E Proteins and ID Proteins: Helix-Loop-Helix Partners in Development and Disease

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Lan-Hsin Wang1 and Nicholas E. Baker1,2,3,* 1Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA 2Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA 3Department of Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2015.10.019

The basic Helix-Loop-Helix (bHLH) proteins represent a well-known class of transcriptional regulators. Many bHLH proteins act as heterodimers with members of a class of ubiquitous partners, the E proteins. A widely expressed class of inhibitory heterodimer partners—the Inhibitor of DNA-binding (ID) proteins—also exists. Genetic and molecular analyses in humans and in knockout mice implicate E proteins and ID proteins in a wide variety of diseases, belying the notion that they are non-speciﬁc partner proteins. Here, we explore relationships of E proteins and ID proteins to a variety of disease processes and highlight gaps in knowledge of disease mechanisms.

E proteins and Inhibitor of DNA-binding (ID) proteins are widely conferring DNA-binding specificity and transcriptional activation expressed transcriptional regulators with very general functions. on heterodimers with the ubiquitous E proteins (Figure 1). They are implicated in diseases by evidence ranging from Another class of pervasive HLH proteins acts in opposition to confirmed Mendelian inheritance, association studies, and E proteins. These ID proteins lack a basic DNA-binding region so mouse models that resemble human disorders. Here, we briefly that heterodimers of ID proteins with E proteins or with class II recap the properties of E proteins and ID proteins in develop- bHLH proteins are unable to bind DNA or activate transcription ment and differentiation, cell proliferation, and cancer that (Benezra et al., 1990). ID protein levels may be low in differenti- have been gleaned from decades of study (Massari and Murre, ated cells but are abundant in proliferating, multipotent cells 2000; Murre, 2005; Belle and Zhuang, 2014; Lasorella et al., including stem cell populations (Ling et al., 2014). Like the E pro- 2014; Ling et al., 2014; Nair et al., 2014), before describing dis- teins, ID proteins are represented by a single homolog in ease associations where the mechanisms of E protein and ID Drosophila, the extra macrochaetae (emc) gene, which was iden- protein contributions are incompletely understood. tified in parallel with mammalian ID proteins. emc encodes a negative regulator of proneural bHLH proteins including Achaete HLH Proteins in Differentiation and Scute and is a negative regulator of neurogenesis so that The helix-loop-helix (HLH) domain was first recognized in emc mutants exhibit ectopic neural differentiation (Ellis et al., dimeric transcription factors that regulate the IgG enhancer in 1990; Garrell and Modolell, 1990). Figure 1 summarizes the basic B cells. Since these bind to Ephrussi-box (E-box) sequences outline of HLH function in cell-type specification and differentia- (CANNTG), the first HLH proteins were named E proteins (Murre tion, in which specific expression of particular class II bHLH pro- et al., 1989). Mammalian E proteins include E12 and E47, which teins must compete with ID proteins to heterodimerize with arise through alternative splicing of the E2A (HGNC:11633) gene, E proteins and form tissue- and cell-type-specific transcription E2-2 (HGNC:11634), and HEB (HGNC:11623). Renaming these factors that drive specification and differentiation. bHLH proteins proteins TCF3, TCF4, and TCF12, respectively, has been sug- are now known to regulate differentiation in too many additional gested (Table 1), but caution must be used to avoid confusion situations to be listed here. For example, E proteins and ID pro- since the acronym TCF had already been assigned to unrelated teins regulate Sertoli cell differentiation (Chaudhary et al., 2005), proteins. There is a single E protein in Drosophila, the Daughter- possibly through the class II bHLH proteins POD1/Epicardin less (Da) protein. (TCF21) and Scleraxis (Muir et al., 2006; Bhandari et al., 2011). E proteins have many functions as heterodimer partners of the Expression of Da, the only E protein, in Drosophila seems to be tissue-specific basic Helix-Loop-Helix (bHLH) proteins that are ubiquitous, whereas particular mammalian E protein and ID pro- sometimes called class II bHLH proteins. The diverse class II tein genes have various overlapping patterns of expression. Id1 bHLH proteins include the myogenic transcription factors exem- and Id3 in particular are often upregulated by BMP signals, and plified by MYOD, a factor sufficient to transform cultured fibro- Id1 by cytokines (Ling et al., 2014). TGFb,FGF and TCR signaling blasts into myoblasts, the conserved proneural proteins such also regulate Id gene expression (Lasorella et al., 2014; Ling as Achaete and Scute from Drosophila, and hematopoiesis pro- et al., 2014). Both Drosophila and mammals exhibit cross-talk tein T cell acute lymphocytic leukemia 1 (TAL1) (Massari and between ID proteins and Notch signaling, consistent with their Murre, 2000; Powell and Jarman, 2008). The paradigm that common regulation of bHLH-mediated differentiation (Baonza was soon established is that tissue-specific class II bHLH factors et al., 2000; Adam and Montell, 2004; Bhattacharya and Baker, whose specific expression defines specification and differentia- 2009; Lasorella et al., 2014; Ling et al., 2014). It is increas- tion programs such as myogenesis or neurogenesis act by ingly recognized that Id genes can be transcriptional targets of

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Table 1. HLH Protein Nomenclature Protein Name Old Gene Name New Gene Name E12, E47 E2A TCF3 E2-2 E2-2 TCF4 HEB HEB TCF12

E proteins, in Drosophila and in mammals. This makes ID proteins feedback inhibitors of E protein activity, which may be particularly important as a brake on E protein autoregulation (Bhattacharya and Baker, 2011; Schmitz et al., 2012)(Figure 2). ID proteins are also regulated at the level of protein stability (Lingbeck et al., 2005; Lasorella et al., 2006; Kim et al., 2009; Wil- liams et al., 2011b; Lasorella et al., 2014). Since mouse knockouts of each of the E proteins and ID proteins have mutant phenotypes, neither E proteins nor ID proteins can be completely redundant. The example of Id1 and Id3 double null (Id1À/À; Id3À/À) mice, which exhibit premature neuronal differentiation, forebrain vascular abnormalities, cardiac defects, and embryonic lethality not seen in the single mutants (Lyden et al., 1999; Lasorella et al., 2014), illustrates significant functional overlap that may also occur for other ID proteins and similarly between E proteins (Table 2). Non-overlapping expression accounts for some non-redundancy, although unique protein functions may also exist, especially in the case of E proteins that differ in basic region sequence and DNA-binding preference. It is thought that any unique functions of individual proteins are superimposed on the overall ratio of total E proteins to total ID proteins within a cell that is an important index of competence in differentiation and other processes. For example, elevating Da levels, which are controlled by feedback through Emc, affects the capacity of Figure 1. Transcriptional Modalities of HLH Proteins (A) Transcriptional activation of Drosophila proneural genes requires the Ato or Sc to induce neurons in Drosophila (Bhattacharya and ubiquitously expressed E protein (Da) forming heterodimers with tissue- Baker, 2011), and elevated E protein levels are thought to act specific bHLH proteins (e.g., Ac/Sc) and binds to promoter E boxes. similarly in the postnatal mouse brain (Fischer et al., 2014). (B) Transcription of myogenic genes is controlled by the binding of E protein- myogenic-specific bHLH protein (MyoD) heterodimers to promoter E boxes. (C) E protein (or Da) and ID (or Emc) protein heterodimers fail to bind DNA and HLH Proteins in the Cell Cycle and Senescence cannot activate transcription. The commitment of progenitor cells to begin cell differentiation is often associated with cell-cycle withdrawal. MYOD provided the regulate proliferation in many important tissues, for example, first example connecting bHLH proteins to cell-cycle arrest, in- during the expansion of mammary epithelia during pregnancy hibiting cell cycling as myoblasts differentiate (Gu et al., 1993; (de Candia et al., 2004). Because of their dual effects on differen- Halevy et al., 1995; Guo et al., 1995; Zhang et al., 1999). Subse- tiation and proliferation, ID protein expression is associated with quent studies have uncovered a common relationship between stem cell maintenance, and, in tumors, anaplasia (Nam and Ben- E and ID proteins and cell-cycle progression. In many cells, ezra, 2009; Anido et al., 2010; Barrett et al., 2012; Niola et al., E12 or E47 homodimers activate transcription of the CDK inhib- 2012; Lasorella et al., 2014; Ling et al., 2014; Nair et al., 2014). itors (CDKIs) p15, p16 (also known as INK4a), p21, p27, and p57 Although this general relationship is seen in many cells, there (Sloan et al., 1996; Prabhu et al., 1997; Pagliuca et al., are examples of different effects. Id1 is reported to protect 2000)(Figure 3). Through this mechanism, E proteins inhibit mammary epithelia cells from senescence independently of cell-cycle progression and are also implicated in cellular senes- CDKI expression (Swarbrick et al., 2008), and rapidly dividing cence (Ling et al., 2014). In fact, E47 is necessary for senescence transit amplifying cells surprisingly express less Id1 than their of human fibroblasts, which is prevented by E protein knock- quiescent neural stem cell progenitors (Nam and Benezra, down (Zheng et al., 2004). 2009). The poor growth of Drosophila cells lacking emc is As one might expect, the role of E proteins is blocked by ID also due to Da hyperactivity, not attributed to CDKI expression proteins (Peverali et al., 1994). ID proteins can promote retino- but to repression of the phosphatase Cdc25/string or to over- blastoma (RB) protein inactivation and activate E2F-mediated expression of the gene expanded within the Salvador-Warts- transcription and cell-cycle progression indirectly because of Hippo pathway of tumor suppressors (Bhattacharya and Baker, their inhibition of E proteins and consequently inhibition of 2011; Andrade-Zapata and Baonza, 2014; Wang and Baker, CDKIs’ expression, while ID2 can be sequestered by RB directly 2015)(Figure 4). It is also envisaged that ID functions indepen- (Lasorella et al., 2014; Ling et al., 2014)(Figure 3). ID1 and ID3 are dent of dimerization with HLH proteins may exist (Ling et al., degraded during senescence (Kong et al., 2011). ID proteins 2014).

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metastasis and angiogenesis(Slattery et al., 2008; Lasorella et al., 2014; Ling et al., 2014; Nair et al., 2014). ID proteins are required to maintain glioma stem cells in the perivascular niche by inhibiting E protein transcription of the RAP1GAP gene, since RAP1 controls cancer stem cell adhesion to the niche through in- tegrin signaling (Niola et al., 2013). Unexpectedly, ID3 and ID4 sometimes behave as tumor suppressors (Yu et al., 2005; Chen et al., 2011; Love et al., 2012; Richter et al., 2012; Schmitz et al., 2012). For example, recurrent mutations in the E47 (TCF3) and ID3 genes occur in Burkitt’s lymphoma and may allow unre- strained E47 expression to activate both cyclin D3 and PI3K survival signaling (Love et al., 2012; Richter et al., 2012; Schmitz et al., 2012)(Figure 2B).

Postmitotic ID Functions: Neuronal Morphogenesis In addition to roles in cell fate specification and in the regulation of the cell cycle and senescence, in postmitotic neurons ID1, ID2 and ID4 are ubiquitinated by the Anaphase Promoting Complex/ Cyclosome (APC/C), and their subsequent turnover affects axon and dendrite development (Lasorella et al., 2006; Kim et al., 2009). Mutations of the APC/C interaction motif of ID2 enhance axonal growth in mouse cerebellar granule neurons both in vitro and in the cerebellar cortex, suggesting that ID2 degradation maintains axon morphology by restraining E protein- dependent axon growth (Lasorella et al., 2006). On the other hand, knockdown of Id1 in the cerebellar cortex or primary hip- pocampal neurons stimulates dendrite morphogenesis, indi- cating an inhibitory role of ID1 in dendrite development (Kim et al., 2009). APC/C is important in cell-cycle exit (Peters, 2006) and therefore might couple cell-cycle exit to dendrogene- sis in differentiating neurons. It remains unknown at present Figure 2. Autoregulation and Feedback Inhibition in Normal and Disease Conditions whether ID protein downregulation contributes to terminal cell- (A) Emc expression requires Da activity. Once Emc level is upregulated, it will in cycle withdrawal in neurons and how E proteins affect dendrites. turn block autoregulation of da and Da activity (Bhattacharya and Baker, 2011). These questions are of considerable interest, as neurological This regulation may ensure the steady state of E protein and ID protein levels in normal progenitor cells. disorders are associated with ID and E proteins (see below). (B) ID proteins are transcriptional targets of E proteins in Burkitt lymphoma cells. Both gain-of-function mutations of E protein (indicated by + symbol) and Beyond Developmental Defects and Cancer: loss-of-function mutations of ID protein (indicated by X symbol) found in Burkitt Connections to Disease lymphoma interrupt the feedback network between E and ID proteins. Cyclin D3 is also a target gene of E proteins in Burkitt lymphoma cells and may E proteins and ID proteins have been implicated in a number of contribute to the excessive proliferation of tumor cells (Schmitz et al., 2012). human diseases, which may be related to the roles of E and ID proteins in differentiation and cycle regulation, or perhaps reflect E Proteins and ID Proteins in Cancer other mechanisms (Table 3). It is interesting to catalog these There is a well-established connection between cancer and E potential disease relationships because their wide range argues proteins (Murre, 2005; Belle and Zhuang, 2014). In addition to persuasively that E and ID proteins represent central regulators lymphoma predisposition in E2a/Tcf3 mutant mice, E2A/TCF3 active in most or all cells, rather than ubiquitous heterodimer mutations have been found in many human lymphomas and leu- partners of little intrinsic interest, and also so that gaps in knowl- kemias (Murre, 2005; Tijchon et al., 2013; Belle and Zhuang, edge can be identified. The following summaries are ordered 2014; Cozen et al., 2014). As would be expected, ID proteins according to the strength of the evidence linking to human mostly have the opposite properties (Lasorella et al., 2014; Nair disease, beginning with unambiguous Mendelian genetic et al., 2014). ID levels are elevated in human tumors ranging disease in humans and progressing through other human data from melanoma to neuroblastoma (Perk et al., 2005). ID1 and to mouse models that are suggestive but for which direct evi- ID3 are clearly implicated in lung cancer initiation, and ID4 is dence in human disease is wanting. overexpressed by a t(6;14)(p22;q32) chromosomal translocation Pitt-Hopkins Syndrome in leukemia and amplified in ovarian cancer, qualifying it as a Haploinsufficiency for the E protein transcription factor gene proto-oncogene (Perk et al., 2005; Lasorella et al., 2014; Nair E2-2/TCF4 due to heterozygous mutations or deletions is et al., 2014). responsible for Pitt-Hopkins syndrome (PTHS), an autosomal No doubt many oncogenic and tumor suppressor roles of ID dominant disorder characterized by severe intellectual disability, proteins and E proteins reflect their roles in cell proliferation, global developmental delay, recurrent seizure, and hyperventila- stemness, and senescence, but there are also contributions to tion (Zweier et al., 2007; Amiel et al., 2007; Giurgea et al., 2008;

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Table 2. Phenotypes of HLH Protein Knockout Mice Genotype Phenotype References E2a(Tcf3)À/À disrupted B cell development; lymphopoietic developmental Belle and Zhuang, 2014 defects with high frequency of T cell lymphomas Heb(Tcf12)À/À reduced pro-B cells; developmental arrest of thymocyte Zhuang et al., 1996 development E2-2(Tcf4)À/À reduced pro-B cells; normal mammary gland development; Zhuang et al., 1996; Flora et al., 2007; abnormal hindbrain development in the pontine nucleus Itahana et al., 2008 Id1À/À altered lipid and glucose metabolism; osteoporotic phenotype; de Candia et al., 2004; Chan et al., 2009; normal mammary gland development; impaired cancer stem Satyanarayana et al., 2012; Lasorella et al., 2014 cell properties in glioma Id2À/À altered lipid and glucose metabolism; hydronephrosis (smooth Aoki et al., 2004; de Candia et al., 2004; muscle hypertrophy at the ureteropelvic junction); Parkinson’s Havrda et al., 2008; Park et al., 2008; disease-like features (impaired dopaminergic system); reduced Hou et al., 2009; Mathew et al., 2013; proliferation in mammary epithelia (lactation defect); reduced Tripathi et al., 2012; Havrda et al., 2013; body size; absence of lymph nodes and Peyer’s patches; absence Lasorella et al., 2014 of NK cells and Langerhans cells Id3À/À symptoms of Sjo¨ gren’s syndrome (impaired tear and saliva secretion de Candia et al., 2004; Li et al., 2004 due to defective autoimmune system); normal mammary gland development Id4À/À premature neuronal differentiation; altered lipid and glucose Dong et al., 2011; Murad et al., 2010; metabolism; reduce osteoblast differentiation; enhance adipocyte Tokuzawa et al., 2010; Lasorella et al., 2014 differentiation; reduced proliferation and increased apoptosis in mammary cells Id1À/À Id3À/À premature neuronal differentiation; cardiac developmental defects; Lyden et al., 1999; Lasorella et al., 2014 brain vascular abnormalities; embryonic lethality Id1À/+ Id3À/À decreases of proliferation and mineralization in osteoblasts; vascular Maeda et al., 2004; Lasorella et al., 2014 defects and growth failure in tumor cell xenografts and genetic models of cancer Id3À/ÀApoEÀ/À or enhanced atherosclerosis Doran et al., 2010; Lipinski et al., 2012 Id3À/À LdlrÀ/À de Pontual et al., 2009). Most of the mutations found in PTHS hallmark of this disease is the increasing density of tiny bumps, patients disrupt E2-2/TCF4 transcriptional activity (Forrest termed guttae, that form on the cornea, loss of the ﬂuid-pump- et al., 2012; Sepp et al., 2012). In the developing and mature ing function of the endothelium cells, decrease in corneal trans- CNS, E2-2/TCF4 is a heterodimer partner for class II bHLH pro- parency, and resultant loss of vision. The lack of Fuchs corneal neural and neuronal precursor proteins. Hence, heterozygous dystrophy in PTHS patients that have heterozygous loss-of- loss of function affects a large number of target genes, including function E2-2/TCF4 mutations suggests that Fuchs corneal the neuronal adhesion receptor genes contactin associated dystrophy might involve E2-2/TCF4 gain of function. Interest- protein-like 2 (CNTNAP2) and neurexin 1 (NRXN1)(Forrest ingly, endothelial samples from Fuchs corneal dystrophy pa- et al., 2012). Both CNTNAP2 and NRXN1 mutations have been tients exhibit reduced ID1 expression, which could be another associated with PTHS-like disorder, schizophrenia, and autism route to elevate E2-2/TCF4 activity (Matthaei et al., 2014) (Alarco´ n et al., 2008; Zweier et al., 2009; Kirov et al., 2009). (Figure 1). Since corneal endothelium is normally non-prolifera- PTHS could reﬂect: defective expression of such neuronal differ- tive, cell-cycle arrest is unlikely to cause disease, but altered entiation genes that depend on E2-2/proneural gene hetero- expression of senescence-related genes has been reported dimers; defective cell-cycle withdrawal (since cell-cycle re-entry (Matthaei et al., 2014). In Drosophila, elevated Da expression can be associated with defective neuronal differentiation; can be toxic and activates the Salvador-Warts-Hippo pathway Ruggiero et al., 2012); or defects in dendrite and axon morpho- of tumor suppressors (Wang and Baker, 2015), but it is not genesis. It is also noteworthy that a particular E2-2/TCF4 muta- known whether this occurs in Fuchs corneal dystrophy. A tion has been found in Rett syndrome (see below), suggesting different view is suggested, however, by studies of SNP that related molecular mechanisms lead to PTHS and Rett rs613872 associated with an intronic trinucleotide repeat syndrome. expansion (Breschel et al., 1997; Wieben et al., 2012). This Fuchs Corneal Dystrophy expansion causes mis-splicing and RNA toxicity, similar to Multiple single nucleotide polymorphisms (SNPs) in E2-2/TCF4 other trinucleotide expansion disorders such as myotonic dys- are associated with Fuchs corneal dystrophy (Baratz et al., trophy type 1 and 2, fragile X syndrome, and frontotemporal 2010; Forrest et al., 2014; Lau et al., 2014), a common, domi- dementia (Du et al., 2015). RNA toxicity affects expression of nant, progressive, late onset disease in which endothelial cells many other genes, and the effects of this SNP might have little are gradually lost from the internal surface of the cornea. The to do with E2-2/TCF4 function.

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function and how they differ from those associated with Fuchs corneal dystrophy. Rett Syndrome Rett syndrome is an X-linked dominant disorder primarily caused by loss-of-function mutations in the methyl-CpG-binding protein 2 (MeCP2) gene, which lead to intellectual disability including language deficits, epileptic seizures, and respiratory dysfunction (Bedogni et al., 2014). Microarray and chromatin immunoprecipitation analyses identified ID1, ID2, ID3, and ID4 genes as primary targets of MeCP2 (Peddada et al., 2006). MeCP2 is a repressor, and upregulation of all four IDs has been found in MeCP2 null mouse brain (MeCP2-/Y hemizy- gotes) as well as brain tissue from Rett syndrome patients (Peddada et al., 2006). The MeCP2 null mutant mouse also has reduced expression of the neural precursor gene NeuroD1 (Peddada et al., 2006). Since E47/TCF3 stimulates NeuroD1 transcription (Sharma et al., 1999), high ID protein levels could reduce NeuroD1 expression. Interestingly, a frameshift mutation of E2-2/TCF4 was recently discovered in a variant Rett syndrome patient who did not have mutations in MeCP2 (Armani et al., 2012). E2-2/TCF4 loss of function is expected to mimic effects of ID protein overexpression. Other E2-2/ TCF4 loss-of-function mutations cause Pitt-Hopkins syndrome (see above), which shares symptoms with Rett syndrome. Although the cellular basis of Rett syndrome is not yet clear, Figure 3. Mammalian HLH Proteins and Cell-Cycle Control altered neural differentiation as a consequence of reduced Cell-cycle regulation involving HLH proteins in multiple ways. E protein- NeuroD expression, defects in neuronal cell-cycle withdrawal, mediated activation of the CDK inhibitors p16, p21, p27, and p57 is shown. or altered axon or dendrite morphogenesis are all plausible Cell-cycle arrest is promoted by these CDK inhibitors in G1-S transition, S, and G2-M phases. This regulation is negatively modulated by ID proteins candidates. through the association with E proteins. Rb can antagonize ID2 by direct A further connection between HLH proteins and autism spec- interaction. trum disorders is that ID3 has been reported as a target of miR- 29b, a brain-specific miRNA that is expressed more highly in autism spectrum disorders. ID3 expression is significantly down- Schizophrenia regulated in miR-29b-transfected cells (Sarachana et al., 2010). In addition to linkage to PTHS and Fuchs corneal dystrophy, Whether ID3 is really involved in autism, whether its function in several SNPs in E2-2/TCF4 are significantly associated with this regard is related to that of E2-2/TCF4, and whether differen- schizophrenia (Stefansson et al., 2009; Forrest et al., 2014; tiation, cell cycle, neuronal morphology, or other defects are Quednow et al., 2014). E2-2/TCF4 is also a target of microRNA involved remain to be elucidated. Intriguingly, one trait that is (miRNA) 137, which has been mapped as a schizophrenia sus- strongly associated with autism is circadian rhythm dysfunction ceptibility locus (Schizophrenia Psychiatric Genome-Wide Asso- (Hu et al., 2009). ID proteins play essential roles in controlling ciation Study (GWAS) Consortium, 2011; Wright et al., 2013), and circadian rhythm by sequestering the bHLH transcription factors predicted to bind other miRNAs associated with schizophrenia, CLOCK and BMAL that are central components of the circadian autism, and other CNS disorders (Perkins et al., 2007; Talebiza- pacemaker (Duffield et al., 2009). deh et al., 2008). However, none of the variants affect the Atherosclerosis E2-2/TCF4 protein, so how E2-2/TCF4 activity is affected in ID3 maps to atherosclerosis susceptibility loci in both humans schizophrenia remains uncertain (Williams et al., 2011a). This and mice (Lusis et al., 2004). A SNP (rs11574) substitutes Thr has not been resolved by mouse studies. E2-2/Tcf4 homozy- for Ala105 in the human ID3 protein and is associated with gous null (E2-2À/À) mice displayed abnormal hindbrain develop- increased carotid intima-media thickness (IMT) (Doran et al., ment in the pontine nucleus (Flora et al., 2007)(Table 2). Pontine 2010). This mutation decreases ID3 function as measured by nucleus development requires heterodimers of E2-2/TCF4 with binding to E12 in NIH 3T3 cells (Doran et al., 2010). ATOH-1 (Flora et al., 2007). Thus, schizophrenia could have a Atherosclerosis is strongly enhanced when Id3 is deleted in developmental origin caused by reduction of E2-2/TCF4 func- ApoE or Ldlr knockout mice (i.e., in Id3À/À ApoEÀ/À or Id3À/À tion. On the other hand, E2-2/TCF4 overexpressing transgenic LdlrÀ/À genotypes) (Doran et al., 2010; Lipinski et al., 2012) mice have defects in fear conditioning and sensorimotor gating (Table 2). Bone marrow transplantation from Id3+/+ ApoEÀ/À (Brzo´ zka et al., 2010). Sensorimotor gating has been used for a donors is atheroprotective, pointing to a role in bone marrow- long time in animal model studies because it resembles the over- derived cell types (Doran et al., 2012). These mice have sensitivity to sensory stimulation seen in schizophrenic patients reduced B cell numbers but increased macrophage numbers (Braff and Geyer, 1990). Clearly, it would be very informative to in the aorta (Doran et al., 2012; Lipinski et al., 2012). Chemo- resolve how schizophrenia-associated SNPs affect E2-2/TCF4 kines and vascular cell adhesion molecule 1 (VCAM-1) are

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Figure 4. Drosophila HLH Proteins in Growth Control When Drosophila ID protein (emc) is mutated, excess Drosophila E protein (Da) homodimers activate expanded (ex) transcription via binding to E boxes in the cis-regulatory element. The subsequent activation of Salvador-Warts-Hippo signaling pathway inhibits Yki/YAP activity, thereby blocking cell growth and survival. High levels of Da are also involved in blocking cell-cycle progression in G2-M transition through inhibiting stg/cdc25 transcription (Andrade-Zapata and Baonza, 2014; Wang and Baker, 2015).

vascular abnormalities, and embryonic lethality (Lyden et al., 1999)(Table 2). Bone Marrow Failure A connection between HLH proteins and bone marrow failure has been suggested based only on ID2 upregulation and E47 and HEB/TCF12 downregulation in Dia- mond Blackfan anemia (DBA) patients (Zhang et al., 1997). E47/TCF3, HEB/ TCF12, and a tissue-restricted expressed bHLH protein, SCL, regulate early elevated and may promote these changes in leukocyte adhe- erythroid differentiation and are reduced during maturation sion and recruitment into the arterial wall. The VCAM-1 pro- when ID2 is upregulated so that elevated ID2 would be moter is regulated by E12 and could be activated by E12 expected to decrease the expression of erythroid speciﬁc genes following a reduction in ID3 (Lipinski et al., 2012). One model, and erythroid differentiation. This could be re-evaluated now that therefore, is that ID3 protects against atherosclerosis by it is known that approximately 65% of patients harbor mutations altering leukocyte recruitment to artery walls (Doran et al., in ribosomal protein genes (Farrar et al., 2011) and that many 2010; Lipinski et al., 2012; Doran et al., 2012). aspects of DBA have been attributed to p53 hyperactivity The effect of ID proteins on the vasculature is complicated, caused by nucleolar stress (Ellis, 2014). Although p53 can regu- however (Yang et al., 2014). ID3 inhibits adiponectin expression late ID1 and ID2 transcription in neural stem cells, repression is by preventing E47 from binding to E boxes in the adiponectin observed, contrary to the notion that ID2 is upregulated in DBA promoter (Doran et al., 2008). Adiponectin is an adipocyte- patients (Paolella et al., 2011). It is not impossible that differenti- derived cytokine that attenuates plaque formation: low adipo- ation defects can contribute to DBA, however, as suggested by nectin levels are associated with coronary artery disease, mutations in the hematopoietic transcription factor GATA1 that whereas adiponectin overexpression reduced atherosclerosis were recently found in some DBA cases (Sankaran et al., 2012). in ApoE-deﬁcient mice. Atherosclerosis might also be impacted Diabetes, Glucose, and Lipid Metabolism by the roles of ID proteins in vascular smooth muscle cells and Insulin affects glucose and lipid metabolism, and both are dis- endothelial cells (see below). For example, alternative splicing rupted in diabetes. b cells in the islets of Langerhans of diabetic of Id3 in vascular lesions may produce a protein with a uniquely mice or isolated b cells exposed to hyperglycemia or lipid have atheroprotective role (Forrest et al., 2004). elevated ID1/ID3 expression (Wice et al., 2001; Busch et al., Arterial Vascular Disease 2002; Kjørholt et al., 2005; Billestrup, 2011). Consistent with Mutations in the BMPR2 gene that encodes the BMP type II re- the notion that ID proteins impair glucose and lipid metabolism, ceptor are found in most cases of heritable pulmonary arterial hy- islets from Id1À/À mice are protected against high-fat diet- pertension (PAH), in which smooth muscle cells proliferate to induced repression of b cell genes, such as pancreatic duodenal occlude the vessel (Lane et al., 2000; Machado et al., 2006). homeobox-1, NeuroD1 (also called Beta2), Glut2, pyruvate ID1 and ID3 are targets of BMP signaling in pulmonary artery carboxylase, and Gpr40 (Akerfeldt and Laybutt, 2011). Id1À/À smooth muscle cells, as in other cell types, and a recent study mice show less insulin resistance in skeletal muscle, liver, and suggests that ID proteins mediate anti-proliferative effect of white adipose tissue and increased Uncoupling Protein 1 and BMP signaling (Yang et al., 2013). The mechanism is unclear, Peroxisome proliferator-activated receptor gamma coactivator however, because both Id2 and Id3 are also reported to inhibit 1 in brown adipose tissue (corresponding to increased energy p21 expression in proliferating vascular smooth muscle cells, expenditure) (Satyanarayana et al., 2012). Mice overexpressing as in many other cell types (Matsumura et al., 2002; Forrest E proteins revealed similar phenotypes (Zhao et al., 2014). et al., 2004; Taylor et al., 2006)(Figure 3). ID proteins also have Although Id1 expression is downregulated and the protein roles in endothelial cells (Ling et al., 2014; Yang et al., 2014). apparently degraded during adipocyte differentiation, adipogen- Id1À/À;Id3À/À mice exhibit cardiac developmental defects, brain esis appears normal in Id1À/À mice, suggesting that it is effects

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Table 3. HLH Protein Diseases and Mechanisms Affected Organ or Disease Tissue HLH Protein Evidence Process Affected Pitt-Hopkins syndrome brain, face E2-2/TCF4 human mutation differentiation? cell cycle? neuronal morphogenesis? Fuchs corneal dystrophy eye E2-2/TCF4 human gene association senescence? Schizophrenia brain E2-2/TCF4 human gene association differentiation? cell cycle? neuronal morphogenesis? Rett syndrome brain E2A/TCF3, ID1–4 gene expression differentiation? cell cycle? neuronal morphogenesis? Atherosclerosis arteries E2A/TCF3, ID3 human, mouse gene differentiation? association Diamond Blackfan anemia bone marrow E2A/TCF3, gene expression differentiation? HEB/TCF12, ID2 Diabetes, glucose and multiple ID1-2,4 mouse mutant models differentiation? lipid metabolism Osteoporosis bone ID1, ID4 mouse mutant models differentiation? Arterial vascular disease arteries ID1, ID3 mouse mutant models cell cycle? Congenital hydronephrosis kidney ID2 mouse mutant models differentiation? Polycystic kidney disease kidney E2A/TCF3, ID2 gene expression cell cycle? Parkinson’s disease brain, nerves ID2 mouse mutant models differentiation? Sjogren’s syndrome lachrymal, salivary ID3 mouse mutant models differentiation? glands, skin on oxygen consumption and thermogenesis that protect them Osteoporosis and age-related osteopenia also result from dif- against a high-fat diet (Satyanarayana et al., 2012). E2A/TCF3 ferentiation of MSCs into bone marrow adipocytes rather than and E2-2/TCF4 proteins can also directly increase insulin osteoblasts. Adipocytes additionally suppress osteogenesis expression in b cells (Vierra and Nelson, 1995). because secreted adipogenic signals influence osteoclast differ- Other ID proteins also regulate metabolism. Mice with homo- entiation by HSCs. Id4À/À mice reduce osteoblast differentiation zygous deletions of Id2 or Id4 have less body fat and gain and enhance adipocyte differentiation (Tokuzawa et al., 2010) much less weight on a high-fat diet (Murad et al., 2010; Mathew (Table 2). ID4 competes with the bHLH factor Hes1 to dimerize et al., 2013). Id2À/À mice have altered lipid metabolism, impaired with Hey2, allowing Hes1 to activate osteoblast-specific gene adipogenesis, and reduced gonadal white adipose deposits and transcription and to promote osteogenesis through the key oste- liver lipid content (Park et al., 2008; Hou et al., 2009; Mathew oblast differentiation factor Runx2 (Tokuzawa et al., 2010). It is et al., 2013)(Table 2). Male Id2À/À mice also reveal increased in- not known how Hes1 regulates Runx2 levels (Ikawa et al., sulin sensitivity, increased glucose uptake by skeletal muscle 2006). Other ID proteins also affect osteoblasts, which show and brown adipose tissue, and reduced intramuscular triacylgly- reduced proliferation and mineralization in Id1+/À; Id3À/À mice cerol and diacylglycerol levels (Mathew et al., 2013). Apparently, (Maeda et al., 2004). Taken together, these findings make both therefore, ID1, 2, and 4 play a variety of roles in insulin secretion ID and perhaps E proteins potential targets for the prevention and energy metabolism. and treatment of osteoporosis because of their roles in osteo- Osteopenia and Osteoporosis clast and osteoblast specification. Id1À/À mice have an osteoporotic phenotype, i.e., reduced bone Renal Disease density and increased bone fragility (Chan et al., 2009)(Table 2). Id2+/À or Id2À/À mice develop congenital hydronephrosis, which Healthy bone volume is controlled by the balance between bone is common in humans and may result in early-onset renal failure deposition by osteoblasts and bone resorption by osteoclasts. (Aoki et al., 2004; Tripathi et al., 2012). The cause of hydroneph- Excess of bone resorption over bone formation results in rosis is frequently obstruction at the ureteropelvic junction. osteoporosis and its milder potential precursor, osteopenia. Id2 mutant mice display irregular, hypertrophic muscle layers Osteoclasts and osteoblasts are derived from distinct lineages: at the ureteropelvic junction, suggesting that smooth muscle osteoclasts differentiate from hematopoietic stem cell (HSC) hypertrophy at the ureteropelvic junction could be the cause of precursors (Suda et al., 1992), while osteoblasts arise hydronephrosis (Aoki et al., 2004). E protein-dependent myo- from mesenchymal stem cells (MSCs). The bone phenotype of genesis could be stimulated when ID2 levels are decreased, Id1À/À is due to excess osteoclastogenesis (Chan et al., 2009). but the usual role of E proteins is to inhibit proliferation, not pro- Genes required for osteoclastogenesis are upregulated, including mote it (Figures 1 and 3). Trap, Oscar, and Ctsk; overexpressing ID1 downregulates these A role for ID proteins was also suggested in autosomal domi- genes (Chan et al., 2009). It would be reasonable to anticipate a nant polycystic kidney disease (PKD), which is mostly caused by positive role of one or more E proteins in osteoclast differentiation mutations in the PKD1 and PKD2 genes (Peters and Sandkuijl, and maturation, although none seems to have been reported. 1992). In this disease, bilateral renal cysts replace the normal

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renal parenchyma, often resulting in end-stage renal disease. dronephrosis, Parkinsonism, or Sjogrens’ syndrome in humans, Cysts are lined by a single layer of epithelium that is character- but their mouse mutations provide models of these disorders ized by increased cellular proliferation and decreased differenti- and suggest they may be involved. This variety of diseases ation (Nadasdy et al., 1995). It was reported that Polycystin-2, and models reinforces the notion that E and ID proteins and their the product of the PKD2 gene, sequestered ID2 in the cytoplasm relative expression levels constitute an important aspect of the in a PKD1-dependent manner, suppressing inappropriate prolif- cellular regulatory environment that remains important eration through E47-dependent p21 transcription (Li et al., 2005; throughout life, not only during early development. The mecha- Zhou, 2009). More recent publications focus on defects in Ca2+ nisms established for HLH protein regulation in normal develop- regulation at the cilium as a cause of PKD, however, and the rela- ment provide a useful starting point for disease mechanisms tionship to cell-cycle regulation by ID and E proteins is unclear (Figures 1 and 2). For example, do ID proteins regulate dendrite (Fliegauf et al., 2007; Fedeles et al., 2011). growth through E proteins and transcription, and is this relevant Parkinsonism to neurological conditions? More still remains to be learned Id2 homozygous null mutant mice display features of Parkin- concerning normal E and ID protein expression and function, son’s disease, such as fewer dopaminergic neurons in the olfac- especially at the post-transcriptional level. Post-translational tory bulb and reduced olfactory discrimination (Havrda et al., modification and changes in subcellular localization have all 2008). Id2À/À mice also have reduced dopamine transporter been reported (Lingbeck et al., 2005; Kurooka and Yokota, expression, activated caspase-3, as well as glial infiltration in 2005; Sun et al., 2005; Rollin et al., 2009; Nio-Kobayashi et al., the substantia nigra pars compacta of brain (Havrda et al., 2013; Lasorella et al., 2014). Protein turnover and stability may 2013)(Table 2). These observations suggest that ID2 is required be a regulatory factor and therapeutic target. Recent work for the development of midbrain dopaminergic neurons and raise shows that the Drosophila E protein Da is destabilized by Notch the question of how ID2 might be involved in Parkinson’s disease signaling, and this may be one of the mechanisms by which and other disorders of the dopaminergic system such as atten- Notch regulates so many differentiation processes (Kiparaki tion deficit hyperactivity disorder, schizophrenia, and drug et al., 2015). These studies exemplify the broad effects and po- abuse. Intriguingly, significant inductions of ID1, ID2, and ID3 tential therapeutic opportunities expected to accrue from modu- expression by decreased dopamine levels have been detected lating these very widely expressed and important HLH proteins. in rodents under continuous stress conditions (Konishi et al., 2010). At present, it is not known whether E proteins play specific ACKNOWLEDGMENTS roles in dopaminergic neurons, other than their general role in neuronal differentiation (Figure 1). We thank A. Bhattacharya and K. Li for contributions to work on HLH proteins Sjogren’s Syndrome in our laboratory and J. Hebert, H. Lachman, B. Morrow, N. Sibinga, and the ID3 homozygous null (Id3À/À) mice develop many symptoms anonymous reviewers for comments on the manuscript. Our work on HLH proteins is supported by the NIH (GM047892) and by an Unrestricted Grant from similar to those found in Sjo¨ gren’s syndrome, an autoimmune Research to Prevent Blindness to the Department of Ophthalmology and Vi- disease in which immune cells chronically attack the lachrymal sual Sciences. and salivary glands, resulting in impaired tear and saliva secretion (Li et al., 2004)(Table 2). The effect of Id3 gene inactivation REFERENCES may be on lymphocyte differentiation because adoptive transfer À/À of Id3 bone marrow cells is sufficient to induce symptoms of Adam, J.C., and Montell, D.J. (2004). A role for extra macrochaetae down- Sjo¨ gren’s syndrome (Li et al., 2004; Guo et al., 2011) and stream of Notch in follicle cell differentiation. Development 131, 5971–5980. because CD20 antibody treatment, which depletes B cells, re- Akerfeldt, M.C., and Laybutt, D.R. (2011). 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