Genomics of the OLIG family of a bHLH transcription factor associated with oligo dendrogenesis

The glial cell neoplasms are not fully classified by using cellular morphology. However, this is possible using known molecular markers in glial development. Oligo-dendrocyte lineage gene induces differentiation of neural progenitors and putative immature progenitor cells of the adult central nervous system. These oligo-dendrocyte lineage genes OLIG1 and OLIG2 encode basic helix-loop-helix transcription factors. The murine bHLH transcription factors found in chromosome 21 are essential for oligo-dendrocyte development. Moreover, OLIG3 of the OLIG family is known to be linked with the brain and spinal cord development. Therefore, it is of interest to analyse oligodendrocyte lineage genes in the OLIG family of bHLH domain for the understanding of oligo-dendrogenesis in eukaryotes. Several bHLH domain linked basic-helix-loop-helix transcription factors in Homo sapiens and Mus musculus from this analysis are reported. Thus, genomics data analysis of OLIG family of bHLH transcription factors help explain observed similarity and differences within the molecular evolutionary context and hence assess the functional significance of the distinct genetic blueprints


Background:
The primary tumors of the human brain are thought to be glial cell origin. The glial cell neoplasm cannot be fully classified by cellular morphology or conventional markers for astrocyte or oligodendrocyte. The diagnostic potential OLIG markers identified oligodendroglial tumors. The oligodendrocyte lineage transcription factors originally identified in rodent encoded bHLH transcription factors. In a rodent central nervous system, they are exclusively expressed in oligodendrocyte. OLIG1 promote the formation of chondroitin sulfate proteoglycan-positive glial progenitors. It is suggested that novel molecular markers are found among factors that have roles in glial development. The markers for different types of cells were known and it is stated that Albert Einstein's brain contains significantly more glia than normal brains in the left angular gyrus [1]. The human OLIG1 and OLIG2 express strongly in oligodendroglioma with contrasting low expression in the astrocytoma. These studies show that neoplastic cells of oligodendroglioma resemble oligodendrocyte derived from cells of this lineage. The OLIG1 gene mapped to chromosome 21q22.11 base on sequence alignment in genomic data has roles in the development and maturation of oligodendrocytes especially within the brain. The OLIG1 have an essential role in oligodendrocyte differentiation and consequent remyelination. OLIG1 exhibited failure of remyelination induces lesions and contrasting extensive remyelination of normal controls. A genetic requirement for OLIG1 is repairing the types of lesions occurring in patients with multiple sclerosis [2][3][4][5][6][7]. OLIG2 is essential for the oligodendrocyte and motoneuron development in the spinal cord. OLIG2 encoded BHLHB1 deduced 357 amino acids region has bHLH domain characteristic of transcriptional regulators. OLIG2 positive cells in the late fetal telencephalon primarily develop into astrocyte. OLIG2 expression was upregulated in neoplastic oligodendrocyte yet not in neoplastic astrocyte in brain tumor cells inferring its specific The bHLH (basic helix-loop-helix), one of the largest transcription factors containing protein structural motif is characterized by two α-helices connected by a loop. The bHLH domains contain helix motif to bind specific DNA. The bHLH TFs is dimeric with specific DNA binding functions. The basic helix-loop-helix is conserved and characterizes the largest families of transcription factors in eukaryotes. The bHLH transcription factors are made of 40-50 amino acids with two amphipathic alpha helices separated by a linker region. The peptide sequence in the bHLH domain has specific motifs for binding to DNA sequence [21][22][23][24][25]. Thus, the transcription factors are the key regulatory proteins to bind specific DNA sequences

Materials and Methods: Primary query sequence database and tools:
Primary query sequence was retrieved from different databases (UniProt, EMBL, GenBank and NCBI). The web based application SMART was used for the identification of specific domain in a given sequence. Pfam was used for retrieving protein family information. PROSITE was used for identification of the domain, family, and functional sites as well as associated pattern and profile. PROCHECK was used to examine the stereo-chemical quality of the primary peptide sequence. The genome sequences were downloaded from genomic data in different specialized databases (NCBI, Ensemble and TIGR).

Standalone tools and GO annotation:
Domain specific profile search was completed using HMMER. HMMER is a statistical algorithm, making use of multiple sequence alignment (MSA) for specific domains in profile search. It uses a probabilistic model called hidden Markov model. Standalone BLAST was used for searching homologous gene in Homo sapiens and Mus musculus. The BLAST2GO was used for the accurate retrieval of specific sequences in the genome. BLAST2GO is a tool for high-throughput gene annotation of the novel sequence data. Functional information was retrieved using Gene Ontology (GO) annotation, which is a controlled vocabulary of the functional attributes.

Domain, motif, and phylogeny:
Multiple sequence alignment (MSA) methods were used to calculate the best match for homologous sequences for identities, similarities, and differences between them. MSA of multiple sequence hits was carried out with a web-based tool MultAlin for the identification of conserved bHLH domain. The development of the molecular evolutionary relationship between Homo sapiens and Mus musculus was completed using MEGA7 (a tool for phylogenetic tree using Neighbor-Joining method). The MEME suite is a computational tool for analysis of sequence motifs and thus specific motifs in a given query sequence was retrieved using the MEME web-based Tool.

Gene expression and chromosome location:
Gene expression analysis was carried out using the GENEVESTIGATOR tool, which is a high-performance search engine for gene expression of different biological events. Chromosome location was retrieved using gene card (a database of the human genes provides genomic, proteomic, transcriptomic genetic and functional information on all known and predicted human genes).

Results:
In this study, a survey of OLIG family of the bHLH transcription factor in Homo sapiens and Mus musculus was completed. High confidence bHLH transcription factor data in both the organisms for the understanding of the development of multi cellular organisms are created.

Sequence data analysis:
The primary coding region of the gene and its 972 nucleotide translated 323 residues long peptide sequence with 55 residues long binding region to specific DNA sequence is known. The conserved bHLH domain with 55 amino acids is characterized by two αhelices connected by the loop (Figure 1). The flexibility of the loop allows dimerization, folding and packing against another helix, both amphipathic alpha helices separated by a linker region.

Standalone tools:
The HMMER results gave a total of 59 and 57 bHLH domains. The standalone BLAST results gave a total of 28 and 22 homologous sequences in Homo sapiens and Mus musculus, respectively. The transcription factor data analysis suggested 7 OLIG genes as well as 57, 58 bHLH domains in Homo sapiens and Mus musculus, respectively ( Table 1). The OLIG family of the bHLH transcription factor is essential for the functional redundancy of a specific transcription factor in the genome.

GO annotation:
The gene ontology annotation summary identified the bHLH domain in both the organism's genome (Table: 2).

Multiple sequence alignment:
Multiple sequence alignment (MSA) result showed conserved domain having high consensus with extended basic helix-loophelix domain (Figure 2) indicating specific motifs (Figure 3).

Phylogeny:
The phylogenetic tree demonstrated the molecular evolutionary relationship between OLIG1, OLIG2, and OLIG3 in Homo sapiens and Mus musculus. Some clades define bHLH domain encoding genes in both human and mouse (Figure 4).

Gene expression:
Gene expression analysis of OLIG2 shows that it is highly expressed in neoplasm of the eye, brain, and central nervous system ( Figure 5).

Chromosome location:
The chromosome localization study shows that OLIG2 is located at band 21q22.11 ( Figure 6) in the human. The family-wise classification is essential for a better understanding of the organism's gene contents in this context. Thus, the genome wide analysis identified the genes OLIG1, OLIG2 and OLIG3 with conserved bHLH domain.

Discussion:
The salient features of genes OLIG1, OLIG2 and OLIG3 having conserved bHLH domain that are associated with brain tumor are discussed. The astrocytoma, oligodendroglioma, and oligoastrocytoma collectively referred as diffuse glioma is a common primary brain tumor. These data classified similarity to astrocyte and oligodendrocyte. The lineage markers represent close to morphologic classification. The murine bHLH transcription factors express neural progenitor and oligodendroglia essential for oligodendrocyte development. OLIG2 closely restricted to the normal oligodendroglia in the human brain. OLIG2 is highly expressed in diffuse glioma. The specific group of oligodendroglioma is susceptible to adjuvant therapy and it is important to elucidate the biological characteristic of tumors. High expression of OLIG1 and OLIG2 is also seen in anaplastic oligodendroglioma and astrocytoma. The oligodendroglial lineage associated markers OLIG1 and OLIG2 are expressed in different glioma. The expression of OLIG2 enables oligodendroglioma and distinguishes glioblastoma from other astrocytic glial tumors. OLIG2 markers of diffuse glioma are expressed in astrocytoma preclude glioma. OLIG2 participate in transcriptional system governing cell fate specification in the ventral spinal cord. Selective interaction of OLIG2 directed motor neuron fate further promoting oligodendrocyte production. The oligodendroglial marker OLIG2 is universally expressed in diffuse glioma and lower in other brain tumors. OLIG1 and OLIG2 help to define the real spectrum of oligodendroglial tumors, which may include a wide variety of tumors with different prognoses. The combinatorial interaction between pro-neural transcription factor NEUROG2 involved in the genesis of motor neuron and oligodendrocyte is seen.
Genetic markers and particularly the loss of 1p and 19q chromosomes have been predicted for prognosis and response to treatment. These emerging techniques will be very helpful in the clinical practice for refining classification and as a therapeutic indication of the oligodendroglial tumors [30][31][32][33][34][35]. OLIG3 is a third member of OLIG family of bHLH transcription factor coordinate specification of the dorsal neuron in the spinal cord. The dorsal horn neurons integrate and relay sensory information and arise during development in the dorsal spinal cord and alar plate. The class A and B neurons emerge in dorsal and ventral alar plate and dependence on roof plate signals for specification and settle in the deep superficial dorsal horn. The OLIG3 in progenitor cells generate 'class A' (dI1-dI3) neurons that is important in the development of neuronal cell types. OLIG3 mutant development of 'class A' neuron; dI1 neurons generally reduced the number, whereas dI2 and dI3 neurons are misspecified and assume the identity of class B neurons. Conversely, OLIG3 repress emergence of class B neurons in the spinal cord. OLIG3 was transiently expressed in lateral margin of sub-ventricular zones as three ventral clusters at the level of p3, p2, and p0 domain in the dorsal neural tube. OLIG3 is expressed in different types of progenitors in the embryonic central nervous system and disappear in course of development. OLIG3 was first detected in the dorsal neural tube from midbrain/hindbrain and spinal cord. Those results suggest that OLIG3 distinguishes major classes of progenitors in the dorsal spinal cord and determines a distinct specification program of 'class A' neurons [20,36,37]. In this study, information of OLIG family of the bHLH transcription factor in eukaryotes is discussed using data from available databases and published contents.

Conclusion:
The OLIG family of bHLH domain is associated with oligodendrogenesis in eukaryotes. Several bHLH domain linked basichelix-loop-helix transcription factors in Homo sapiens and Mus musculus from literature are discussed. Genomics data analyses of OLIG family of bHLH transcription factor is helpful to characterize gene contents to explain observed similarity and differences within the molecular evolutionary context and hence assess the functional significance of the distinct genetic blueprints.

Conflict of interest:
The author did not avail of any financial assistance from any source in undertaking the present study. The author declares that there is no conflict of interests regarding the publication of this article.