**1. Introduction**

The bHLH (basic Helix-Loop-Helix) proteins are the important family of transcription factors (TFs) present in all eukaryotes: from yeasts [1,2] and fungi [3] to plants [4] and metazoans [5–10]. All family members contain the HLH domain responsible for dimerization [11]. This domain is usually preceded by a stretch of basic residues which enable DNA binding [12]. The bHLH TFs recognize tissue-specific enhancers containing E-box sequences which regulate expression of a wide range of genes involved in cell differentiation and development [13].

Currently, a few independent classification systems of the bHLH proteins exists: evolutionary classification based on the phylogenetic studies of the bHLH proteins, which classify the bHLH family members into six A-F classes [7,8,14], and a new one based on the complete amino acid sequence analyses, classifying the bHLH proteins into six clades without assumptions about gene function [15]. Contrary to the previous methods, natural method of classification proposed by Murre [12], which divides the bHLH proteins into seven classes, is based on the presence of additional domains, expression patterns and performed transcriptional function [10]. For purposes of clarity, some attempts to revise

and systematize different classification systems were undertaken [16]. In this review we present classification of bHLH proteins according to Murre [12], with some short description of presented classes (Table 1).



Both class I (known as E proteins) and class II of the bHLH TFs do not possess domains additional to the bHLH. Contrary to the class I which is expressed in many tissues, the class II proteins expression is tissue specific. Members of the class II are dimerization partners for the class I transcription factors. Class III comprises proteins possessing Leucine-zipper (LZ) motif in addition to the bHLH. Important members of the class III are proteins belonging to the Myc subfamily, which regulate oncogenic transformation, apoptosis, and cellular differentiation. To class IV belong MAD and MAX which can dimerize with MYC and regulate its activity. Also, MAD/MAX are able to create homo- and heterodimers with each other. Although these TFs do not possess transcription activation domain (TAD), MAD/MAX dimers can influence the transcription in a differentiated way. Class V contains transcriptional inhibitors ID1-3 which are not able to bind DNA and act by the other bHLH proteins

sequestration. Interestingly, the fourth member of this class- ID4 function as inhibitor of ID1-3 [42]. Class VI comprise proteins containing additional Orange domain adjacent C-terminally to the bHLH domain (bHLH-O). Transcription factors from the described classes perform regulatory function in various developmental processes including cells differentiation and maintaining pluripotency. For this reason they are often linked to cancer development. Class VII comprise transcription factors which possess PAS (Period-Aryl hydrocarbon receptor nuclear translocator-Single minded) domain located C-terminally to the bHLH domain. PAS domain is crucial for the bHLH-PAS proteins specifity [43]. Structurally, the C-terminal PAS domain is often associated with PAC (C-terminal to PAS) motif [44,45]. bHLH-PAS transcription factors are responsible for sensing environmental signals like the presence of xenobiotics (AHR), hypoxia (HIF) or setting of circadian rhythms of organism (CLOCK, CYCLE, BMAL). The members of subclass II of bHLH-PAS TFs -ARNT proteins are general dimerization partners of the subclass I members.
