▪ Abstract Eukaryotic translation initiation factor 4F (eIF4F) is a protein complex that mediates recruitment of ribosomes to mRNA. This event is the rate-limiting step for translation under most circumstances and a primary target for translational control. Functions of the constituent proteins of eIF4F include recognition of the mRNA 5′ cap structure (eIF4E), delivery of an RNA helicase to the 5′ region (eIF4A), bridging of the mRNA and the ribosome (eIF4G), and circularization of the mRNA via interaction with poly(A)-binding protein (eIF4G). eIF4 activity is regulated by transcription, phosphorylation, inhibitory proteins, and proteolytic cleavage. Extracellular stimuli evoke changes in phosphorylation that influence eIF4F activity, especially through the phosphoinositide 3-kinase (PI3K) and Ras signaling pathways. Viral infection and cellular stresses also affect eIF4F function. The recent determination of the structure of eIF4E at atomic resolution has provided insight about how translation is initiat...

eIF4 Initiation Factors: Effectors of mRNA Recruitment to Ribosomes and Regulators of Translation

The helix-loop-helix (HLH) family of transcriptional regulatory proteins are key players in a wide array of developmental processes. Over 240 HLH proteins have been identified to date in organisms ranging from the yeast Saccharomyces cerevisiae to humans (6). Studies in Xenopus laevis, Drosophila melanogaster, and mice have convincingly demonstrated that HLH proteins are intimately involved in developmental events such as cellular differentiation, lineage commitment, and sex determination. In yeast, HLH proteins regulate several important metabolic pathways, including phosphate uptake and phospholipid biosynthesis (19, 67, 112). In multicellular organisms, HLH factors are required for a multitude of important developmental processes, including neurogenesis, myogenesis, hematopoiesis, and pancreatic development (12, 86, 127, 179). The purpose of this review is to examine the structure and functional properties of HLH proteins.



E-box sites: elements mediating cell-type-specific gene transcription. 
Gene transcription of the immunoglobulin heavy-chain (IgH) gene has long been known to be regulated, in part, by a cis-acting DNA element known as the IgH intronic enhancer (109, 156). By in vivo methylation protection assays, a number of sites were identified in both the IgH and the kappa light-chain gene enhancers which were specifically protected in B cells but not in nonlymphoid cells (41). These elements shared a signature motif which consisted of the core hexanucleotide sequence, CANNTG, and were subsequently dubbed E boxes (41). A total of five E-box elements are present in the IgH gene enhancer: μE1, μE2, μE3, μE4, and μE5. The Ig kappa enhancer also contains three cannonical E boxes, designated κE1, κE2, and κE3. E-box sites have been subsequently found in B-cell-specific promoter and enhancer elements, including a subset of Ig light-chain gene promoters, the IgH and Ig light-chain 3′ enhancers, and, more recently, the λ5 promoter (110, 118, 156).

E-box elements have also been identified in promoter and enhancer elements that regulate muscle-, neuron-, and pancreas-specific gene expression. For example, in muscle, the muscle creatine kinase gene, acetylcholine receptor genes α and δ, and the myosin light-chain gene all require E-box elements for full activity (27, 51, 85). A number of genes whose expression is limited to the pancreas also require E-box sites for proper expression. The insulin and somatostatin genes, for example, contain E-box sites that, when multimerized, are sufficient to regulate pancreatic β-cell-specific gene expression (168). More recently, E-box regulatory sites have been identified in a number of neuron-specific genes, including the opsin, hippocalcin, beta 2 subunit of the neuronal nicotinic acetylcholine receptor, and muscarinic acetylcholine receptor genes (1, 21, 52, 125).




E-box sites: cognate recognition sequence for HLH proteins. 
Two proteins, termed E12 and E47, were originally identified as binding to the κE2/μE5 site (65, 102). They have a region of homology with the Drosophila Daughterless protein, the myogenic differentiation factor MyoD, members of the achaete-scute gene complex, and the Myc family of transcription factors (102). This stretch of conserved residues, known as the Myc homology region, appeared to be critical for the DNA binding properties of E12 and E47 (102). The E12 and E47 proteins, which differ only within this Myc homology region, arise by alternative splicing of the E2A gene (157). This conserved sequence, which was modeled as two amphipathic alpha helices separated by a flexible loop structure, was named the HLH motif and shown to function as a dimerization domain.




The HLH structure. 
The solution structure of the basic HLH (bHLH)-leucine zipper (LZ) factor Max first confirmed the existence of the HLH motif (44). Subsequently, the three-dimensional structure of the E47 bHLH polypeptide bound to its E-box recognition site, CACCTG, has been solved at 2.8-Å resolution (38). A number of interesting features were revealed from analysis of the E47 crystal structure. The E47 dimer forms a parallel, four-helix bundle which allows the basic region to contact the major groove (38). In addition to the basic region, residues in the loop and helix 2 also make contact with DNA (38). Stable interaction of the HLH domain is favored by van der Waals interactions between conserved hydrophobic residues (38). The E47 dimer is centered over the E box, with each monomer interacting with either a CAC or CAG half-site. A glutamate present in the basic region of each subunit makes contact with the cytosine and adenine bases in the E-box half-site. An adjacent arginine residue stabilizes the position of the glutamate by direct interaction with these nucleotides and additionally the phosphodiester backbone. Both the glutamate and the arginine residues are conserved in most bHLH proteins, consistent with a role in specific DNA binding (6, 38, 102).




Classification of the HLH proteins. 
Owing to the large number of HLH proteins that have been described, a classification scheme that was based upon tissue distribution, dimerization capabilities, and DNA-binding specificities was devised (Fig. ​(Fig.1)1) (101). Class I HLH proteins, also known as the E proteins, include E12, E47, HEB, E2-2, and Daughterless. These proteins are expressed in many tissues and capable of forming either homo- or heterodimers (103). The DNA-binding specificity of class I proteins is limited to the E-box site. Class II HLH proteins, which include members such as MyoD, myogenin, Atonal, NeuroD/BETA2, and the achaete-scute complex, show a tissue-restricted pattern of expression. With few exceptions, they are incapable of forming homodimers and preferentially heterodimerize with the E proteins. Class I-class II heterodimers can bind both canonical and noncanonical E-box sites (103). Class III HLH proteins include the Myc family of transcription factors, TFE3, SREBP-1, and the microphthalmia-associated transcription factor, Mi. Proteins of this class contain an LZ adjacent to the HLH motif (66, 177). Class IV HLH proteins define a family of molecules, including Mad, Max, and Mxi, that are capable of dimerizing with the Myc proteins or with one another (7, 22, 174). A group of HLH proteins that lack a basic region, including Id and emc, define the class V HLH proteins (18, 39, 47). Class V members are negative regulators of class I and class II HLH proteins (18, 39, 47). Class VI HLH proteins have as their defining feature a proline in their basic region. This group includes the Drosophila proteins Hairy and Enhancer of split (76, 141). Finally, the class VII HLH proteins are categorized by the presence of the bHLH-PAS domain and include members such as the aromatic hydrocarbon receptor (AHR), the AHR nuclear-translocator (Arnt), hypoxia-inducible factor 1α, and the Drosophila Single-minded and Period proteins (34). 



FIG. 1

Multiple sequence alignment and classification of some representative members of the HLH family of transcription factors. Shown is a dendrogram created by aligning the sequences of the indicated HLH proteins by the Clustal W algorithm (160).



Recently, another classification method of HLH proteins has been described (6). Based on the amino acid sequences of 242 HLH proteins, a phylogenetic tree was created to group family members according to evolutionary relationships (6). Four major groups, A through D, which comprise more than 24 protein families were identified (6). The groupings were based upon DNA-binding specificity as well as conservation of amino acids at certain positions (6). As the number of HLH proteins continues to grow, this evolutionary or “natural” classification may provide a more accurate and convenient means of categorization.

Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms.

The myoD gene converts many differentiated cell types into muscle. MyoD is a member of the basic-helix-loop-helix family of proteins; this 68-amino acid domain in MyoD is necessary and sufficient for myogenesis. MyoD binds cooperatively to muscle-specific enhancers and activates transcription. The helix-loop-helix motif is responsible for dimerization, and, depending on its dimerization partner, MyoD activity can be controlled. MyoD senses and integrates many facets of cell state. MyoD is expressed only in skeletal muscle and its precursors; in nonmuscle cells myoD is repressed by specific genes. MyoD activates its own transcription; this may stabilize commitment to myogenesis.

https://science.sciencemag.org/content/sci/251/4995/761.full.pdf

The myoD gene family: nodal point during specification of the muscle cell lineage

Chromosomal abnormalities in tumours were recognized at the end of the last century but their significance has only recently become clear. Distinct translocations in leukaemias and in solid tumours lead to the activation of proto-oncogene products or, more commonly, creation of tumour-specific fusion proteins. The proteins in both categories are often transcription factors and thus disruption of transcriptional control plays a major role in the aetiology of cancer. Fusion proteins formed after chromosomal translocations are common in a range of tumour types; these are unique tumour antigens and are therefore potential targets for therapy design.

Chromosomal translocations in human cancer.

Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytc leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n = 278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.

http://www.rticc.org/2006-2012/docs/programas/documento-121381205176b457a69c8cff2223a24563374476707973e5f9.pdf

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program

A new homeobox gene contributes the DNA binding domain of the t(1;19) translocation protein in pre-B ALL.

The DNA binding activities of some basic region and putative helix-loop-helix (bHLH)-containing transcriptional factors can be inhibited by the Id protein. Because Id contains the HLH motif for dimerization but not the basic amino acid region for DNA binding, heterodimers of Id with bHLH transcriptional factors may not bind to DNA. We have isolated and characterized the gene and cDNA clones for a new Id protein, designated Id2. The Id2 protein contains a helix-loop-helix motif similar to that of the previously described Id protein (referred to here as Id1), but the two proteins are different elsewhere. Id1 and Id2 are encoded by two unlinked genes, as shown by chromosome mapping. The two Id proteins have similar inhibitory activities. They selectively bind to and inhibit the function of one set of bHLH proteins, typified by E2A.E47 and E2B.m3, but not that of the other set, including TFE3, USF, and AP4. The Id proteins also homodimerize poorly. Expression of both Id genes is down-regulated during differentiation in a variety of cell types.

Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins

An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers

To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was cotransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

Human immunodeficiency virus tat-activated expression of poliovirus protein 2A inhibits mRNA translation

An intracellular method of inhibiting HIV in mammalian cells, in which a recombinant construct is introduced into the cells. The recombinant construct includes a) the HIV long terminal repeat (LTR) or a portion of the HIV LTR which includes a functional HIV promoter and DNA of non-HIV origin encoding a product which is toxic to HIV-infected cells, when present in such cells alone or in conjunction with a selected substance, or b) all of the HIV LTR or a portion of the HIV LTR which includes a functional HIV promoter and mutated or altered HIV DNA. Compositions for use in inhibiting HIV, which include such recombinant constructs, are also disclosed.

Xiao-Hong Sun

Papers

A new homeobox gene contributes the DNA binding domain of the t(1;19) translocation protein in pre-B ALL.

Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins

An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers

Human immunodeficiency virus tat-activated expression of poliovirus protein 2A inhibits mRNA translation

Intracellular method of inhibiting hiv in mammalian cells