Differential Interactions of Id Proteins with Basic-Helix-Loop-Helix Transcription Factors

Abstract

Dimerization of three Id proteins (Id1, Id2, and Id3) with the four class A E proteins (E12, E47, E2-2, and HEB) and two groups of class B proteins, the myogenic regulatory factors (MRFs: MyoD, myogenin, Myf-5 and MRF4/Myf-6), and the hematopoietic factors (Scl/Tal-1, Tal-2, and Lyl-1) were tested in a quantitative yeast 2-hybrid assay. All three Ids bound with high affinity to E proteins, but a much broader range of interactions was observed between Ids and the class B factors. Id1 and Id2 interacted strongly with MyoD and Myf-5 and weakly with myogenin and MRF4/Myf-6, whereas Id3 interacted weakly with all four MRFs. Similar specificities were observed in co-immunoprecipitation and mammalian 2-hybrid analyses. No interactions were found between the Ids and any of the hematopoietic factors. Each Id was able to disrupt the ability of E protein-MyoD complexes to transactivate from a muscle creatine kinase reporter construct in vivo. Finally, mutagenesis experiments showed that the differences between Id1 and Id3 binding map to three amino acids in the first helix and to a small cluster of upstream residues. The Id proteins thus display a signature range of interactions with all of their potential dimerization partners and may play a role in myogenesis which is distinct from that in hematopoiesis.An increasingly important role is ascribed to protein-protein interactions in the regulation of cellular growth and differentiation pathways. Dimerization serves to convert inactive monomeric molecules into transcriptionally active dimeric complexes at specific times during cellular development. Deletional analysis has identified a number of evolutionarily conserved regions that mediate these interactions. One such region, commonly associated with transcription factors involved in a range of proliferative and differentiation pathways, is the basic-helixloop-helix (bHLH) 1 (1). This domain is conserved from yeast to mammals and is composed of a positively charged basic region followed by two amphipathic ␣-helices separated by a spacer loop. Dimers are stabilized by a series of hydrophobic and electrostatic interactions between the helices of compatible molecules (2-5). The juxtaposition of two basic regions resulting from dimerization forms a DNA binding interface able to insert into the major groove in a sequence-specific manner (2, 6). Although bHLH proteins have no discernible DNA binding activity as monomers, dimers recognize a consensus DNA sequence (CANNTG), termed the E box (4, 7-9).bHLH transcription factors can be broadly placed into two categories (reviewed in Ref. 10). The class A factors, or E proteins, (E2-2, HEB, and the E2A gene products E12 and E47) are expressed in a virtually ubiquitous pattern and are able to dimerize efficiently with tissue-restricted class B factors to activate gene expression (1,(11)(12)(13). Because each factor contributes a specific DNA recognition half-site, class A and class B heterodimeric complexes and class A hetero-or homodimers theoretically provide distinct combinatorial E b...

Id3 modulates cellular localization of bHLH Ptf1‐p48 protein

“…Indeed, although Id proteins are usually believed to bind preferentially the ubiquitous class A bHLH E-proteins such as E47, several reports demonstrated that Id3 could also interact with tissue-specific class B bHLH factors, such as myogenic regulatory factors, or other transcription factors such as Pax5. [31][32][33] Here, we also describe for the first time a mechanism involving Id3 protein as a regulator of bHLH protein cellular distribution. The inhibition of OLIG transcription factor by other members of the Id family has recently been reported.…”

E2A protein degradation by the ubiquitin-proteasome system is stage-dependent during muscle differentiation

“…The E2A proteins are regulated by the dominantnegative Id (inhibitor of differentiation/DNA binding) proteins, which are HLH proteins that lack a basic, DNA-binding domain and function by sequestering E2A proteins through heterodimerization into an inactive state unable to bind DNA. The formation of the transcriptionally active complex between E2A proteins and MyoD is regulated by Id1, which is the most active Id protein in terms of MyoD binding (Langlands et al, 1997).…”

A murine ATFa-associated factor with transcriptional repressing activity

“…Besides these molecules which may be considered as mediators of an epigenetic control of gene expression, repressors acting directly either on enhancer-bound proteins or on the basal transcription machinery have been identi®ed and fall into two broad classes: (i) passive repressors which compete for the target of DNAbinding transcriptional activators or alter their binding activity (MyoD/Id, c-Jun/JunB) (Dias et al, 1994;Langlands et al, 1997;Mechta et al, 1997) or their activating properties (Gal4/Gal80) (Lohr et al, 1995); (ii) active repressors which operate through proteinprotein interactions with components of the basal or activated transcription apparatus, thereby preventing the assembly of functional pre-initiation complexes or mediating the formation of frozen complexes.…”