25/05/24, 18:08 Alternative splicing variants and DNA methylation status of BDNF in inbred chicken lines - ScienceDirect

Brain Research
Volume 1269, 7 May 2009, Pages 1-10

Research Report

Alternative splicing variants and DNA methylation status of

BDNF in inbred chicken lines
Ying Yu a 1, Huanmin Zhang b, Mardi S. Byerly a, Larry D. Bacon b, Tom E. Porter a, George E. Liu c, Jiuzhou Song a

Show more

Share Cite

https://doi.org/10.1016/j.brainres.2009.01.071
Get rights and content

Abstract

Brain-derived neurotrophic factor (BDNF) has multiple alternative splicing variants and plays diverse biological
functions in mammals, including neuronal survival, cholesterol metabolism, cell differentiation and tumor
development. However, genomic structures of chicken BDNF (cBDNF) variants and its potential functions are still
undefined. Here, we characterized two novel alternative splicing variants of cBDNF, cBDNF1 and cBDNF2, via combining
comparative genomics methods and molecular techniques in inbred chicken line 63 and line 72, which have been
developed to be resistant and susceptible, respectively, to Marek's disease tumor since 1939. Both cBDNFs consist of a
bipartite transcript, with different 5′ exons, exon I (298 bp) in cBDNF1 and exon II (286 bp) in cBDNF2, each of which is
spliced to the common 3′ exon IV. Exon I and IV are highly conserved between chicken and mammals, whereas exon II
is unique for chicken. The amino acid sequence of cBDNF1 contains 8 additional amino acids in the N terminal
compared to cBDNF2. cBDNF1 and cBDNF2 were only expressed in the hypothalamus among eight tissues, and cBDNF2
showed lower expression than that of cBDNF1 in both lines. The expression level of cBDNF1 was significantly higher in
line 72 than in line 63 (P < 0.01). Notably, the DNA methylation levels on the cis-regulatory region of cBDNF1 was
negatively correlated with its expression level, which suggests that the mRNA expression level of cBDNF1 may be
related to the DNA methylation status in the chickens. We also discussed a potential role of variant cBDNF1 in MD
tumor resistance and susceptibility.

Introduction

Brain-derived neurotrophic factor (BDNF), one member of the neurotrophic factor family, is highly expressed in brain
and exerts multiple biological functions. BDNF was initially thought to be involved in neuronal survival and
differentiation (Brady et al., 1999, Kernie et al., 2000, Leingartner et al., 1994, Monteggia et al., 2004). Further studies
have shown in mammals that BDNF elicits cholesterol biosynthesis in cultured cortical and hippocampal neurons, and
also plays essential roles in immunity and tumor progression (Chen and von Bartheld, 2004, Katz and Meiri, 2006,
Kruse et al., 2007, Rao et al., 2007, Suzuki et al., 2007, Yang et al., 2006).

One of the main contributors to the diverse functions of the gene is mRNA alternative splicing (Black, 2000, Talavera et
al., 2007). Alternative splicing can generate different mRNA variants that encode distinct protein products, thus
increasing the coding capacity of the gene (Smith and Valcarcel, 2000, Xing and Lee, 2006). The genomic structure of

https://www.sciencedirect.com/science/article/abs/pii/S0006899309001644?via%3Dihub 1/5
25/05/24, 18:08 Alternative splicing variants and DNA methylation status of BDNF in inbred chicken lines - ScienceDirect

the BDNF gene is complex in that it contains multiple promoters that are used to produce different alternative
transcripts (Chen et al., 2003, Liu et al., 2006, Liu et al., 2005, Martinowich et al., 2003). These distinct sets of BDNF
alternative splicing variants are used to achieve precise temporal and spatial expression as well as diverse functions of
BDNF production (Martinowich et al. 2003).

DNA methylation is a key epigenetic modification that provides heritable information not encoded in the nucleotide
sequence (Egger et al., 2004, Meissner et al., 2005, Widschwendter and Jones, 2002). Its role in disease susceptibility,
such as cancers, becomes increasingly evident (Baylin, 2005, Lofton-Day et al., 2008, Shames et al., 2007, Yu et al.,
2008a). 5-methylcytosine status of CpG dinucleotides in promoters is strongly linked to the expression levels of
alternatively spliced genes (Dolzhanskaya et al., 2006, Lande-Diner et al., 2007). The mammalian BDNF gene was
identified as an activity-dependent target of methyl-CpG binding protein 2 (MeCP2) (Martinowich et al. 2003). A
decrease in CpG methylation levels within one of the cognate promoters of the BDNF gene is correlated with an
increased synthesis of BDNF in neurons (Chen et al., 2003, Martinowich et al., 2003). These results suggested that the
transcriptional level of BDNF splicing variants can be predicted by the CpG methylation status of the promoters.

Although the coding region of chicken BDNF (cBDNF) has been annotated (Isackson et al., 1991, Katz and Meiri, 2006),
the alternative splicing variants of the cBDNF and their epigenetic regulatory mechanisms are still undefined. In this
study, we sought to identify novel alternative splicing variants of cBDNF and to gain an insight into the regulatory
effects of DNA methylation status on mRNA expression levels of alternative splicing variants in inbred chicken lines.
Chicken samples used in the study were two highly inbred chicken lines, 63 and 72, which were developed in 1939. Line
63 is resistant to Marek's disease (MD) tumors that are induced by Marek's disease herpesvirus (MDV), while line 72 is
susceptible to MD tumors (Bacon et al., 2000, Bacon et al., 2001, Zhang et al., 2005, Zhang et al., 2006). The two inbred
lines are an ideal model system to explore the association between aberrant gene expression and tumor resistance.

To achieve our goal, we first characterized the gene structures and mRNA expression level of cBDNF splicing variants
through comparative genomics, cloning sequence, and real-time RT-PCR in MD tumor-resistant line 63 and -susceptible
line 72 as well as in ordinary layer. Then we measured the CpG methylation status on the predicted cis-regulatory sites
of cBDNFs with pyrosequencing technology in both lines. We also discussed the regulatory effects of DNA methylation
status on mRNA expression levels of cBDNFs variants. Our results are the first identification of two tissue-specific
alternative splicing variants of cBDNFs, and we discovered a negative correlation between DNA methylation status on
the promoter regions and mRNA expression levels of cBDNF1 in the chickens.

Access through your organization

Check access to the full text by signing in through your organization.

Access through your instit…

Section snippets

Novel alternative splicing variants structures of chicken BDNF

To obtain reference sequences for alternative splicing transcripts of chicken BDNF (cBDNF), we initially aligned human
BDNF (hBDNF) variants to the whole genomic sequence of the chicken (May, 2006, UCSC) with comparative genomics
methods on the UCSC Genome Browser website. As shown in Fig. 1, four exons of cBDNF were discovered in the minus
strand of chicken chromosome 5. Among them, the first cBDNF exon (exon I) is a hidden novel exon. The fourth cBDNF
exon (exon IV) was annotated as a coding…

Discussion

Previous information about the alternative splicing variants of BDNF was mainly derived from mammals and zebrafish.
For mice/rats and humans, nine and thirteen alternative mRNA transcripts of BDNF were respectively discovered (Liu et

https://www.sciencedirect.com/science/article/abs/pii/S0006899309001644?via%3Dihub 2/5
25/05/24, 18:08 Alternative splicing variants and DNA methylation status of BDNF in inbred chicken lines - ScienceDirect

al., 2006, Liu et al., 2005). In this study, we found three alternative splicing variants of chicken BDNF (cBDNF) using
comparative genomics tools. cBDNF1 and cBDNF2 were first validated and characterized in highly inbred chicken line
63 and 72 with RT-PCR and…

Animal samples

Blood and tissue samples of the inbred White Leghorn line 63 and line 72 were collected from ADOL (Avian Disease and
Oncology Laboratory, USDA). Separated red blood cells from plasma were stored at − 20 °C. Eight organs, including the
hypothalamus, pituitary gland, heart, liver, kidney, spleen, abdominal fat and muscle were collected immediately from
five female chickens of each line after euthanasia, and frozen in liquid nitrogen then stored at − 80 °C. Five regions in
brain, including…

Acknowledgments
We thank Dr. Wansheng Liu for his many helpful discussions, Mary Rogier, Fei Tian and Apratim Mitra for manuscript
modifications. The authors are grateful to the foundation for international exchange program of excellent scholar of
China and the support by USDA-NRI 2008-00870.…

Recommended articles

References (42)

BaconL.D. et al.
A review of the development of chicken lines to resolve genes determining resistance to diseases
Poult. Sci. (2000)

BlackD.L.
Protein diversity from alternative splicing: a challenge for bioinformatics and post-genome biology
Cell (2000)

GeorgelP.T. et al.
Chromatin compaction by human MeCP2. Assembly of novel secondary chromatin structures in the
absence of DNA methylation
J. Biol. Chem. (2003)

IsacksonP.J. et al.
Comparison of mammalian, chicken and Xenopus brain-derived neurotrophic factor coding sequences
FEBS Lett. (1991)

KruseN. et al.
Differential expression of BDNF mRNA splice variants in mouse brain and immune cells
J. Neuroimmunol. (2007)

Lande-DinerL. et al.
Role of DNA methylation in stable gene repression
J. Biol. Chem. (2007)

LeingartnerA. et al.
Brain-derived neurotrophic factor increases neurotrophin-3 expression in cerebellar granule neurons
J. Biol. Chem. (1994)

LiuQ.R. et al.
Rodent BDNF genes, novel promoters, novel splice variants, and regulation by cocaine

https://www.sciencedirect.com/science/article/abs/pii/S0006899309001644?via%3Dihub 3/5
25/05/24, 18:08 Alternative splicing variants and DNA methylation status of BDNF in inbred chicken lines - ScienceDirect
Brain Res. (2006)

PearseR.N. et al.
A neurotrophin axis in myeloma: TrkB and BDNF promote tumor-cell survival
Blood (2005)

RomanovM.N. et al.
Construction of a California condor BAC library and first-generation chicken-condor comparative physical
map as an endangered species conservation genomics resource
Genomics (2006)

View more references

Cited by (14)

In ovo Sound Stimulation Mediated Regulation of BDNF in the Auditory Cortex and Hippocampus of
Neonatal Chicks
2019, Neuroscience

Citation Excerpt :
…BDNF expression was evaluated further at the transcript level in response to sound stimulation. Chicken BDNF has three different
isoforms reported, cBDNF1, cBDNF2 and cBDNF3 (Yu et al., 2009). In the present study, cBDNF1 and cBDNF2 were found to be
expressed in the auditory system and hippocampus of neonatal chicks, while cBDNF3 was not found (Fig. 4B).…

Show abstract

Corticosterone is correlated to mediators of neural plasticity and epigenetic potential in the hippocampus
of Senegalese house sparrows (Passer domesticus)
2018, General and Comparative Endocrinology

Citation Excerpt :
…Second, given that DNA methylation typically suppresses gene expression, DNMT1 expression was expected to be negatively
correlated to BDNF expression. A possible explanation here is that epigenetic regulation of hippocampal BDNF expression is highly
complex and context-dependent due, in part, to certain structural attributes of the BDNF gene (e.g. multiple promoter regions,
alternative splicing variants) (Pruunsild et al., 2007; Yu et al., 2009). For example, in rats, exposure to a novel environment was
associated with hypomethylation of BDNF exon I and high BDNF expression in the dentate gyrus (DG), whereas hypermethylation of
BDNF exon I and IV and high BDNF expression was reported in the Cornu Ammonis 3 (CA3) (Roth et al., 2015).…

Show abstract

Selective footprints and genes relevant to cold adaptation and other phenotypic traits are unscrambled in
the genomes of divergently selected chicken breeds
2023, Journal of Animal Science and Biotechnology

Comparative Genomics of the BDNF Gene, Non-Canonical Modes of Transcriptional Regulation, and
Neurological Disease
2021, Molecular Neurobiology

Learning-Dependent Transcriptional Regulation of BDNF by its Truncated Protein Isoform in Turtle

2021, Journal of Molecular Neuroscience

Intronic enhancer region governs transcript-specific bdnf expression in rodent neurons

https://www.sciencedirect.com/science/article/abs/pii/S0006899309001644?via%3Dihub 4/5
25/05/24, 18:08 Alternative splicing variants and DNA methylation status of BDNF in inbred chicken lines - ScienceDirect
2021, eLife

View all citing articles on Scopus

1 Present Address: College of Animal Sciences and Technology, China Agricultural University, Haidian District, Beijing, 100193, China.

View full text

Copyright © 2009 Elsevier B.V. Published by Elsevier B.V. All rights reserved.

All content on this site: Copyright © 2024 Elsevier B.V., its licensors, and contributors. All rights are reserved, including those for text and data mining, AI training, and similar technologies. For all open
access content, the Creative Commons licensing terms apply.

https://www.sciencedirect.com/science/article/abs/pii/S0006899309001644?via%3Dihub 5/5