1 Background

BHLHE41, also called differentiated embryo chondrocyte 2 (DEC2), split and hairy related protein 1 (SHARP1) or BHLHB3, is a nuclear transcription factor (TF) that belongs to the basic helix-loop-helix (bHLH) protein superfamily. Six phylogenic groups (group A to group F) of bHLH proteins have been described [1]. Their molecular structure includes helix loop helix (HLH) domains, that act as protein oligomerization interfaces facilitating protein-protein interactions and the formation of homo or heterodimeric complexes. They are juxtaposed to basic amino acid regions that bind to DNA at consensus hexanucleotide sequences, such as E-box sequences and to a lesser degree, N-box sequences. These sequences are found in the transcriptional regulatory region of many genes. Additional domains act as dimerization regions, including Drosophila Period, human Aryl hydrocarbon receptor nuclear translocator and Drosophila Single-minded (PAS) domains in group C bHLH proteins [2], orange domains in group E bHLH proteins [3] and leucine zipper domains in group B bHLH members [4]. The different combinations of dimeric complexes and the DNA binding sequence heterogeneity determine how bHLH proteins control critical biological processes, through transcriptional regulation from yeast to humans [1, 5].

In this review, we summarize the biological characteristics of BHLHE41, including its structure and expression, its interacting partners and its transcriptional activities. We also review its involvement in physiological processes and its role in solid cancer and hematological malignancies.

2 Biological characteristics of BHLHE41

2.1 Structure

The human BHLHE41 gene is located on chromosome 12, at 12p12.1 [6]. It includes five exons and four introns, with two E-boxes at −0.3 and −1.8 kb [7]. Rat Bhlhe41 was the first cloned in 1997 [8]. The human gene, initially named DEC2, and its mouse homologue were characterized in 2001 [6].

The human BHLHE41 protein is 482 amino acids long, and has a molecular weight of 50 kDa. It contains a bHLH domain at the N terminus that binds to the E box motif CANNTG in the target promoters [9], an orange motif in the central region, and an alanine/glycine-rich sequence in the C-terminal region (Fig. 1).

Fig. 1
figure 1

Structure of the mammalian BHLHE41 protein. bHLH basic helix-loop-helix domain, Rep D Repression domain, DNA BD DNA binding domain, Dim D Dimerization domain, Orange D Orange domain, A/G rich D Alanine/Glycine-rich domain.

Adapted from [10] and created with BioRender.com

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The bHLH domain also contains an LxxLL motif for the interaction with nuclear retinoid receptors [11]. The bHLH region is crucial for BHLHE41 functionality, because it allows the formation of homo/heterodimers before binding to the E-box sequence [5].

BHLHE41 protein structure shares 42% of homology with BHLHE40 (also known as DEC1 or BHLHB2), another member of the bHLH superfamily. Their bHLH domains are highly conserved (97% of homology) and allow the formation of BHLHE40-BHLHE41 heterodimers that regulate the expression of common downstream target genes [10, 12].

2.2 Expression

BHLHE40 is ubiquitously expressed in human tissues. Conversely, the expression of its paralogue BHLHE41 is tissue-restricted (Fig. 2).

Fig. 2
figure 2

Tissue-specific expression of BHLHE41 in humans. In humans, BHLHE41 is highly expressed in skeletal muscle, brain, and retina, found at intermediate levels in the heart, pancreas, and thyroid, and detectable but at low levels in the lung and placenta. Created with BioRender.com

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Specifically, BHLHE41 is highly expressed in skeletal muscle, brain and retina, found at intermediate levels in the heart, pancreas and thyroid, and detectable, but at low levels, in the lung and placenta [6, 8, 13, 14]. The expression of this gene is downregulated through a regulatory feedback loop involving BHLHE41 and BHLHE40 [7]. Indeed, it has been observed that an overexpression of BHLHE41 or BHLHE40 results in BHLHE41 downregulation [15]. This effect is mediated through the binding of BHLHE40 to the E box sequence in BHLHE41 promoter [16]. Additionally, BHLHE41 can repress BHLHE40 transcription by interacting with Specificity Protein 1 (SP1) [9]. BHLHE41 expression is also regulated by different factors including members of the clock family proteins, growth factors, cytokines, transcription factors, nutrients or hormones (Table 1).

Table 1 Factors modulating BHLHE41 expression. Several conditions and biological processes modulate BHLHE41 expression, including hypoxia through clock proteins, metabolism through nutrients or hormones, growth factors, cytokines and transcription factors
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Of note, most of these factors can also up regulate the expression of BHLHE40, as it has been described for Nerve Growth Factor (NGF) [8], Transforming growth factor (TGF) beta [28] or Tumor-Necrosis Factor (TNF) alpha [29]. These parallel modifications of expression potentially influence the fate of BHLHE41/BHLHE40 heterodimer.

Hypoxia is a regulator of BHLHE41 expression. Hypoxia-inducible factor-1 (HIF-1) is a TF regulating oxygen homeostasis and involved in the adaptation to oxygen deprivation [30]. HIF-1 is a protein complex composed of two subunits: HIF-1 alpha and HIF-1 beta (also known as aryl hydrocarbon receptor nuclear translocator 1). HIF-1 beta is constitutively expressed. In normoxic conditions, HIF-1 alpha is rapidly degraded after hydroxylation by prolyl-hydroxylase and ubiquitination by the von Hippel–Lindau protein for proteasomal destruction. In contrast, under hypoxic conditions, HIF-1 alpha is stabilized, activated and translocated to the nuclear compartment, where it heterodimerizes with HIF-1 beta [31]. The resulting complex interacts with the HIF-1 binding site in the Hypoxia Response Element (HRE) of the promoters of hypoxia-inducible genes to modulate their expression. Miyazaki and colleagues reported that BHLHE41 harbors a HRE in its promoter region and that its expression is induced by hypoxia [20]. Moreover, BHLHE41 can interact with HIF-1 alpha and contribute to its proteasomal degradation [32]. In addition, upon induction in hypoxic conditions, BHLHE41 downregulates vascular endothelial growth factor (VEGF), a major cytokine involved in angiogenesis [33].

Besides hypoxia, BHLHE41 expression is also modulated throughout circadian rhythms. In mammals, they are controlled by molecular clockwork systems that involve a series of clock genes, including CLOCK, BMAL1, BMAL2, PER1, PER2, PER3, CRY1, CRY2, CRY3, BHLHE40 and BHLHE41. The protein CLOCK is a transcription factor heterodimerizing with BMAL1, binding to E-box elements upstream of the period (PER1, PER2, PER3) and cryptochrome (CRY1, CRY2) genes and upregulating their transcription. In turn, PER and CRY heterodimerize and repress their own transcription through a feedback loop that involves CLOCK-BMAL1 complexes which also upregulate BHLHE41 [7, 20, 34]. Of note, mouse mutants that were designed to explore the role of BHLHE41 in circadian rhythms regulation and that do not express BHLHE41 were viable [35], suggesting the potential redundancy of bHLH family members.

Several soluble factors also modulate BHLHE41 expression. In an in vitro model to study neurite outgrowth, BHLHE41 expression can be induced by NGF, an essential factor for neuronal development. It has been proposed that BHLHE41 could mediate cellular changes at late stages of the central nervous system development [8]. TGF beta signaling in the bone marrow environment leads to BHLHE41 induction in mice xenografted with HEp3 cells, a model of head and neck squamous cell carcinoma [18]. Moreover, in an in vitro model of synovial fibroblasts, TNF alpha increases BHLHE41 expression in a NF-kappa B dependent manner [19].

BHLHE41 expression is also positively modulated by different TFs and intracellular proteins depending on the cellular context, including RAR-related orphan receptor (ROR) alpha [21], p63 (a member of the p53 family) [22], GLI1 and GLI2 (two members of the family of zinc finger proteins acting as mediators of the sonic hedgehog signal transduction cascade) [23] and GATA3 [24].

In addition, it has been observed that a combination of glucose and amino acids can rapidly modulate the expression of BHLHE41 and clock genes in mouse liver, suggesting an influence of nutrients in the regulation of the liver clock [26]. In rat H4IIE hepatoma cells, insulin rapidly increases BHLHE41 production in a dose-dependent manner, through different signaling pathways, including PI3K/Rac/JNK pathways [27].

2.3 Interacting partners

BHLHE41 homo- or hetero-dimerizes with its paralogue BHLHE40 or other proteins, in particular several TFs, including CCAAT/enhancer-binding protein alpha (CEBPA) and beta (CEBPB) [36], HIF-1 alpha [32, 33], myogenic differentiation 1 (MYOD1) [5], RORA [37], retinoid X receptor (RXR) alpha [10], sterol regulatory element binding protein 1c (SREBP-1c) [38], SP1 [9], and the RNA binding protein NONO [37] (Table 2). Furthermore, BHLHE41 interacts with a variety of proteins including cell cycle controlling factors casein kinase 2 alpha-1 (CSNK2A1), CSNK2A2, CSNK2B, proteins involved in the control of cell growth and division (i.e. PPP2CB, PPP2R1B, PPP2R5E [37], LLGL2 and KLC3) [39], cell signaling molecules (e.g. RASD1, a member of the Ras superfamily of small GTPases and MKNK2) [37, 39], proteins involved in epigenetic mechanisms (e.g. HDAC1, SIRT1 or WDR5) [5, 29], and DNA damage response proteins (ATM, CDCA5, CSNK1E, RTF2 and POLK) [32]. BHLHE41 also interacts with factors implicated in protein trafficking (e.g. VPS26A, a component of the retromer complex involved in the retrograde transport of proteins from endosomes to the trans-Golgi network) [39], protein degradation factors (e.g. FBXW11, a member of the F-box protein family that is a subunit of the ubiquitin protein ligase complex [37] and PSMA4, the core alpha subunit of the 20 S proteosome [28]), GSK3B (a protein implicated in the control of glucose homeostasis [37]), CLOCK proteins (CRY1, CRY2, PER2 [37]), and with MYH13 (a myosin chain involved in muscle contraction [39]). BHLHE41 also forms heterodimers with nuclear factor interleukin 3 regulated (NFIL3, also known as E4BP4), a basic leucine zipper TF. This heterodimer can bind to the PER2 EE-element (E-box and E-box-like sequences) to modulate the circadian oscillation [40].

Table 2 BHLHE41 known or predicted interacting partners. A large diversity of interacting partners has been described for BHLHE41, including other TFs, factors involved in transcriptional regulation and RNA splicing, cell cycle, cell growth and division, cell signaling, epigenetic mechanisms, DNA damage response, protein trafficking, ubiquitination, proteasomal degradation, metabolism, circadian rhythms, muscular activity
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2.4 Transcriptional activities

Although most bHLH members act as transcriptional activators, BHLHE41 functions as a nuclear transcriptional repressor through different mechanisms [9, 41] (Fig. 3).

Fig. 3
figure 3

BHLHE41 transcriptional repressive activities through different mechanisms. The BHLHE41 repressive activity first relies on direct DNA binding-dependent mechanisms through E-box sites of target promoters leading to recruitment of chromatin regulators or through interaction with other TF. It can also be exerted through heterodimerization leading to non-functional complexes, to sequestration of TF or to proteasomal degradation of TF.

Adapted from [45] and created with BioRender.com

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First, this repressive activity relies on the direct binding of BHLHE41 to the E-box sequence of target promoters. A competition with E-box binding trans-activators then occurs. Finally, BHLHE41 recruits co-repressors, including Histone DeACetylase 1 (HDAC1), sirtuin 1 (SIRT1) and Euchromatic Histone lysine Methyltransferase 2 (EHMT2, also known as G9a) that mediate the deposition of repressive chromatin marks [45].

Second, this inhibitory activity can occur through direct protein-protein interactions, independently of DNA binding. These interactions involve other TFs, such as SP1 [9]. BHLHE41 can heterodimerize with bHLH factors to form non-functional complexes or to sequester these factors, as observed for MYOD1 [5]. It also antagonizes the transcriptional activity of other TFs, such as HIF-1 alpha, by promoting their proteasome-mediated degradation [32]. In addition, BHLHE41 regulates the expression of other TFs of the bHLH family that display repressor activity, such as TWIST1, involved in the regulation of myogenesis and osteogenesis [46]. Furthermore, in HEK293 cells, BHLHE41 suppresses ligand-induced RXR alpha transactivation activity, repressing target gene expression mediated by the RXR-LXR heterodimer [11].

2.5 Involvement of BHLHE41 in physiological processes

TFs of the bHLH superfamily contribute to the regulation of embryo development, homeostasis of different organs/tissues and differentiation of various cell types, including neuronal, skeletal muscle and hematopoietic cells [5, 47,48,49,50]. BHLHE41 also is implicated in various biological processes (Fig. 4).

Fig. 4
figure 4

BHLHE41 is involved in major biological processes during development and homeostasis and in specific cell types. BHLHE41 is involved in various biological processes including DNA repair, circadian rhythms, myogenesis, adipogenesis or spermatogenesis. It can also display major roles in lymphoid lineage, mesenchymal stem cells or macrophages cell fate. Created with BioRender.com

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2.5.1 Myogenesis regulation

BHLHE41 is expressed in developing skeletal muscles where it heterodimerizes with the myogenic regulatory factor MYOD1. This factor belongs to bHLH family, is expressed by myoblasts and is involved in skeletal muscle differentiation, driving transcription of muscle-specific genes. BHLHE41 is expressed in proliferating myoblasts and is down-regulated during myogenic differentiation. It thus acts as a negative regulator of myogenesis, its overexpression inhibiting myogenic differentiation [51].

In addition, BHLHE41 interacts with the methyltransferase G9a that belongs to the SET domain-containing Su(var)3–9 family of proteins including Suv39h1/h2, SETDB1, SETDB2 and DIM-5. G9a mediates transcriptional repression activities by mono- and di-methylation of histone H3 lysine-9. It can also methylate MYOD1, resulting in the inhibition of its transcriptional activity and the negative regulation of myoblast differentiation [52].

BHLHE41-G9a interactions enhance BHLHE41 inhibition of skeletal myogenesis [44]. Interestingly, sumoylation of BHLHE41 by SUMO1 is required for the interaction with G9a and its full transcriptional repression activity during skeletal muscle differentiation [53, 54]. BHLHE41 also modulates skeletal muscle regeneration by regulating TGF-beta signaling and by counteracting SMAD-3-dependent expression of collagens and TIMP1 (tissue inhibitor of metalloproteinase 1) [55]. SREBP-1, which is involved in myogenesis regulation, can induce BHLHE41 expression that in turn mediates the repression of muscle genes [25]. BHLHE41 is a HDAC4 target in satellite cells, a specific type of muscle stem cells. During proliferative stage of myogenesis HDAC4 represses BHLHE41 expression, allowing satellite cell differentiation and fusion [56].

2.5.2 Adipogenesis regulation

BHLHE41 interacts with CEBPA and CEBPB, two transcriptional master regulators of adipogenic differentiation. It inhibits their transcriptional activity by retaining HDAC1 and G9a at the regulatory sites of their promoters [45], as well as for Peroxisome Proliferator-Activated Receptor gamma (PPARG), another master transcriptional regulator of adipogenesis [36]. SUMOylation enhances BHLHE41 repression of PPARG expression. This effect can be reversed by SENP1, a protease of the Sentrin/SUMO-specific Protease (SENP) family that de-SUMOylates BHLHE41, thus suppressing BHLHE41 inhibition of PPARG expression and enhancing adipogenesis [57].

2.5.3 Spermatogenesis

Spermatogenesis is a continuous process that relies on self-renewal and differentiation of spermatogonial stem cells [58]. Several factors, including SOHLH1, a testis-specific bHLH TF, are needed during the gonocyte-to-spermatogonia transition. BHLHE41 can inhibit spermatogonial differentiation in neonatal murine germ cells by repressing SOHLH1 expression [59]. The mechanisms that might inhibit the activity of BHLHE41 to allow the progress of spermatogenesis remain unexplored.

2.5.4 Regulation of circadian rhythms

In mammals, circadian rhythms are regulated by the suprachiasmatic nucleus of the hypothalamus and by other areas in the brain and peripheral tissues. At cellular level, several clock gene families are involved in circadian rhythms generation. They act in cooperation in the so-called “molecular clock”, through an autoregulatory transcription-translation feedback loop. CLOCK and BMAL1, two major clock genes, encode proteins that heterodimerize in the cytoplasm and then translocate to the nucleus, where they bind to E-box enhancer sequences and activate the transcription of other clock genes [60], including BHLHE40 and BHLHE41 [7, 12]. Honma et al. showed, in a murine model, that BHLHE40 and BHLHE41 are expressed in the suprachiasmatic nucleus in a circadian way. They mighty repress the CLOCK-BMAL1-induced transactivation of the PER1 promoter via direct protein interactions with BMAL1 or through E-box motif competition. Therefore, BHLHE40 and BHLHE41 are two regulators of the mammalian molecular clock and are thus considered as clock genes [42]. Of note, BHLHE41 gene mutations affect sleep duration in humans [61, 62]. A missense mutation in exon 5 of BHLHE41 inducing a replacement of proline at position 384 by an arginine was described in individuals with short sleep duration. This mutation resulted in the inability of the BHLHE41 protein to interact properly with the circadian clock transcription factors CLOCK and BMAL1 [61].

BHLHE41 is also implicated in the interplay between circadian and metabolic rhythms. BHLHE41 contributes to the circadian control of liver metabolism by regulating the expression of P450 cytochromes [63], through its interaction with CEBPA and the formation of a complex with HDAC1 [64]. Moreover, BHLHE41 expression is decreased by glucose deprivation [65], whereas it is increased by insulin [27].

2.5.5 DNA repair mechanisms

BHLHE41 influences DNA repair mechanisms. Liu and colleagues observed that BHLHE41 is upregulated in response to genotoxic agents in mouse NIH3T3 cells, resulting in S and G2/M cell cycle arrest. BHLHE41 upregulation led to increased expression of BRCA1, which is required for cell cycle arrest and DNA repair, and GADD45A, a p53 target gene also involved in cell cycle arrest. In addition, upon BHLHE41 overexpression, cells are protected from DNA damage-induced apoptosis and p53, p21 and BAX expression levels are reduced [66]. This indicates that BHLHE41 is involved in survival mechanisms when cells are treated by DNA-damaging agents. Hypoxia can reduce the expression of different genes involved in the DNA damage response, leading to impaired DNA repair and genome instability [67,68,69,70,71,72]. In hypoxic conditions, BHLHE41 downregulates DNA damage response genes through the repression of their promoter activities [71].

2.5.6 Homeostasis of mesenchymal stem cells

BHLHE41 is highly expressed in mesenchymal stem cells (MSC) that can differentiate into several cell types, including adipocytes and smooth muscle cells. BHLHE41 suppresses MSC differentiation into muscle cells and adipocytes by inhibiting the transcriptional activities of MYOD1 and CEPB. This suggests that BHLHE41 may be involved in maintaining MSCs in an undifferentiated state [36, 73].

2.5.7 Regulation of tissue-specific macrophage properties

Macrophages are innate immune cells that contribute to the front-line immune defense and tissue homeostasis. Macrophages also display tissue-specific profiles through tissue-specific molecular programs, including transcriptional networks [74]. Rauschmeier et al. observed that BHLHE41 and its paralogue BHLHE40 can repress transcriptional activities that are crucial for defining the identity of lung alveolar macrophages, likely occurring at least in part through histone deacetylation. In this context, BHLHE41 is involved in their final stages of differentiation and maturation [75]. In addition, macrophages can be involved during repair and remodeling after myocardial infarction. Xu et al. reported that macrophages expressing BHLHE41 could limit fibrosis and expansion of developing infarct area [76].

2.5.8 BHLHE41 in the lymphoid lineage

The differentiation of B lymphocytes into plasma cells (PC) is driven by a complex network of TFs. Among them, the transcriptional repressor PRDM1/BLIMP1 is the master regulator of terminal B cell development [77]. During PC differentiation, the expression of PRDM1/BLIMP1 is significantly increased from the proliferating plasmablast stage. PRDM1/BLIMP1 directly represses several genes, including BCL6, PAX5, SPIB/Spi-B, ID3, CTIIA and MYC [78, 79]. PAX5 is essential for B-lineage commitment and activates the expression of B cell identity genes, including CD19. It represses the expression of genes activated in PCs, such as IGH immunoglobulin heavy locus and XBP1. PRDM1/BLIMP1 blocks immunoglobulin class switching by suppressing the expression of AID, KU70, KU86, DNA-PKCs and STAT6 [5]. In 2006, De Vos et al. reported that BHLHE41 was highly induced during PC differentiation, particularly at the plasmablast and PC stages. More recently, using an in vitro model of naive B cell differentiation, Santamaria et al. found that BHLHE40 is preferentially associated with non-differentiating CD23+ cells, whereas BHLHE41 is part of a terminal differentiation gene signature expressed in a subset of CD23- pre-plasmablast cells [80]. In addition, BHLHE41 appears in signatures of memory B cell (MBC) differentiation and activation [81, 82]. It is expressed in memory B cells (MBCs) that are poised for activation [83], as well as in more mature quiescent MBCs [84]. In addition, it might regulate the pre-MBC cell fate [85].

Furthermore, Kreslavsky et al. described a high expression of Bhlhe41 in B-1a murine cells. These long-lived, self-renewing innate-like B cells are localized in peritoneal and pleural cavities and contribute to the first-line defense against pathogens and self-antigens. Interestingly, during murine B cell development, Bhlhe41 is weakly expressed in PC, compared with B-1a cells. In Bhlhe41-/- mice, B cell development is unaffected, but the number of B-1a cells is significantly reduced. In these cells, Bhlhe41 acts as a transcriptional repressor, targeting cell cycle regulators, restricting B-1a cell proliferation and affecting their self-renewal. Therefore, Bhlhe41 is considered essential for B-1a cell development and functionality [86].

During B-1a differentiation, Bhlhe41 expression is under the control of ARID3A, a member of the ARID (AT-rich interaction domain) superfamily of DNA binding proteins involved in large chromatin-modulating complexes, with major roles in embryogenesis [87].

Upon activation after antigen recognition, naive CD4+ T cells can differentiate into distinct effector subsets, including T helper 2 (Th2) cells. These T lymphocytes secrete interleukin (IL)-4, IL-5 and IL-13, which play a significant role in the defense against parasites, allergic diseases and immunoglobulin production by B cells [88]. The differentiation into Th2 cells is tightly regulated and IL-2 has a key role in this process [89]. BHLHE41 is highly and selectively expressed in Th2 cells among all T helper cell subsets [24]. Its induction is particularly high during the late phase of Th2 cell differentiation, and seems to require IL-4, which activates STAT6, which in turn induces GATA3 expression [90]. In addition, BHLHE41 can induce the upregulation of CD25, the alpha subunit of the IL-2 receptor, resulting in hyper-responsiveness to IL-2 stimulation [91].

2.6 BHLHE41 in cancer

According to the role of BHLHE41 as a transcriptional repressor and its interactions with epigenetic enzymes participating in normal cell differentiation, deregulation of BHLHE41 may contribute to tumorigenesis [92]. Epigenetic modifications, miRNA, epitranscriptomic regulation and post translational modification may regulate BHLHE41 expression [93]. Furthermore, since BHLHE41 is involved in DNA damage response and of immune cell differentiation, defects may contribute to DNA damage response abnormalities and immune cell dysfunction promoting tumor formation [94].

Several studies have shown that, in solid tumors, BHLHE41 is involved in the regulation of cell proliferation, apoptosis, hypoxia response, invasion and metastasis. However, depending on the tumor type, BHLHE41 acts as an oncogene or a tumor suppressor (Fig. 5).

Fig. 5
figure 5

Different BHLHE41 roles in cancer development/progression according to the tumor type. Depending on the tumor type, BHLHE41 exhibits either protumoral or antitumoral properties, affecting proliferation, apoptosis, invasiveness or metastatic potential. Created with BioRender.com

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2.6.1 Endometrial carcinoma

In solid tumors, cancer cells are often exposed to hypoxia that can modulate the expression of various genes and contributes to aggressive cancer phenotypes [95]. Yunokawa et al. described in endometrial carcinoma cell lines cultured in hypoxic conditions an increase in the expression levels of HIF and its target genes, including BHLHE41. In addition, they observed that BHLHE41 expression was higher in complex atypical endometrial hyperplasia and endometrial carcinoma samples, compared with normal endometrium, suggesting a contribution of this TF to carcinogenesis [96]. However, other findings are in favor of BHLHE41 anti-tumor activity. Specifically, in the human endometrial carcinoma cell lines Ishiwaka and HEC-1B, overexpression of BHLHE41 inhibited their migration, invasion, and metastatic potential by attenuating NOTCH1 signaling [97]. Another study reported that BHLHE41 expression was significantly lower in endometrial carcinoma than in normal endometrium samples. In addition, BHLHE41 expression was inversely correlated with both myometrial invasion and lymph node invasion. In agreement, BHLHE41 expression in endometrial carcinoma samples progressively decreased with the increasing stage and histological grade. In addition, BHLHE41 upregulation inhibited tumor angiogenesis by decreasing HIF-1 alpha, leading to tumor growth inhibition [98]. Through the association with SP1, BHLHE41 might suppress the expression of TWIST1, a gene involved in Epithelial to Mesenchymal Transition (EMT) [99]. Of note, MIR130 microRNA family members can suppress the expression of BHLHE41 and BHLHE40 and promote EMT and tumor cell invasion in the HHUA endometrial cancer cell line [100].

2.6.2 Breast cancer

Several studies performed in breast cancer cell lines suggest that BHLHE41 has anti-apoptotic effects. Liu et al. observed that BHLHE41 knock-down (KO) in the human MCF-7 cell line enhanced apoptosis, affecting FAS, MYC, CASP8 (caspase 8), PARP (poly (ADP-ribose) polymerase) and BAX gene expression [101]. Similarly, Wu et al. described an increase in cleaved PARP in this cell line after BHLHE41 deletion [102]. BHLHE41 might also suppress the cell invasion potential of MCF-7 and MDA-MB-231 cells [103]. Montagner et al. showed that in MDA-231 cells, BHLHE41 reduces HIF activity and limits the effects of hypoxia, thus controlling the migratory and invasive properties. This effect is based on BHLHE41 binding to HIF-1 alpha, leading to its proteasomal degradation [32]. Conversely, Wu et al. found that BHLHE41 contributes to hypoxia-induced MCF-7 cell proliferation [104]. Bexarotene, a selective RXR agonist, used as chemopreventive agent, represses CCDN1 (cyclin D1) transcription, leading to cell cycle progression arrest. In MCF-7 cells, BHLHE41, but not BHLHE40, was induced by incubation with bexarotene. Subsequently, BHLHE41 bound the CCDN1 promoter and recruited HDAC1, resulting in the repression of CCDN1 transcription [105]. Indeed, tumors appeared earlier and were significantly larger in mice xenografted with MCF-7 cells in which BHLHE41 expression was inhibited by siRNAs compared to control siRNA. Interestingly, BHLHE41 was found in a breast cancer dormancy gene signature based on expression profile studies performed in dormant breast cancer cells [106]. In a large cohort of patients with triple-negative breast cancer, a low BHLHE41 expression level was correlated with a higher probability of metastasis development and reduced survival. It was also associated with a gene expression signature linked to TGF-beta activity, p53 mutations, and high HIF activity [32]. Fang et al. observed a lower BHLHE41 expression in primary breast cancer samples compared with normal tissues. They also found that a lower BHLHE41 expression level was correlated with a more aggressive phenotype and worse overall survival [107].

2.6.3 Oral cancers

Bhawal et al. observed that BHLHE41 gene expression increased after BHLHE40 silencing in the human tongue squamous cell carcinoma HSC-3 cell line. In turn, BHLHE41 inactivation enhances BHLHE40 expression. In addition, siRNA-mediated BHLHE41 silencing decreased the cyclin D1 level [16], a major regulator of the cell cycle G1/S transition [108]. Conversely, BHLHE41 overexpression in the HSC-3 cell line resulted in the inhibition of the pro-apoptotic factor BIM and of cisplatin-induced apoptosis, suggesting an anti-apoptotic effect [109]. Similar results were obtained in TE-11 cells (human esophageal squamous cell carcinoma) [110].

Salivary adenoid cystic carcinoma (SACC) is a cancer characterized by a substantial risk of recurrence and metastasis. It was observed that BHLHE41 might contribute to tumor cell dormancy in the SACC-83 cell line and primary SACC cells [111].

2.6.4 Gastric cancer

In a series of 49 human gastric cancer samples evaluated by immunohistochemistry, Li et al. observed a lower BHLHE41 expression compared with adjacent non-tumoral tissues. Moreover, this expression level was negatively correlated with clinical data, including lymph node invasion, TNM staging (Tumor, lymph Node, Metastasis) and survival. In vitro, the overexpression of BHLHE41 in the gastric cancer cell lines MGC803 and MKN-45 inhibited cell proliferation and increased apoptosis [112]. BHLHE41 also increased the sensitivity of gastric cancer cells to 5-fluorouracil through STAT5A inhibition [113]. In patient-derived xenograft (PDX) mouse models, BHLHE41 suppressed tumor growth and metastasis of human gastric cancer cells [112].

2.6.5 Colon cancer

In HCT116 and Lovo colon cancer cell lines, BHLHE41 overexpression decreased cell viability, migration and invasion. It also blocked the cell cycle and induced apoptosis. In mice xenografted with BHLHE41-overexpressing HCT116 cells, tumor weight was decreased compared to control cells. Of note, BHLHE41 reduced the level of HIF-1 alpha and EMT-related proteins in tumor tissues, suggesting that a high BHLHE41 expression attenuates the malignant behavior of colon cancer cells [114].

2.6.6 Pancreatic cancer

In BxPC-3 human pancreatic cancer cell line, TGF-beta increased the expression of BHLHE41, resulting in impaired cancer cell migration and invasive properties. BHLHE41 could limit pancreatic cancer progression by inhibiting the transcriptional repressor SNAIL family transcriptional repressor 2 (SNAI2, also known as SLUG) and BHLHE40 expression [115].

In a pancreatic ductal adenocarcinoma cell line, the expression of MLH1, a gene encoding a protein member of the DNA mismatch repair system, was suppressed by BHLHE41 following its induction by GLI1, a zinc finger TF that inhibits the activity of the mismatch repair system in these cells [23], this finding linking BHLHE41 to the DNA damage response.

2.6.7 Thyroid cancer

At mRNA and protein levels, BHLHE41 was significantly downregulated in thyroid cancer samples compared with normal tissues. In addition, in thyroid cancer cell lines, the overexpression of BHLHE41 demonstrated inhibitory effects on their viability, migration and invasive properties, concurrently reducing the expression of HIF-1 alpha [14].

2.6.8 Lung cancer

In lung adenocarcinoma, BHLHE41 expression was downregulated compared with normal lung tissue [116]. Moreover, a higher BHLHE41 expression was associated with a favorable prognosis [117]. In the A549, NCI-H520 and NCI-H596 lung cancer cell lines, colony formation was inhibited by BHLHE41 overexpression, mainly through the downregulation of CCDN1, leading to the inhibition of cell proliferation in NCI-H520 cells [116]. BHLHE41 also enhanced autophagy in the A549 and H2030 adenocarcinoma cell lines [117].

2.6.9 Osteosarcoma

While BHLHE41 has been reported to promote HIF-1 alpha degradation in several cancer models, it exhibits a contrasting role by stabilizing this factor in the osteosarcoma cell lines U2OS, MNNG and 143B. Elevated levels of both BHLHE41 and HIF-1 alpha in osteosarcoma cells have been associated with heightened invasiveness and metastatic potential. In addition, the activation of HIF-1 alpha upregulated the expression of BHLHE41, inducing a feedback loop in these cells [118]. It is noteworthy that, in osteosarcoma cells, BHLHE41 expression was negatively modulated by microRNA-138, which also promoted cell apoptosis [119].

2.6.10 Prostate cancer

In the PC-3 human prostate cancer cell line, Liu et al. observed opposite effects of BHLHE41 and BHLHE40. In the presence of TGF-beta, a pivotal factor for epithelial-mesenchymal transition (EMT) induction, BHLHE41 expression decreased, whereas BHLHE40 expression increased. Furthermore, BHLHE40 enhanced migratory capacities, whereas BHLHE41 impaired them in the PC-3 cell line. BHLHE41 and BHLHE40 also differentially regulated the expression of EMT-related factors in PC-3 cells [120]. Notably, BHLHE41 inhibited paclitaxel-induced apoptosis of human prostate cancer cells [121].

2.6.11 Renal cell carcinoma

Renal cell carcinoma (RCC) is one of the most common urologic cancer types, and clear cell RCC is the main subtype. In a series of 50 clear-cell RCC samples, Shen et al. observed that BHLHE41 was highly expressed in 94% of tumor tissue samples compared to normal adjacent tissues. In vitro, BHLHE41 gene silencing in A498 and in CAKI-1 RCC cell lines impaired cell proliferation and migration, indicating that it might act as a tumor-promoting factor in RCC [122]. However, other studies showed that BHLHE41 expression decreased during RCC stage progression [123]. A study reported that BHLHE41 promoted RCC cell growth without HIF1 expression [124]. However, there is no reported link between BHLHE41 expression and overall survival in patients with RCC using TCGA database investigations [125].

2.6.12 Glioblastoma

In the U87 malignant glioma cell line and U251 glioblastoma cell line, BHLHE41 overexpression promoted cell proliferation through the ERK/CCDN1 pathway and colony formation. Its deletion had the opposite effect in these tumor cells [126].

2.6.13 Expression and role of BHLHE41 in hematological malignancies

2.6.13.1 Acute Myeloid Leukemias

The KMT2A gene (previously known as MLL for Mixed Lineage Leukemia) is located on chromosome 11q23 and encodes a large histone methyltransferase. In acute leukemia, the 11q23 translocation, in which a region of KMT2A is fused with a partner gene, is one of the most common chromosomal abnormalities. Fusion with AF6 results in the fusion protein KMT2A-AF6 that can recruit elongation assisting protein (EAP) and disruptor of telomeric silencing 1-like (DOT1L, a histone methyltransferase that methylates lysine-79 of histone H3) to activate the transcriptional elongation of target genes [127,128,129]. Interestingly, Numata et al. found that BHLHE41 was overexpressed in KMT2A::AF6 acute myeloid leukemia (AML) cells, and that BHLHE41 was a direct transcriptional target of KMT2A-AF6. They did not observe BHLHE41 overexpression in other AML subtypes and in normal CD34+ bone marrow cells. In addition, PDX human BHLHE41-deleted KMT2A::AF6 AML cells showed a prolonged survival compared to control condition. This effect was linked to increased apoptosis, reduced growth and decreased colony formation of blast cells in the absence of BHLHE41 expression. Of note, BHLHE41 deletion did not affect normal hematopoiesis. Therefore, BHLHE41 is described as crucial to maintain clonogenic growth and to prevent apoptosis in KMT2A::AF6 AML cells [130].

2.6.13.2 Plasma cell disorders

Multiple myeloma is characterized by accumulation of malignant plasma cells in the bone marrow compartment. A high BHLHE41 expression was observed in malignant plasma cells [131]. In addition, in a Korean cohort of 67 patients with newly diagnosed multiple myeloma, BHLHE41 somatic mutations were associated with poor outcome [132]. Similarly, BHLHE41 was found upregulated in Waldenström macroglobulinemia samples [133], Waldenström macroglobulinemia being a rare indolent B-cell lymphoma with bone marrow infiltration by lymphoplasmacytic tumoral cells and IgM monoclonal gammopathy.

2.6.13.3 BHLHE41 is an essential gene in hematological malignancies

We used public datasets of RNAi viability assay screening (Dependency Map data, Broad Institute, www.depmap.org) [134] to determine whether BHLHE41 is an essential gene in several cancers. We observed that BHLHE41 was associated with a significantly lower DEMETER2 score (a measure of gene dependency) in Ewing sarcoma (P=1E-9, n=10), multiple myeloma (P=1.6E-5, n=16), non-Hodgkin lymphoma (P=6.1E-6, n=26) and AML (P=6E-5, n=22) cell lines (Fig. 6A). Furthermore, using the Genomicscape webtool (http://www.genomicscape.com) and publicly available cohorts of patients with hematological malignancies, we identified that a high expression of BHLHE41 is associated with a poor survival in patients with AML and MM (Fig. 6B), supporting the results of the RNAi screening (Fig. 6A).

Given that BHLHE41 could govern the expression of genes required for the development of germinal center B cells and plasma cells, its deregulation could be involved in the biology of non-Hodgkin lymphoma and multiple myeloma. In mice, loss of BHLHE41 impedes plasma cell differentiation at an activated B cell stage, regulating IGH and key plasma cell TFs, including PRDM1/BLIMP1 and XBP1. A study reported that BHLHE41 is required for the induction and maintenance of open chromatin regions [135], suggesting a potential role in the pathophysiology of malignant cells. A more recent study reported that, in Ewing sarcoma, ID2, a member of the ID1-4 family which binds and sequesters bHLH TFs, is a critical regulator of developmental-related genes and tumor growth in vitro and in vivo [136]. Altogether, these findings underscore the need for further studies to decipher the precise role of BHLHE41 in cancer, and its interest as a potential therapeutic target.

Fig. 6
figure 6

A. BHLHE41 is an essential gene in several cancer cell types. Boxplots showing the dependency scores calculated using data from the Cancer Dependency Map project (https://depmap.org). RNAi-based viability assays in Ewing sarcoma (n=10), multiple myeloma (n = 16), non-Hodgkin lymphoma (n = 26) and acute myeloid leukemia (n = 22) cell lines. DEMETER2 scores were calculated using data from RNAi-based screens. The mention “all” refers to the 974 cell lines included in the projects Achilles, DRIVE and Marcotte. A DEMETER score = 0 indicates that the gene is not essential. A lower score indicates that the gene is more likely to be essential in a given cell line. The figure shows the scores for the BHLHE41 gene in the indicated samples. B. A high expression of BHLHE41 is associated with a poor survival in patients with AML and MM. The link between BHLHE41 and outcome in hematological malignancies was investigated using GenomicScape webtool (http://www.genomicscape.com/microarray/survival.php). High BHLHE41 expression (red) is associated with a significant poor overall survival in a cohort of newly-diagnosed cytogenetically normal AML (GSE12417—Gene expression omnibus, n=163) and a cohort of newly diagnosed MM patients (HM cohort, n=206, Array Express public database (E-MTAB372)) compared to patients with low BHLHE41 expression (blue)

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3 Conclusion

The complexity of BHLHE41 activity as a transcriptional repressor relies on the multitude of pathways that regulate its expression and on the diversity of interaction partners. Its activity contributes to the regulation of basic physiological mechanisms including transcriptional regulation, DNA damage response and immune cell differentiation. Defects in DNA damage response and immune response play a major role in cancer development. Recent investigations underlined a BHLHE41 overexpression in several cancers including ovarian serous adenocarcinoma, stomach adenocarcinoma and thyroid cancer [125]. Furthermore, genetic changes involving BHLHE41 with missense mutation, amplification, gene fusion and deletion have been identified in solid cancer, suggesting that BHLHE41 deregulations may be associated with processes related to cancer development [125]. Accordingly, BHLHE41 was identified as a factor involved in many solid cancer types and in some hematological malignancies. Its contribution to tumor mechanisms, as a pro-tumoral factor or as an anti-tumoral factor, is not fully characterized in the different cancer types and require further investigations. However, RNAi screening uncovers BHLHE41 as an essential gene in different cancers including Ewing sarcoma, multiple myeloma, Hodgkin lymphoma and acute myeloid leukemia. Since BHLHE41 could govern the expression of genes required for the development of germinal center B cells and plasma cells, its deregulation could play a role in the pathophysiology of non-Hodgkin lymphoma and multiple myeloma. Using publicly available cohorts of patients, a high expression of BHLHE41 is associated with a poor outcome in multiple myeloma and acute myeloid leukemia hematological malignancies, supporting the results of RNAi screening and suggesting it could represent a potential therapeutic target. The biological functions of BHLHE41 and their interactions with other proteins are complex in normal and malignant cells. BHLHE41 might be a new candidate biomarker in cancer, and possibly a new therapeutic target.