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.

Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia

https://lymphochip.nih.gov/Shaffer_Blimp_Immunity2002.pdf

Blimp-1 Orchestrates Plasma Cell Differentiation by Extinguishing the Mature B Cell Gene Expression Program

Id proteins may control cell differentiation by interfering with DNA binding of transcription factors. Here we show that targeted disruption of the dominant negative helix–loop–helix proteins Id1 and Id3 in mice results in premature withdrawal of neuroblasts from the cell cycle and expression of neural-specific differentiation markers. The Id1–Id3 double knockout mice also display vascular malformations in the forebrain and an absence of branching and sprouting of blood vessels into the neuroectoderm. As angiogenesis both in the brain and in tumours requires invasion of avascular tissue by endothelial cells, we examined the Id knockout mice for their ability to support the growth of tumour xenografts. Three different tumours failed to grow and/or metastasize in Id1+/-Id3-/- mice, and any tumour growth present showed poor vascularization and extensive necrosis. Thus, the Id genes are required to maintain the timing of neuronal differentiation in the embryo and invasiveness of the vasculature. Because the Id genes are expressed at very low levels in adults, they make attractive new targets for anti-angiogenic drug design.

Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 is a major player in cell cycle control and it is mainly regulated at the transcriptional level. Whereas induction of p21 predominantly leads to cell cycle arrest, repression of p21 may have a variety of outcomes depending on the context. In this review, we concentrate on transcriptional repression of p21 by cellular and viral factors, and delve in detail into its possible biological implications and its role in cancer. It seems that the major mode of p21 transcriptional repression by negative regulators is the interference with positive transcription factors without direct binding to the p21 promoter. Specifically, the negative factors may either inhibit binding of positive regulators to the promoter or hinder their transcriptional activity. The ability of p21 to inhibit proliferation may contribute to its tumor suppressor function. Because of this, it is not surprising that a number of oncogenes repress p21 to promote cell growth and tumorigenesis. However, p21 is also an inhibitor of apoptosis and p21 repression may also have an anticancer effect. For example, c-Myc and chemical p21 inhibitors, which repress p21, sensitize tumor cells to apoptosis by anticancer drugs. Further identification of factors that repress p21 is likely to contribute to the better understanding of its role in cancer.

https://cancerres.aacrjournals.org/content/canres/65/10/3980.full.pdf

Lost in transcription: p21 repression, mechanisms, and consequences.

The helix-loop-helix transcription factor E2A plays important roles not only in promoting cellular differentiation but also in suppressing cell growth. Id proteins, the inhibitors of E2A, have opposite effects on cell differentiation and growth. To understand the mechanisms by which E2A suppresses cell growth, we examined the role of E2A in regulating the expression of the cyclin-dependent kinase inhibitor p21CIP1/WAF1/SD11, which prevents cell cycle progression upon overexpression. By using transient-cotransfection assays of luciferase reporter constructs in HeLa cells, we have found that overexpression of E2A can transcriptionally activate the p21 gene. To identify the sequences that mediate this activation in the promoter of the p21 gene, we carried out mutational analyses. Out of the eight putative E2A-binding sequences (E1 to E8) in the promoter, the E1 to E3 sequences located close to the transcription start site are found to be essential. In addition, loss of the E boxes in the promoter also reduces p21 expression without cotransfection with E2A in HIT pancreatic cells, where the endogenous E2A-like activity is high. Furthermore, we have also shown that overexpression of E2A in 293T cells activates expression of the endogenous p21 gene at both the levels of mRNA and protein. In correlation with the finding that E47 overexpression leads to growth arrest in NIH 3T3 cells, we have shown that Id1 overexpression in NIH 3T3 cells accelerates cell growth and inhibits p21 expression. Taken together, these results provide insight into the mechanisms by which E2A and Id proteins control cell growth.

Regulation of the Expression of Cyclin-Dependent Kinase Inhibitor p21 by E2A and Id Proteins

B-lymphocyte activation and proliferation induced by the B-cell receptor (BCR) signals are important steps in the initiation of humoral immune responses. How the BCR signals are translated by nuclear transcription factors into cell cycle progression is poorly understood. Id3 is an immediate-early gene responding to growth and mitogenic signals in many cell types including B cells. The primary function of the Id3 protein has been defined as that of inhibitor of basic-helix-loop-helix (bHLH) transcription factors. The interaction between Id3 and bHLH proteins, many of which are essential for cellular differentiation, has been proposed as a key regulatory event leading to cellular proliferation instead of differentiation. To further investigate the role of Id3 in tissue and embryo development and the mechanism of Id3-mediated growth regulation, we generated and analyzed Id3-deficient mice. While these mice display no overt abnormality in tissue and embryo development, their humoral immunity is compromised. The amounts of immunoglobulins produced in Id3-deficient mice immunized with a T-cell-dependent antigen and a type 2 T-cell-independent antigen are attenuated and severely impaired, respectively. Further analysis of lymphocytes isolated from Id3-deficient mice reveals a B-cell defect in their proliferation response to BCR cross-linking but not to lipopolysaccharide or a combination of BCR cross-linking and interleukin-4. Analyses of cultured lymphocytes also suggest involvement of Id3 in cytokine production in T cells and isotype switching in B cells. Finally, the proliferation defect in Id3-deficient B cells can be rescued by ectopic expression of Id1, a homologue of Id3. Taken together, these results define a necessary and specific role for Id3 in mediating signals from BCR to cell cycle progression during humoral immune responses.

Impaired Immune Responses and B-Cell Proliferation in Mice Lacking the Id3 Gene

Id1 is an inhibitor of a group of basic helix-loop-helix transcription factors, collectively called E proteins, which includes E12, E47, E2-2, and HEB. We have generated transgenic mice in which Id1 is specifically expressed in T cells. The total number of thymocytes in these mice is less than 4% of that in wild-type mice. The majority of the transgenic thymocytes are CD4 and CD8 double negative and bear the cell surface markers of multipotent progenitor cells. A small number of thymocytes, however, differentiate into CD4 or CD8 single-positive T cells, which also display different characteristics from their wild-type counterparts. More importantly, apoptotic cells constitute about 50% of the total thymocytes. These apoptotic thymocytes have rearranged their T-cell receptor genes, suggesting that they are differentiating T cells. This finding has raised the possibility that the T-cell deficiency in Id1 transgenic mice is the result of a massive apoptosis of differentiating T cells triggered by Id1 expression as opposed to a developmental block at the earliest progenitor stage. The progenitor cells accumulated in the transgenic mice might have survived because they are not susceptible to the apoptotic signals. Despite the massive cell death of the thymocytes at young ages, Id1 transgenic mice frequently develop T-cell lymphoma later in their life span, and lymphomagenesis appears to occur at different stages of T-cell development. Taken together, our data suggest that E proteins, being the targets of Id1, are essential regulators for normal T-cell differentiation and tumor suppression.

Massive apoptosis of thymocytes in T-cell-deficient Id1 transgenic mice.

The Tal1 Oncoprotein Inhibits E47-mediated Transcription MECHANISM OF INHIBITION

The Id1 protein acts as a negative regulator in early-B-cell differentiation by antagonizing the function of the basic helix-loop-helix transcription factors. Expression of the Id1 gene during B-cell development is governed at the transcriptional level primarily by a pro-B-cell-specific enhancer (PBE) located 3 kb downstream of the gene. We report here the identification of CAAT/enhancer binding protein beta (C/EBPbeta) as a component of the two major PBE-binding complexes (PBEC1 and PBEC2) found in pro-B cells by gel mobility shift assays. Formation of the PBECs is abolished when a classic C/EBP binding site is used as a competitor, and binding complexes similar to the PBECs are formed when the classic C/EBP site is used as a probe. We show that CHOP, a negative regulator of C/EBPs, specifically inhibits PBE binding in vitro and its enhancer activity in vivo. In pro-B cells, C/EBPbeta binds to the PBE site not as apparent homodimers but possibly in association with at least one other polypeptide, which might determine the pro-B-cell-specific expression of the Id1 gene. Although isoforms of C/EBPbeta are expressed in various B cells, they bind to DNA only in LyD9 and Ba/F3 pro-B cells. We show that CHOP is expressed in 70Z/3 and WEHI-231 cells. We also demonstrate that CHOP is associated with C/EBPbeta in WEHI-231 cells, which may provide an additional mechanism to control the function of C/EBPbeta and the expression of the Id1 gene.

Xiao Hong Sun

Papers

Regulation of the Expression of Cyclin-Dependent Kinase Inhibitor p21 by E2A and Id Proteins

Impaired Immune Responses and B-Cell Proliferation in Mice Lacking the Id3 Gene

Massive apoptosis of thymocytes in T-cell-deficient Id1 transgenic mice.

The Tal1 Oncoprotein Inhibits E47-mediated Transcription MECHANISM OF INHIBITION

Regulation of the pro-B-cell-specific enhancer of the Id1 gene involves the C/EBP family of proteins.