I-POU: a POU-domain protein that inhibits neuron-specific gene activation

Abstract

A novel, structurally distinct POU-domain protein has been identified that inhibits activation by another positive POU-domain regulator of neuron-specific transcription units. Two Drosophila POU-domain proteins, I-POU and Cf1-a, are coexpressed in overlapping subsets of neurons during development. Because I-POU lacks two basic residues in the N terminus of its homeodomain, it cannot bind DNA, but it does form a stable heterodimeric complex with Cf1-a, preventing Cf1-a from binding to DNA recognition elements and from transactivating the dopa-decarboxylase gene. The inhibition by I-POU provides a potential strategy by which the activation of genes in development is controlled by a homeodomain-containing protein that does not bind DNA.

The Id proteins and angiogenesis

“…Since Id lacks a basic DNA binding domain, heterodimers between Id and bHLH proteins cannot bind DNA. This dominant negative mode of inhibition of DNA binding activity is widely used in the cell as it is also employed by members of the leucine zipper and homeodomain protein families (Ron and Habener, 1992;Treacy et al, 1991).…”

A human POU domain gene, mPOU, is expressed in developing brain and specific adult tissues

“…The most significant feature of its sequence is the lack of two basic amino acid residues at the N-terminus of POU,,. Using this criterion BDOM234 was assigned to class IV-1, which was established after the discovery of the Drosophila inhibitory POU protein named I-POU [24]. For Drosophila I-POU, it has been demonstrated that the lack of two residues in the highly conserved basic pentapeptide leads to loss of DNAbinding activity.…”

“…Isolated DNA fragments were end labeled with [y-"PIATP to a specific activity of 1 X 10'cpdpg. Binding reactions (15 pl) contained 25 mM KC1, 1 mM EDTA, 4% , Bm-1 [37], I-POU [24], Unc86 [38], Oct-3A [25], Emb [26]. r, rat; h, human; m, mouse; d, Drosophila; c, Cuenorhubditis elegans.…”

Zfy1 encodes a nuclear sequence‐specific DNA binding protein

“…The sequences of these oligonucleotides were: Random sequence 76-mer oligonucleotide (R76), 5'-CAGGTCAGATCAGCG-GATCCTGTCG(N)26GAGGCGAATTCAGTGCATGTGCAGC-3' , 'Forward' primer, 5'-GCTGCACATGCACTGAATTCGCCTC-Y; 'Back' primer, 5'-CAGGTCAGATCAGCGGATCCTGTCG-Y. Random oligonucleotide binding assays were performed essentially as previously described [29,30]. Briefly, lug of purified renatured protein was electrophoresed on a 10% SDS-PAGE gel, electroblotted onto a nitrocellulose filter and the filter incubated at 4°C with the 3~p-labelled pool of random oligonucleotides (107108 cpm/gg) prepared by primed synthesis [28].…”

“…Recombinant ZfylC protein was purified by preparative gel electrophoresis, then denatured and re-folded in the presence of zinc cations and used in random oligonucleotide binding assays [30]. Oligonucleotides bound to the Zfyl protein were eluted and amplified by PCR, then used in further rounds of binding, elution and amplification, to select for sequences that bound specifically.…”