Am

ber Graham

, Hans R

indt, Jeffrey Bryan and C

arol Reinero

College of V

eterinary Medicine, U

niversity of Missouri

Acknow

ledgements

Funding for the project and student supportfor the V

RS

P program

was provided

by the Morris A

nimal F

oundation.

Hypothesis

•B

ased on knowledge of the im

portance of epigenetic regulation of the prom

oter region of the genes coding for Th2 cytokines in hum

an asthm

atics and murine asthm

a models, w

e will investigate m

ethylation patterns in the analogous prom

oter region of the feline IL-4, IL

-5 and IL-

13 genes.

Introduction•

Epigenetics is the study of heritable changes that alter gene expression

without altering D

NA

sequence. It is a relatively new field of study,

which focuses on the relationship betw

een genetics and environmental

influences on disease. •

Several studies have demonstrated a relationship betw

een methylation

status in the promoter region of several genes and the developm

ent of asthm

a in humans and m

ouse models.

•A

reas rich in a cytosine nucleotide followed by a guanine nucleotide

(CpG

dinucleotides) can be found in gene promoter regions and are

referred to as CpG

islands. •

Asthm

a is a chronic respiratory disease of the lower airw

ays that is m

ediated by a T helper type 2 (T

h2) imm

une response to inhaled aeroallergens. C

ats spontaneously develop allergic asthma and share

similar pathological features as hum

ans, including eosinophilic airway

inflamm

ation, airway hyperresponsiveness

and airway rem

odeling. •

Currently no research has been done to explore if epigenetic regulation

present in humans and m

ice translates to the cat. •

In humans and m

ice, several genes for key cytokines that play a role in the T

h2 response have shown differences in C

pGisland m

ethylation of the prom

oter region. (Figure 1)•

In both humans and in m

ouse models of asthm

a, epigenetic changes at the prom

oter region of the gene for Interleukin (IL)-13 have been

detected in asthmatics and correlate w

ith the severity of symptom

s. •

Further, we w

ill explore the epigenetic regulation of cytokines IL-4 and

IL-5 based on their role in the T

h2 imm

une mediated response.

Results

•C

omparison of hum

an, mouse and cat D

NA

showed differences in

CpG

island location and presence within the gene prom

oter regions. •

Based on the finding of C

pGislands in IL

-13 as well as IL

-5, we

designed primers to am

plify sequences that spans the CpG

island found in each prom

oter region. (Fig. 5)•

Further work is needed to develop prim

ers to analyze IL-13 using

MSP

and to amplify the IL

-4 promoter region.

Methods

•D

NA

sequence for feline IL-4, IL

-5 and IL-13

promoter region w

as attained through the NC

BI

Nucleotide database

(http://ww

w.ncbi.nlm

.nih.gov/nuccore) from a w

hole genom

e shotgun sequence of Cinnam

on, an A

byssinian cat. •

Maverix

Genom

e B

rowser(https://w

edge.maverixbio.com

) was utilized

to detect any single nucleotide polymorphism

s (SNP

), w

hich have been identified from 14 cats that have

been sequenced in that database. Those SN

Ps, w

hich w

ere identified, were excluded from

primer design to

decrease the likelihood of primer failure.

•C

pGislands w

ere located using Methprim

er, a program

that locates CpG

islands in DN

A sequence.

•M

ethprimer

was used to design prim

ers for two types

of methylation P

CR

, Methylation-Specific P

CR

(M

SP) and C

ombined B

isulfite-Restriction A

nalysis (C

OB

RA

). •

Bisulfite C

onversion was perform

ed by treating DN

A

with bisulfite. (Fig. 2)

•B

isulfite converted DN

A is used in C

OB

RA

and MSP

. (Fig. 3)

•A

temperature gradient P

CR

was conducted to

determine the optim

al annealing temperature for each

primer in order to ensure reproducibility of the P

CR

and optim

ize the PC

R product. (Fig. 4)

•Splenic D

NA

was used in all initial P

CR

tests. DN

A

was extracted using an alcohol extraction m

ethod and w

as used for the development of the prim

ers and PC

R

protocol for IL-4, IL

-5 and IL-13.

Figure 1. Several promoter regions for T

h2 cytokine genes including IL-4, IL

-5 and IL-13 w

ere compared across hum

an, mouse

and cat to look for the presence of CpG

islands using MethP

rimer

software. C

pGislands w

ere identified as areas that have a greater than expected ratio of cytosine and guanine as w

ell as at least 50% of the sequence consisting of guanine-cytosine pairs. T

here were

differences in the presence of CpG

islands in all species suggesting that the cat will have specific and different patterns of

methylation than the hum

an and mouse.

Future Directions

•E

pigenetics is a rapidly growing field of research and w

ill have im

portant clinical applications. •

Currently, the diagnosis of asthm

a in feline patients has presented considerable challenges. T

he identification of specific methylation

profiles in asthmatic cats could lead to the developm

ent of a non-invasive diagnostic tool.

•D

etermining the role of epigenetic regulation in asthm

a will increase

understanding of the pathogenesis and expand future research avenues for novel therapeutics.

Developm

ent of a Novel PC

R Protocol to Study E

pigenetic Regulation

of IL-4, IL-5 and IL-13 in Cats

Human

FelineMouse

IL-13

IL-5

IL-4

Figure 2. DN

A is treated w

ith bisulfite which w

ill convert unm

ethylatedcytosine to uracil. In subsequent P

CR

amplification, a

thymine w

ill be incorporated in place of the uracil in the amplified

sequence. Methylated cytosine w

ill remain unconverted.

Figure 3. Both C

OB

RA

and MSP

are run with bisulfite converted D

NA

. C

OB

RA

uses restriction enzymes to distinguish differences in

methylation w

ithin the sequence of interest by only cleaving at sites of m

ethylation. MSP

primers used for P

CR

are designed to include CpG

dinucleotides close to their 3’ ends. Prim

ers are designed to amplify

either methylated C

pGor unm

ethylatedC

pGafter bisulfite conversion

to TpG

, but not both. If product is formed and a band is seen w

hen using the m

ethylated-specific primers that w

ould indicate that the sequence w

as methylated; conversely, a band w

ould not be expected w

ith use of the unmethylated-specific prim

ers and vice versa.

Figure 5. PC

R products w

ere generated with prim

ers designed to am

plify an area within a C

pGisland in the prom

oter region of feline IL-

13 and IL-5. T

wo different prim

er sets were used to generate the

products shown for IL

-5.

IL-13

IL-5

Figure 4. PC

R w

ith an annealing temperature gradient

of 53-63 degrees Celsius w

as performed to determ

ine the optim

al annealing temperature for the prim

ers used. For this prim

er pair the optimal annealing

temperature w

as determined to be 61 degree C

elsius. A

gold arrow points to the expected product.

63°62.3°61.1°

59.3°57°

55.3°54°

53°