Ubiquitous bHLH Proteins Research Articles

Nonsense mutations of the bHLH transcription factor TWIST2 found in Setleis Syndrome patients cause dysregulation of periostin

Setleis Syndrome (OMIM ID: 227260) is a rare autosomal recessive disease characterized by abnormal facial development. Recently, we have reported that two nonsense mutations (c.486C > T [Q119X] and c.324C > T [Q65X]) of the basic helix-loop-helix (bHLH) transcription factor TWIST2 cause Setleis Syndrome. Here we show that periostin, a cell adhesion protein involved in connective tissue development and maintenance, is down-regulated in Setleis Syndrome patient fibroblast cells and that periostin positively responds to manipulations in TWIST2 levels, suggesting that TWIST2 is a transactivator of periostin. Functional analysis of the TWIST2 mutant form (Q119X) revealed that it maintains the ability to localize to the nucleus, forms homo and heterodimers with the ubiquitous bHLH protein E12, and binds to dsDNA. Reporter gene assays using deletion constructs of the human periostin promoter also reveal that TWIST2 can activate this gene more specifically than Twist1, while the Q119X mutant results in no significant transactivation. Chromatin immunoprecipitation assays show that both wild-type TWIST2 and the Q119X mutant bind the periostin promoter, however only wild-type TWIST2 is associated with higher levels of histone acetylation across the 5′-regulatory region of periostin. Taken together, these data suggest that the C-terminal domain of TWIST2, which is missing in the Q119X mutant form of TWIST2, is responsible for proper transactivation of the periostin gene. Improper regulation of periostin by the mutant form of TWIST2 could help explain some of the soft tissue abnormalities seen in these patients therefore providing a genotype–phenotype relationship for Setleis Syndrome.

A nonsense mutation of the bHLH transcription factor TWIST2 found in Setleis Syndrome patients causes dysregulation of the human periostin gene

Setleis Syndrome (SS, MIM #227260) is a rare autosomal recessive disorder characterized by abnormal facial and skin development. Recently, we have shown that nonsense mutations in the basic helix‐loop‐helix (bHLH) transcription factor TWIST2 cause Setleis Syndrome. Microarray analysis suggests that periostin (POSTN), a cell adhesion protein involved in connective tissue development and maintenance, is down regulated in Setleis Syndrome patient fibroblasts. Overexpression and knockdown of TWIST2 resulted in increased and decreased expression of POSTN mRNA, respectively. Functional analysis of the Q119X TWIST2 mutant form revealed that it maintains the ability to localize to the nucleus, form homo and heterodimers with the ubiquitous bHLH protein E12 and shows binding to dsDNA. Chromatin immunoprecipitation assays suggest that both wild‐type TWIST2 and its mutant form can bind the POSTN regulatory region, however only wild‐type TWIST2 is associated with higher levels of acetylated histone H3 of the POSTN promoter. Reporter gene assays using deletion constructs of the POSTN regulatory region also reveal that wild‐type TWIST2 can activate this gene, but not the Q119X TWIST2 mutant. This data suggests that the C‐terminal domain of TWIST2, which is missing in Q119X TWIST2, is responsible for proper transactivation of POSTN. This research is supported by MBRS RISE Grant R25GM061838 and RCMI Grant G12RR03051.

Expression of Id and ITF-2 genes in the mammary gland during pregnancy

Id-1 is one of the four related helix-loop-helix Id proteins that act as inhibitors of the basic helix-loop-helix (bHLH) transcription factors that control cell differentiation, development and carcinogenesis. We previously found that Id-1 regulated the growth, differentiation, apoptosis and invasion of mouse mammary epithelial cells in culture. Using the techniques of immunohistochemistry and in situ hybridization, we now show that Id-1 gene expression is specifically detected in the epithelial cells of growing ductal structures during early pregnancy. Using adjacent sections, we determined that Id-1 was expressed in keratin 8/18 positive cells. We also demonstrated that the up-regulation of Id-2 during late pregnancy correlated with the down-regulation of Id-1. Using the yeast-two hybrid system, we identified the bHLH transcription factors, ITF-2A and ITF-2B, as the Id-interacting proteins. The levels of expression of these two splice variants did not change during the transition from growing ductal structures to differentiated alveoli. Therefore Id-1 and Id-2, but not the ubiquitous bHLH proteins, appear to represent the key factors whose expression is modulated during different stages of pregnancy in mouse mammary glands.

Role of the basic helix‐loop‐helix protein ITF2 in the hormonal regulation of Sertoli cell differentiation

Sertoli cells are a post-mitotic terminally differentiated cell population that forms the seminiferous tubules in the adult testis and provides the microenvironment and structural support for developing germ cells. During pubertal development, Sertoli cells are responsive to follicle-stimulating hormone (FSH) to promote the expression of differentiated gene products. The basic helix-loop-helix (bHLH) and inhibitors of differentiation (Id) transcription factors are involved in the differentiation of a variety of cell lineages during development. Both bHLH and Id transcription factors have been identified in Sertoli cells. A yeast two-hybrid screen was conducted using a rat Sertoli cell cDNA library to identify bHLH dimerization partners for the Id1 transcription factor. The ubiquitous bHLH protein ITF2 (i.e., E2-2) was identified as one of the interacting partners. The current study investigates the expression and function of ITF2 in Sertoli cells. ITF2 was found to be ubiquitously expressed in all testicular cell types including germ cells, peritubular myoid cells, and Sertoli cells. Stimulation of cultured Sertoli cells with FSH or dibutryl cAMP resulted in a transient decrease in expression of ITF2 mRNA levels followed by a rise in expression with FSH treatment. ITF2 expression was at its highest in mid-pubertal 20-day-old rat Sertoli cells. ITF2 was found to directly bind to negative acting Id HLH proteins and positive acting bHLH proteins such as scleraxis. Transient overexpression of ITF2 protein in cultured Sertoli cells stimulated transferrin promoter activity, which is a marker of Sertoli cell differentiation. Co-transfections of ITF2 and Id proteins sequestered the inhibitory effects of the Id family of proteins. Observations suggest ITF2 can enhance FSH actions through suppressing the inhibitory actions of the Id family of proteins and increasing the actions of stimulatory bHLH proteins (i.e., scleraxis) in Sertoli cells.

E47 phosphorylation by p38 MAPK promotes MyoD/E47 association and muscle-specific gene transcription

Selective recognition of the E-box sequences on muscle gene promoters by heterodimers of myogenic basic helix-loop-helix (bHLH) transcription factors, such as MyoD, with the ubiquitous bHLH proteins E12 and E47 is a key event in skeletal myogenesis. However, homodimers of MyoD or E47 are unable of binding to and activating muscle chromatin targets, suggesting that formation of functional MyoD/E47 heterodimers is pivotal in controlling muscle transcription. Here we show that p38 MAPK, whose activity is essential for myogenesis, regulates MyoD/E47 heterodimerization. Phosphorylation of E47 at Ser140 by p38 induces MyoD/E47 association and activation of muscle-specific transcription, while the nonphosphorylatable E47 mutant Ser140Ala fails to heterodimerize with MyoD and displays impaired myogenic potential. Moreover, inhibition of p38 activity in myocytes precludes E47 phosphorylation at Ser140, which results in reduced MyoD/E47 heterodimerization and inefficient muscle differentiation, as a consequence of the impaired binding of the transcription factors to the E regulatory regions of muscle genes. These findings identify a novel pro-myogenic role of p38 in regulating the formation of functional MyoD/E47 heterodimers that are essential for myogenesis.

Distinct Gene Expression Programs Function in Progenitor and Mature Islet Cells

Homeodomain transcription factor Nkx2.2 is required for the final differentiation of the beta-cells in the pancreas and for the production of insulin. Nkx2.2 is expressed in islet cell precursors during pancreatic development and persists in a subset of mature islet cells including all beta-cells. To understand the mechanisms regulating the expression of Nkx2.2 in these different cell populations, we outlined the structure of the mouse nkx2.2 gene and identified regions that direct cell type-specific expression. The nkx2.2 gene has two noncoding alternative first exons (exons 1a and 1b). In transgenic mice, sequences upstream from exon 1a directed expression predominantly in mature islet cells. Within this exon 1a promoter, cooperative interactions between HNF3 and basic helix-loop-helix factors neurogenin-3 or NeuroD1 binding to adjacent sites played key roles in its islet cell-specific expression. In contrast, sequences upstream from exon 1b restricted expression specifically to islet cell precursors. These studies reveal distinct mechanisms for directing the expression of a key differentiation factor in precursors versus mature islet cells.

Characterization of a Dominant Inhibitory E47 Protein That Suppresses C2C12 Myogenesis

Skeletal muscle formation is controlled through the coordinated actions of the muscle regulatory factors (MRFs). The activities of these basic helix–loop–helix proteins is mediated in part through heterodimer formation with a family of ubiquitous bHLH proteins, referred to as E-proteins. The primary E-protein in skeletal muscle is the E2A splice variant, E47. To further address the role of E47 during skeletal myogenesis, we created a chimeric E47 repressor protein by replacing the transcriptional activation domain with the Drosophila Engrailed transcriptional repressor domain. The dominant inhibitory E-protein (EnΔE47) formed homodimers capable of binding DNA and abolished E47-directed gene transcription. Stable expression of EnΔE47 in mouse C2C12 myoblasts effectively blocked the cells' ability to differentiate into mature myofibers. Closer examination of the molecular basis for the inhibition of myogenesis revealed that EnΔE47 preferentially forms heterodimers with myogenin. Interestingly, the chimeric repressor did not form DNA-binding heterodimers with MyoD in C2C12 myocytes. The failure to detect MyoD:EnΔE47 heterodimers in myoblasts was not due to protein conformational defects as both wild-type E47 and EnΔE47 readily formed DNA binding complexes with MyoD in vitro. These results indicate that E47 plays a crucial role in C2C12 myogenesis by serving as the preferred heterodimer partner of the myogenin protein.

Remodeling of the hypertrophied human myocardium by cardiac bHLH transcription factors

The basic helix-loop-helix transcription factors eHAND and dHAND are involved in developmental cardiac growth and differentiation. We investigated HAND gene expression in the normal and in the hypertrophied right and left ventricle of patients with tetralogy of Fallot (ToF) and hypertrophic obstructive cardiomyopathy (HOCM). HAND mRNA was constitutively expressed in the hypertrophied heart and increased in the hypertrophic tissue of both patient groups. HAND genes had a complementary left-right cardiac asymmetry of expression with dHAND predominantly in the right and eHAND in the left ventricle. The two cardiac bHLH factors have the ability to form heterodimers with the ubiquitous bHLH protein E12, subsequently recognizing E-boxes in the promoter region of target genes like ALC-1. We found a highly significant positive correlation between HAND and ALC-1 mRNA. The total ALC-1 protein level in ToF was smaller than in HOCM, although ALC-1 mRNA as well as HAND mRNA levels were significantly higher. ToF patients expressed around four times more ALC-1 mRNA for similar amounts of ALC-1 than HOCM patients. Suggesting disturbed ALC-1 translation in ToF, we found ALC-1 antisense mRNA expression in the hypertrophied, but not in the normal, ventricles. The higher the antisense/sense ALC-1 mRNA ratio, the lower ALC-1 protein was expressed. J. Cell. Biochem. 74:551–561, 1999. © 1999 Wiley-Liss, Inc.

Remodeling of the hypertrophied human myocardium by cardiac bHLH transcription factors.

The basic helix-loop-helix transcription factors eHAND and dHAND are involved in developmental cardiac growth and differentiation. We investigated HAND gene expression in the normal and in the hypertrophied right and left ventricle of patients with tetralogy of Fallot (ToF) and hypertrophic obstructive cardiomyopathy (HOCM). HAND mRNA was constitutively expressed in the hypertrophied heart and increased in the hypertrophic tissue of both patient groups. HAND genes had a complementary left-right cardiac asymmetry of expression with dHAND predominantly in the right and eHAND in the left ventricle. The two cardiac bHLH factors have the ability to form heterodimers with the ubiquitous bHLH protein E12, subsequently recognizing E-boxes in the promoter region of target genes like ALC-1. We found a highly significant positive correlation between HAND and ALC-1 mRNA. The total ALC-1 protein level in ToF was smaller than in HOCM, although ALC-1 mRNA as well as HAND mRNA levels were significantly higher. ToF patients expressed around four times more ALC-1 mRNA for similar amounts of ALC-1 than HOCM patients. Suggesting disturbed ALC-1 translation in ToF, we found ALC-1 antisense mRNA expression in the hypertrophied, but not in the normal, ventricles. The higher the antisense/sense ALC-1 mRNA ratio, the lower ALC-1 protein was expressed.

MyoR: a muscle-restricted basic helix-loop-helix transcription factor that antagonizes the actions of MyoD.

Skeletal muscle development is controlled by a family of muscle-specific basic helix-loop-helix (bHLH) transcription factors that activate muscle genes by binding E-boxes (CANNTG) as heterodimers with ubiquitous bHLH proteins, called E proteins. Myogenic bHLH factors are expressed in proliferating undifferentiated myoblasts, but they do not initiate myogenesis until myoblasts exit the cell cycle. We describe a bHLH protein, MyoR (for myogenic repressor), that is expressed in undifferentiated myoblasts in culture and is down-regulated during differentiation. MyoR is also expressed specifically in the skeletal muscle lineage between days 10.5 and 16.5 of mouse embryogenesis and down-regulated thereafter during the period of secondary myogenesis. MyoR forms heterodimers with E proteins that bind the same DNA sequence as myogenic bHLH/E protein heterodimers, but MyoR acts as a potent transcriptional repressor that blocks myogenesis and activation of E-box-dependent muscle genes. These results suggest a role for MyoR as a lineage-restricted transcriptional repressor of the muscle differentiation program.

Differential Regulation of the Glucagon and Insulin I Gene Promoters by the Basic Helix-Loop-Helix Transcription Factors E47 and BETA2

The insulin and glucagon genes are expressed in the beta and alpha cells of the islets of Langerhans, respectively. The factors controlling their cell- and islet-specific expression are poorly known. Insulin-enhancer factor-1 (IEF1) has previously been shown to interact with the E boxes of the rat insulin I and II genes and was proposed to play a critical role in beta cell-specific expression. BETA2, a recently identified basic helix-loop-helix (bHLH) protein, binds with high affinity and transactivates the rat insulin II gene upon dimerization with the ubiquitous bHLH protein E47. We show here that the heterodimer E47/BETA2 also binds and transactivates the rat insulin I and glucagon genes and exhibits the same characteristics as IEF1. In transfection experiments, the E boxes of the insulin I and glucagon genes confer transcriptional activity in both insulin- and glucagon-producing cells, which is increased by overexpression of E47 and BETA2. However, overexpression of E47 inhibits only E box-mediated glucagon gene expression, whereas it activates insulin gene transcription, indicating that the E boxes of the insulin and glucagon genes display gene-specific characteristics. We conclude that the heterodimer E47/BETA2 represents an islet-specific factor that controls both insulin and glucagon gene transcription and that the E47/BETA2 ratio may be important for regulated gene expression.

Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.

We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected.

Restricted Expression of a Novel Murineatonal-Related bHLH Protein in Undifferentiated Neural Precursors

Tissue-specific bHLH proteins play important roles in the specification and differentiation of neural cell lineages in invertebrate and vertebrate organisms. Two groups of bHLH proteins, atonal and achaete-scute, have proneural activities inDrosophila,and the mouse achaete-scute homolog MASH1 is required for the differentiation of several neural lineages. In a screen for proteins interacting with MASH1, we have isolated a novel bHLH protein related to atonal, named MATH4A, which is broadly expressed in neural precursor cells in the mouse embryonic CNS and PNS. Interaction assays in yeast andin vitrodemonstrate that MATH4A interacts efficiently with both MASH1 and the ubiquitous bHLH protein E12. MATH4A-E12 heterodimers, but not MATH4A-MASH1, bind to a consensus E-box sequence.Math4Aexpression is restricted to undifferentiated neural precursors and is complementary to that ofMash1in most regions of the nervous system. In particular,Math4Ais transcribed at high levels in the cerebral cortex, dorsal thalamus, and epibranchial placodes, which present little or noMash1expression. However, expression of the two genes shows limited overlap in certain CNS regions (retina, preoptic area of the hypothalamus, midbrain, hindbrain). Its structure and expression pattern suggest that MATH4A may regulate an early step of neural development, either as a partner of ubiquitous bHLH proteins or associated with other neural-specific bHLH proteins.

Dermo-1: A Novel Twist-Related bHLH Protein Expressed in the Developing Dermis

Transcription factors belonging to the basic helix–loop–helix (bHLH) family have been shown to control differentiation of a variety of cell types. Tissue-specific bHLH proteins dimerize preferentially with ubiquitous bHLH proteins to form heterodimers that bind the E-box consensus sequence (CANNTG) in the control regions of target genes. Using the yeast two-hybrid system to screen for tissue-specific bHLH proteins, which dimerize with the ubiquitous bHLH protein E12, we cloned a novel bHLH protein, named Dermo-1. Within its bHLH region, Dermo-1 shares extensive homology with members of the twist family of bHLH proteins, which are expressed in embryonic mesoderm. During mouse embryogenesis, Dermo-1 showed an expression pattern similar to, but distinct from, that of mouse twist. Dermo-1 was expressed at a low level in the sclerotome and dermatome of the somites, and in the limb buds at Day 10.5 post coitum (p.c.), and accumulated predominantly in the dermatome, prevertebrae, and the derivatives of the branchial arches by Day 13.5 p.c. As differentiation of prechondrial cells proceeded, Dermo-1 expression became restricted to the perichondrium. Expression of Dermo-1 increased continuously in the dermis through Day 17.5 p.c. and was also detected in the dermis of neonates, but became downregulated in adult tissues. The Dermo-1 protein bound the E-box consensus sequence in the presence of E12, but transcriptional activity was not detectable. Instead, Dermo-1 repressed transcriptional activity of myogenic bHLH proteins. The expression pattern of Dermo-1 suggests that it functions as a regulator of gene expression in a subset of mesenchymal cell lineages including developing dermis.

Scleraxis: a basic helix-loop-helix protein that prefigures skeletal formation during mouse embryogenesis

Members of the basic helix-loop-helix (bHLH) family of transcription factors have been shown to regulate growth and differentiation of numerous cell types. Cell-type-specific bHLH proteins typically form heterodimers with ubiquitous bHLH proteins, such as E12, and bind a DNA consensus sequence known as an E-box. We used the yeast two-hybrid system to screen mouse embryo cDNA libraries for cDNAs encoding novel cell-type-specific bHLH proteins that dimerize with E12. One of the cDNAs isolated encoded a novel bHLH protein, called scleraxis. During mouse embryogenesis, scleraxis transcripts were first detected between day 9.5 and 10.5 post coitum (p.c.) in the sclerotome of the somites and in mesenchymal cells in the body wall and limb buds. Subsequently, scleraxis was expressed at high levels within mesenchymal precursors of the axial and appendicular skeleton and in cranial mesenchyme in advance of chondrogenesis; its expression pattern in these cell types foreshadowed the developing skeleton. Prior to formation of the embryonic cartilaginous skeleton, scleraxis expression declined to low levels. As development proceeded, high levels of scleraxis expression became restricted to regions where cartilage and connective tissue formation take place. Scleraxis bound the E-box consensus sequence as a heterodimer with E12 and activated transcription of a reporter gene linked to its DNA-binding site. The expression pattern, DNA-binding properties and transcriptional activity of scleraxis suggest that it is a regulator of gene expression within mesenchymal cell lineages that give rise to cartilage and connective tissue.

Dimerization through the helix-loop-helix motif enhances phosphorylation of the transcription activation domains of myogenin.

The muscle-specific basic helix-loop-helix (bHLH) protein myogenin activates muscle transcription by binding to target sequences in muscle-specific promoters and enhancers as a heterodimer with ubiquitous bHLH proteins, such as the E2A gene products E12 and E47. We show that dimerization with E2A products potentiates phosphorylation of myogenin at sites within its amino- and carboxyl-terminal transcription activation domains. Phosphorylation of myogenin at these sites was mediated by the bHLH region of E2A products and was dependent on dimerization but not on DNA binding. Mutations of the dimerization-dependent phosphorylation sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. The ability of E2A products to potentiate myogenin phosphorylation suggests that dimerization induces a conformational change in myogenin that unmasks otherwise cryptic phosphorylation sites or that E2A proteins recruit a kinase for which myogenin is a substrate. That phosphorylation of these dimerization-dependent sites diminished myogenin's transcriptional activity suggests that these sites are targets for a kinase that interferes with muscle-specific gene expression.

Dimerization through the helix-loop-helix motif enhances phosphorylation of the transcription activation domains of myogenin.

The muscle-specific basic helix-loop-helix (bHLH) protein myogenin activates muscle transcription by binding to target sequences in muscle-specific promoters and enhancers as a heterodimer with ubiquitous bHLH proteins, such as the E2A gene products E12 and E47. We show that dimerization with E2A products potentiates phosphorylation of myogenin at sites within its amino- and carboxyl-terminal transcription activation domains. Phosphorylation of myogenin at these sites was mediated by the bHLH region of E2A products and was dependent on dimerization but not on DNA binding. Mutations of the dimerization-dependent phosphorylation sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. The ability of E2A products to potentiate myogenin phosphorylation suggests that dimerization induces a conformational change in myogenin that unmasks otherwise cryptic phosphorylation sites or that E2A proteins recruit a kinase for which myogenin is a substrate. That phosphorylation of these dimerization-dependent sites diminished myogenin's transcriptional activity suggests that these sites are targets for a kinase that interferes with muscle-specific gene expression.

Alternative multimeric structures affect myogenin DNA binding activity.

The native molecular weight of the basic helix-loop-helix (bHLH) proteins myogenin, MyoD, and E12 was calculated from their mobilities on sucrose gradients and molecular sieve chromatography. The muscle bHLH proteins associate to form a variety of higher order complexes, most of which are larger than dimers. Homodimers bind to DNA sequences such as the MEF-1 site in the creatine kinase enhancer whereas homotetramers and larger forms do not recognize this DNA sequence. The ubiquitous bHLH protein E12 forms monomers or homodimers with little evidence for higher order complexes. Mixtures of myogenin and E12 show some heterodimeric structures, but most of the myogenin remains in large complexes. This result using purified proteins is also obtained in nuclear extracts from differentiated myotubes, in which most of the myogenin is present in large complexes that do not bind to the creatine kinase enhancer. A fusion protein containing only the myogenin HLH region forms large homomeric complexes. A model is presented in which each helix associates with a different subunit to form chains or ring structures to explain these observations. The partition of myogenin in nuclear extracts into dimers that recognize known DNA sequences and higher order complexes that do not raises important new issues concerning the regulation of skeletal muscle bHLH protein activity during myogenesis.