bHLH transcription factors and their role during neuronal

Transcription factors of the basic helix-loop-helix (bHLH) transcription factors are involved in many developmental processes including muscle development (Weintraub, 1993), mesodermal determination (Burgess et al., 1995), skeletal development (Cserjesi et al., 1995) and neural development (Lee, 1997;

Bertrand et al., 2002). Members of the bHLH transcription factor family can be categorized into three classes (Murre et al., 1989). Class A bHLH proteins form homo or heterodimers and are ubiquitously expressed genes such as E12, E47 or daughterless. Class B bHLH transcription factors form heterodimers with class A bHLH proteins and show a tissue specific expression pattern, while class C bHLH transcription factors do not interact with either of the class A or class B bHLH transcription factors (Murre et al., 1989). The formed homo- or heterodimers bind to an E-box motif (CANNTG) on the DNA, usually through the basic region, while the HLH domain is involved in the dimerization (Murre et al., 1989). The activity and DNA target choice of bHLH transcription factors can be regulated via the interaction with co-factors (Ma et al., 2008; Mattar et al., 2008; Rodolosse et al., 2009; Li et al., 2011).

Neuronal differentiation is initiated by the expression of proneural genes, which drive the determination and differentiation of progenitor cells to a neuronal cell fate (Bertrand et al., 2002). The proneural genes are mainly class B bHLH transcription factors (Bertrand et al., 2002) belonging to the drosophila archaete-scute and atonal family. Proneural bHLH transcription factors can be categorized in two distinct subgroups according to their function during neuronal differentiation and based on the timing of their expression: neuronal determination genes and neuronal differentiation genes (Lee, 1997; Farah et al., 2000; Bertrand et al., 2002). Neuronal determination genes such as neurog2 or ascl1 are expressed early during neurogenesis in the mitotically active progenitors, which in the neural tube, are found in the inner ventricular zone (Lee, 1997; Bertrand et al., 2002). Neuronal differentiation genes such as neurod1 are expressed slightly later compared to the determination genes and are positively regulated by the proneural determination genes (Lee, 1997;

Bertrand et al., 2002). During neuronal differentiation, bHLH transcription are

involved in the regulation of several processes (Fig.1.3), which in the following will be demonstrated by the multiple activities of the proneural bHLH transcription factor Neurog2.

Figure 1.2: Overview over Archaete-Scute and Atonal family members of proneural bHLH transcription factors

Scheme showing the relationship between different families of bHLH proteins involved in neuronal differentiation. bHLH proteins are mainly divided in ubiquitously expressed E-proteins, which are orthologs to drosophila Daughterless and the proneural families that are homologue to the drosophila Atonal and Achaete-Scute.

The Atonal family can be further divided in several subfamilies, which includes the Neurogenin family, the Olig family and the Atonal family. Blue shading highlights the invertebrate family members, while red highlights vertebrate family members.

(Modified from Bertrand et al. (2002).

Neurog2 is a member Neurogenin family of the atonal-like bHLH transcription factors. This family consists of three distinct members (Neurog1, Neurog2 and Neurog3) (Sommer et al., 1996; Nieber et al., 2009), all of which are expressed during gastrulation in X. laevis. (Nieber et al., 2009). Transcripts of neurog1 and neurog2 are present in the three longitudinal domains of primary neurogenesis in open neural plate stages, with the expression of neurog2

being broader than neurog1 (Ma et al., 1996; Nieber et al., 2009). Neurog3 expression, in comparison, is restricted to the medial domain (Nieber et al., 2009).

Among the members of the Neurogenin family, the function of neurog2 is best characterized in X. laevis. Overexpression of neurog2 is sufficient to drive neuronal differentiation of the non-neural ectoderm (Ma et al., 1996).

Furthermore, Neurog2 induces the expression of other later expressed proneural bHLH transcription factors such as neurod1, which on the other hand, is not capable of inducing neurog2 (Ma et al., 1996). Other early expressed bHLH transcription factors such as Ascl1, which activates neurod1 expression (Cau et al., 1997), while Neurod1 levels do not affect ascl1 expression (Gao et al., 2009).

It has been demonstrated that in mammalian cell lines, Neurog2 can act as a pioneer transcription factor, initiating target gene transcription on nucleosome bound regions (Chen and Dent, 2014). Often the binding of a pioneer transcription factor will initiate events that lead to an opening of the chromatin and the recruitment of active histone marks stabilizing target gene transcription (Zaret and Carroll, 2011; Zaret and Mango, 2016). Neurog2, for example, binds to regions of non-accessible chromatin (Smith et al., 2016) and promotes the removal of repressive histone marks and the addition of active histone marks at the neurod1 and tubb2b promoters by interacting with the H3 lysine 9 demethylase Kdm3a (Lin et al., 2017). Several other proneural bHLH transcription factors such as Ascl1 or Neurod1 have been demonstrated to act as pioneer transcription factors in murine ES cells and fibroblasts (Wapinski et al., 2013; Pataskar et al., 2016), indicating a general role of proneural bHLH transcription factors in initiating and stabilizing the onset of neuronal gene expression.

Figure 1.3: Multiple roles of the proneural bHLH transcription factor Neurog2 during neurogenesis. bHLH transcription factors like Neurog2 are involved during many processed in regulating neurogenesis. Neurog2, for example is activating a cascade of proneural genes driving neuronal determination and differentiation (Bellefroid et al., 1996; Ma et al., 1996). It is furthermore involved in activating cell cycle regulators and Notch ligand dll1 and thereby controls the cell cycle length and lateral inhibition (de la Calle-Mustienes et al., 2002; Kiyota and Kinoshita, 2002;

Souopgui et al., 2002; Bray, 2006). In addition, Neurog2 activates genes involved in the specification of distinct neuronal subtypes (Bertrand et al., 2002).

The proneural bHLH proteins lead to the transcription of other bHLH transcription factors that are involved in the differentiation to neuronal cells, such as the HLH transcription factors ebf2 (Dubois et al., 1998) and ebf3 (Pozzoli et al., 2001), the atonal family members atoh1 (Kim et al., 1997) and atoh7 (Kanekar et al., 1997), as well as the neuronal differentiation factors neurod1 (Ma et al., 1996) and neurod4 (Perron et al., 1999). For example, the overexpression of the bHLH transcription factor Atoh1 is sufficient to program ectodermal cells to express neuronal differentiation markers in X. laevis without inducing early neuronal differentiation marker genes such as neurog2 or neurod1 (Kim et al., 1997).

Besides driving neuronal differentiation, proneural bHLH transcription factors also influence the length of the cell cycle. Neurog2 indirectly represses cyclins D, E1 and E2 involved in G1-phase progression and G/S-phase transit in chick embryos (Lacomme et al., 2012; Pfeuty, 2015). Furthermore, Neurog2 induces the cell cycle inhibitor genes gadd45-γ and pak3 in X. laevis, leading to cell cycle withdrawal (de la Calle-Mustienes et al., 2002; Souopgui et al., 2002).

The involvement of proneural bHLH proteins in regulating the cell cycle is further supported by studies in mouse, Ascl1 activates several cell cycle regulators that are essential for G1/S transition (e2f1, cdk1, cdk2, and skp2) or entry into mitosis (cdk1 and cdc25b) (Castro et al., 2006; Castro et al., 2011),

but also other factors associated with cell cycle arrest (fbxw7, gadd45g, ccng2h, and prmt2), indicating that Ascl1 has a role in regulating progenitor proliferation as well as in cell cycle exit of progenitor cells (Castro et al., 2011).

Furthermore, many bHLH transcription factors are involved in the regulation of lateral inhibition mediated by the Notch pathway, which controls the number of cells that undergo neuronal differentiation (Lewis, 1998; Bray, 2006;

Kageyama et al., 2008; Ahnfelt-Rønne et al., 2012).