chain gene enhancer. Methylation interference and protection experiments reveal strong specific contacts only with a purine rich track on the late coding strand of the early proximal part of the palindrome. Deletion analysis show that the minimal sequences necessary for binding include only the first domain. The Ig homology contributes only weakly to the binding. The minimal core is similar to the core of the B enhancer defined in vivo. The interactions we observe here are reminiscent of those of TFIIIA positive transcription factor and the 5SRNA gene of Xenopus. INTKRODUIONDNA-protein interactions play an essential role in the regulation of gene expression and DNA replication. Characterization of these interactions can help unravel the mechanisms involved in these processes. Repressor-operator interactions in prokaryotes were the first to be analyzed in great detail: in essence, a repressor dimer interacts with both parts of a palindromic sequence in a symmetric way; a conserved c-helix-turn-a-helix motif fits into the large groove of right-handed B-DNA (1). This model was found to be applicable to activator proteins like the CAP protein (2) or even yeast proteins involved in mating type control (3).In higher eukaryotes only a few cases of specific DNA-protein interactions have been studied in detail so far. The positive transcription factor TF III A of Xenopus laevis contacts the DNA mainly along the non coding strand (4) and the helical configuration of the DNA may be altered by this binding (5). Knowledge of the primary structure of the protein has led to a model in which small Zn++ binding, fingerlike domains interact with the DNA in a way quite different from bacterial repressors or activators (6). Another

The need for enhancers in gene expression first appears during mouse development with formation of the zygotic nucleus.

“…The essential origin sequences are shown (striped bars). DNAinding sites for putative enhancer-specific proteins (open boxes) include PEAl, 2, and 3 (Martin et al 1988); EFC (Fujimura 1986;Ostapuck et al 1986); PEBl (Bohnlein and Gruss 1986;Piette and Yaniv 1986); and F441 (Kovesdi et al 1987). The enhancer region of three PyV host-range mutants selected for growth in undifferentiated mouse embryonal carcinoma F9 cells (Fujimura et al 1981) contains a point mutation at nucleotide 5233 (solid bar, F441), a 31-bp tandem duplication of the mutated region (crosshatched box.…”

Multiple subelements within the polyomavirus enhancer function synergistically to activate DNA replication.

“…PEB1 and EBP20 also bind to sequences adjacent to the primary binding site. The binding of PEB1 to the secondary site is sequence independent (31), and the binding of EPB20 to its secondary sites only occurs at high protein-to-DNA ratios (13 The sequences which make up the a and p enhancer elements correspond to Py enhancer elements 2 and 3. The Py enhancer comprises three enhancer elements, which we have named 1, 2, and 3 (22).…”

“…The auxiliary subelement and core subelement acted synergistically to activate 5000 Py ENHANCER ELEMENTS AND SUBELEMENTS 5001 DNA replication. The subelements share few sequences and interact in vitro with different DNA-binding proteins (2,6,13,28,(30)(31)(32). These results suggest that a number of different enhancer-binding proteins can activate Py DNA replication, presumably by a common mechanism.…”

The effects of DNA intercalators on chromatin of chicken red blood cells --- differential extraction on nonhistone proteins

“…The proteins binding to Z -DNA or cruciform DNA binding proteins have been substantially purified from eukaryotic cells [11][12] . Hence, it has been generally believed that alteration of DNA conformation can influence the stability of DNA -protein complexes and vice versa [13][14][15] . In eukaryotic genome, chromatin is built up with DNA and heterogeneous proteins, and has tremendous capacity of varying its conformation at various hierarchies to meet different functional requirements in.…”

“…cell . The characterization of functionally active regions in the eukaryotic genome have been done in fair detail [11][12][13][14][15][16][17], and its less compact structure can be conveniently measured by assessing their sensitivity to DNase I.…”