Sequence Characterization of Forebrain Embryonic Zinc Finger-like (FEZL) Gene in Indian Zebu (Bos indicus) Cattle and their Crossbreds

Mastitis is one of the most common diseases affecting dairy cattle causing huge economic losses to dairy farmers (Shook, 2006). Selective breeding of dairy cattle for reduced susceptibility/increased resistance to mastitis is difficult, as it is a polygenic trait with very low heritability. However, indirect selection based on somatic cell count and candidate gene markers can help to increase the efficiency of such breeding programs. Bovine forebrain embryonic zinc finger like (FEZL) gene is one of the candidate genes associated with resistance to mastitis in dairy cattle. Forebrain embryonic zinc finger-like (FEZL) factor is involved in transcriptional regulation of neuronal development (Hirata et al., 2004). Heyen et al., (1999) identified seven putative QTLs for SCS, including two on chromosome BTA22 that involve the region covering FEZL gene. It has already been established that there exists a crosstalk between neuronal development and immunity via downstream cytokine expression (Wu et al., 2001). Sugimoto et al., (2006, 2011, 2013) reported single glycine insertion into the glycine stretch of FEZL has large effect on its downstream pathway which makes the cows susceptible to mastitis. However, very little information is available on FEZL gene in Bos indicus cattle and their crossbreds. The present study was undertaken with the objectives of characterizing the promoter, 5' flanking and coding DNA sequence regions of FEZL gene in Sahiwal (B. indicus) and Karan Fries (B. indicus × B. taurus) cattle.

Sampling and DNA extraction
 
Blood samples were collected from one representative Sahiwal and Karan Fries cattle maintained at Cattle Farm, National Dairy Research Institute (NDRI), Karnal. About 10 ml of blood was collected in a sterile 0.5% EDTA (10 µl/ml of blood) coated vacutainers by jugular venipuncture method. The collected blood was stored in the refrigerator at 4^oC till further processing for extraction of DNA. DNA was isolated from blood using standard phenol-chloroform method as described in Sambrook and Russell (2001) with minor modifications. Quality of extracted DNA was checked by loading 2-5 µl DNA on ethidium bromide stained 0.8% agarose gel electrophoresis using 1X TAE as running buffer. The quality of DNA was estimated by comparing the bands with reference DNA and the quantity was determined using Nanodrop.
 
Primer designing, PCR amplification and sequencing
 
Sequence characterization of bovine FEZL gene was carried out by primer walking method. This method works by PCR amplification of whole gene in fragments that overlap with each other at 5' and 3' ends. FEZL gene was sequence characterized by synthesizing ten overlapping fragments to generate the complete sequence of about 3.7 kb covering all exons and 5' upstream regulatory and flanking regions.Ten sets of oligonucleotide primers were designed using PRIMER 3 (Untergasser et al., 2012) for generating overlapping PCR fragments of complete FEZL gene. The details of oligonucleotide primer sequences, their annealing temperature and size of the amplicon are presented in Table 1. Polymerase chain reaction was carried out in a final reaction volume of 20 µL, containing ~50ng of genomic DNA, 5 pmole of each primer, 200 µM of each dNTP, 2µL of 10X buffer with 1.5 mM MgCl₂ and one unit of Taq DNA polymerase (Bangalore Genei Pvt. Ltd., Bangalore India). Amplification was performed using programmable thermal cycler (PTC-200, MJ Research, USA) with an initial denaturation at 95^oC for 2.5 min followed by 35 cycles of 94^oC for 30s, respective annealing temperature for 30s and 72^oC for 1min, with a final extension for 5 min at 72^oC. The amplified PCR products were checked by running on 2% agarose gels in 1X TAE along with suitable standard size DNA marker. The amplified products were visualized, size estimated and documented using gel documentation system. The amplicons from each of the overlapping fragments were subjected to custom DNA sequencing from both ends (5¢ and 3¢ ends) using forward and reverse primers (M/S Chromous Biotech, India).
 

 
Sequence data analysis
 
Raw sequence data were edited using Chromas (Ver. 1.45, http://www.technelysium.com.au/chromas.html). The edited sequences were saved in EditSeq module of LASERGENE software (DNASTAR Inc, Madison, WI, USA) for subsequent analysis. The forward and reverse sequences from each of the fragments from FEZL gene were assembled together manually after multiple alignment with Bos taurus sequence template. Multiple alignments of different sequences were performed using Clustal W program of BIOEDIT and CLC Free Workbench. Sequences from different overlapping fragments were further assembled to derive the contiguous and complete sequence of FEZL gene. The structure of Bos indicus FEZL gene including its exon-intron junctions were predicted using GENSCAN web server available at http://genes.mit.edu/GENSCAN.html (Burge and Karlin, 1997).
       
The sequence homology among the coding DNA sequences of FEZL gene belonging to different species was estimated by Basic Local Alignment Search Tool Analysis (BLAST) at National Centre for Biotechnology Information (NCBI) website (http://blast.ncbi.nlm.nih.gov/ Blast.cgi). The phylogenetic analysis of FEZL gene sequences from various species was performed using CLC Free Workbench. Unweighted paired group method with arithmetic mean (UPGMA) algorithm was followed to derive the dendrogram with bootstrap re-sampling of 1000 subsets of sequence data under analysis. Various binding sites for putative transcription factors in the 5¢ flanking region were identified using TESS (Transcription Element Search Software) and MATCH (Kel et al., 2003). The transcription factor binding sites were predicted from the publicly available database of sequence matrices corresponding to different transcription factors. The option to minimize both error rates in predicting transcription factor binding sites were used for MATCH analysis. The factors with scores below 1.0 for core match and 0.9 for matrix match were not considered for subsequent analysis.

Forebrain embryonic zinc finger like protein is involved in the control and development of monoaminergic neurons and plays an important role in the neuronal development (Matsuo-Takasaki et al., 2000). Later, Wu et al., (2001) showed that there existed a crosstalk between neuronal development and immunity with the neuronal repellent ‘slit’ regulating both neuronal and leukocyte migration. Polymorphisms within coding DNA region of bovine FEZL gene has been reported to have significant association with somatic cell score in Holstein Friesian cattle (Sugimoto et al., 2006, 2011; Ali et al., 2019). Bovine FEZL gene has been mapped to chromosome 22 in cattle (UCSC Cow genome chr22:39720731-39723530). In the present study, the complete FEZL gene sequences of Sahiwal and Karan Fries cattle were generated with a total contig length of 3.772 kb and 3.789 kb respectively. This also included 0.75 kb of 5' flanking region covering the basal promoter complex of FEZL gene. The complete FEZL gene sequences generated in the present study were submitted to NCBI-GenBank and are available at accession numbers JX312831 and JX312832.
 
Structure of bovine FEZL gene
 
Annotation details of the sequence data generated with respect to FEZL gene in Sahiwal and Karan Fries cattle are presented in Table 2. The structure of FEZL gene was found to be conserved among Sahiwal and Karan Fries cattle without any variation in the conceptualized coding DNA regions. The coding DNA region of about 1.38 kb was observed to spread over four exonic regions. A total of eight nucleotide variations including three INDELS and five substitution mutations were observed among FEZL gene sequences of Bos taurus, Bos indicus (Sahiwal) and Bos taurus × Bos indicus (Karan Fries) cattle. Among the eight nucleotide variations, four mutations including three INDELS were found to be within exon 1, while two mutations were observed within each of intron 1 and intron 3 respectively (Table 3).
 

 

       
The three nucleotide INDEL mutation within exon1 region resulted in additional Glycine residue within a stretch of 12G (Glycine) region. This additional Glycine residue i.e. 13G stretch has been reported to be associated with higher somatic cell score as compared to 12G in case of Holstein Friesian cattle (Sugimoto et al., 2006, 2011, 2013; Ali et al., 2019). Association of FEZL allelic variant with higher somatic cell score and consequently susceptibility/resistance to mastitis in cattle is interesting. It would be further interesting to screen Sahiwal and Karan Fries cattle for 12G/13G polymorphism so as to estimate the frequency distribution of resistant/susceptible alleles in the Indian zebu and crossbred cattle populations. Another mutation, Thymidine-Cytidine (T-C) within exon region at position 544 from start codon was non-synonymous, resulting in change of amino acid from serine (S) to proline (P). This change corresponded to amino acid position 181 in Bos taurus cattle and position 182 in Karan Fries and Sahiwal cattle (Fig 1). This non-synonymous mutation was observed in both Karan Fries and Sahiwal cattle. Within intron 1, a G-A mutation was observed in Karan Fries cattle at position 1005 from start codon while a T-C mutation was observed at position 1154 in Karan Fries and Sahiwal cattle. Similarly, G-A substitution mutation was observed within intron 3 at positions 2048 and 2295 in Karan Fries and Sahiwal cattle.
 

 
Sequence homology and phylogenetic analysis of bovine FEZL gene
 
Basic alignment search tool analysis (BLAST) analysis was performed to understand the sequence homology of coding DNA region of bovine FEZL gene with that of other species. The percent identity of FEZL gene of Sahiwal and Karan Fries cattle was 99% with that of Bos taurus, 95% with that of dog, horse and pig, 94% with that of human, 93% with that of rabbit, 92% with that of marmoset, 89% with that of rat and 79% with that of chicken (Table 4). In order to understand the relationship between FEZL gene sequences of different species, phylogenetic analysis was performed on coding DNA sequences of FEZL gene using UPGMA algorithm (unweighted paired group method with arithmetic mean) (Fig 2). FEZL gene sequences of Karan Fries and Sahiwal cattle clustered together followed by that of Bos taurus sequence. Interestingly, sequences of marmoset and human clustered together along with rabbit while horse and dog FEZL gene sequences clustered together in a separate node. The sequences of mouse and pig joined the above mentioned clusters and subsequently by chicken. Chicken FEZL gene was found to be the outgroup and is understandable as all the other sequences belong to mammalian species. The phylogenetic analysis of conceptualized amino acid sequences of FEZL gene was observed to follow a similar trend (Fig 3).


 

 

 
Characterization of FEZL gene promoter and 5' flanking region
 
About 719 bp and 701 bp region upstream to FEZL gene respectively of Sahiwal and Karan Fries cattle including basal promoter and 5¢ flanking region was sequenced. The multiple alignment of nucleotide sequence of 5' flanking region of FEZL gene of different Bos species are presented in Fig 4. Surprisingly, no nucleotide changes were observed in this region among different bovine species viz. Bos taurus, Bos indicus and Bos taurus × Bos indicus. The results of the present study suggests that the basal promoter and 5' flanking region of FEZL gene being conserved among different sub-species of bovines. The 5' flanking region was subjected to TESS (Transcription Element Search Software) and MATCH analysis to predict the putative transcription factor binding sites involved in the transcriptional machinery of FEZL gene. A total of six putative sites for transcription factor binding sites were predicted including Elk-1, Oct-1, HNF4, Lmo2 complex, GATA-3 and Nkx2-5 (Table 5). All these putative sites had a core match of 1.0 and matrix match above 0.90. Among these, three putative sites viz. Oct-1, GATA-3 and Nkx2-5 at positions -138 to -123, -521 to -511 and -560 to -553 were predicted from sense (+) strand while the other three (Elk-1 (-75 to -61); HNF-4 (-166 to -147); Lmo2 complex (-376 to -367)) were predicted from the anti-sense strand (-). Nkx2-5 motif binds to the homeobox protein that play critical role in regulating tissue specific gene expression, essential for tissue differentiation as well as determining the temporal and spatial patterns of development. Elk1 is a member of Ets family of transcription factors involved in cell proliferation and with multiple functions in brain (Besnard et al., 2011). Hepatocyte nuclear factors (HNF4) are a group of phylogenetically unrelated transcription factors that regulate the transcription of a diverse group of genes involved in glucose, cholesterol and fatty acid transport and metabolism. Oct-1 refers to octamer transcription factor while LMO2 protein is a cysteine rich protein and has a central and crucial role in hematopoietic development. GATA-3 belongs to the GATA family of transcription factors and contains two GATA-type zinc fingers and is an important regulator of T- cell development. Although, it seems unlikely that all the putative transcription factors identified in the promoter region exert their individual effects, some may actually influence the regulation of bovine FEZL expression. For example, GATA-3 plays a significant role in the differentiation and proliferation of Th2 cells. The Th2 cells produce cytokines like IL-4, IL-5 and IL-13 that are involved in humoral immunity against extracellular pathogens (Zhu et al., 2006; Zhu 2017). Such motifs may play a significant role in regulating the expression of FEZL and othercytokine genes and consequently influencing thehost immune response to pathogens.


 

       
In summary, we have sequence characterized bovine FEZL gene and its 5' flanking region in Bos indicus cattle. The results revealed eight nucleotide variations among Bos species, of which three nucleotide INDELs and one nucleotide substitution mutations were found within coding DNA region. The 5' flanking region of FEZL gene was conserved among taurine, indicine and crossbred cattle. The study showed the presence of certain putative motifs that could potentially regulate genes involved inimmune response. Elucidation of Bos indicus FEZL gene will further form the basis to identify candidate gene markers for association with mastitis resistance/susceptibility in cattle.

The authors are thankful to the Director, National Bureau of Animal Genetic Resources and Director, National Dairy Research Institute, Karnal, Haryana, India, for providing necessary facilities to the study.