(1) The study aimed to define the structure of the MLIP gene and its tissue-specific expression patterns. (2) PCR and western blot experiments showed that MLIP exon 1a is expressed in heart and skeletal muscle, while exon 1b is expressed in the brain. Neither exon is expressed in the pancreas or liver. (3) This suggests MLIP uses different promoters in different germ layers - exon 1a promoter in mesoderm, exon 1b promoter in ectoderm, and unknown promoters in endoderm tissues like pancreas and liver.

1. Mapping the gene encoding the muscle A-type lamin interacting protein
Anastasia Pimenova1,2, Marie-Elodie Cattin1, Cassandra Roeske1, Patrick G. Burgon1,2,3
1University of Ottawa Heart Institute, 2Department of Cellular & Molecular Medicine
3Department of Medicine(Cardiology), Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada
1. Introduction
Most mammalian cells contain a nucleus, the structure of which is supported by many proteins and fibers. One of the main contributors to
nuclear membrane stability and integrity is the lamin protein which polymerizes into intermediate filaments, forming the nuclear lamina1.
Over 400 mutations have been identified within the lamin gene, resulting in tissue specific diseases such as dilated cardiomyopathy and
atherosclerosis2. In the Burgon lab, 230 amino acids from the N-terminus of A-type lamin were screened for new interactors. The muscle-
enriched A-type lamin-interacting protein (MLIP) was identified in this region which is rich in mutations associated with skeletal and
cardiac muscle laminopathies, and MLIP was shown to co-localize with lamin A in the nucleus2. MLIP was found to be ubiquitously
expressed; its biological function remains elusive but it may be of high relevance for muscle function due to abundant expression in the
heart, skeletal and smooth muscle. The MLIP gene is also heavily spliced in different tissues, with at least 7 known variants in the heart.
This project aims to define the exact structure of the MLIP gene and investigating its expression in different tissues.
pimenova.anastasia.k@gmail.com
MLIP+/+ MLIP+/- MLIP-/-
3. Research plan and methods
4. PCR on heart and brain cDNA
5. PCR on liver, pancreas and skeletal muscle cDNA
Similarly to the heart and brain, PCR on cDNA from mouse liver, pancreas and skeletal muscle
was performed using the following primer combinations:
•MLIPexon1a/MLIPexon1b + MLIPexon9 in liver and skeletal muscle (Figure 7)
•MLIPexon1a/MLIPexon1b + MLIPexon3(rvs) in liver and skeletal muscle (Figure 8)
•MLIPexon1a/MLIPexon1b/MLIPexon3(fwd) + MLIPexon9 in the pancreas (Figure 9)
Figures 7 and 8 display both +/+ and -/- mouse cDNA PCRs.
6. Western Blot of various tissues
8. Conclusions and significance
2. Project rationale and purpose
9. Further stepsReferences
Initially, a Western blot (Figure 1) was performed on various mouse tissues along with a 5’-3’
RACE on the mouse heart2. In the heart, all the protein bands were accounted for by sequencing
the RACE product. However in the brain, the protein band at 58 kDa could not be accounted for.
This led to the hypothesis that:
there must be an additional exon and/or start site in the brain to account for the
large band at ~58kDa.
To test this theory:
•5’3’ RACE was done on the mouse brain
•An alternate promoter was found in the brain, now named the “1b” promoter
•The promoter in the heart now named “1a” promoter
For this honours project, the purpose was to:
•Confirm that exon 1a and its promoter is heart specific and exon 1b and its
promoter is brain specific
•Examine the expression of these 2 exons in different tissues
Figure 1. Western blot
of MLIP protein in
various mouse tissues.
MLIP gene gives rise
to many different
isoforms with a tissue-
specific pattern of
expression2.
Several tools were used in this project:
•MLIP K/O mouse with only exon 1a removed
(Figure 2)
•3 MLIP antibodies (Figure 3)
The following experiments were performed:
•Designing primers for PCR (Figure 4)
•PCR on cDNA in heart, brain, skeletal muscle,
pancreas and liver
•Western blot of mouse tissue lysates, targeting
MLIPC and MLIP1b
21
x CMV-Cre
2
PGKneo
mChr 9
~
~
LoxP
Frt
Figure 2. (Above) Schematic diagram showing the
targeting vector leading, after Cre-recombinase
mediated recombination, to the removal of MLIP
promoter and exon 1a from 129SV mice.2
1a 2 3 4 5 6 7 8 9 10
MLIP1a MLIPC
1b 2 3 4 5 6 7 8 9 10
MLIP1b
MLIPC
Figure 3. (Above) Diagram of the MLIP protein showing 2
forms: one containing 1a exon (top) and another
containing 1b exon (bottom). On the protein are shown the
3 different antibodies used for Western blots. MLIPC Ab
targets antigen in exon 9, and MLIP1a and MLIP 1b Ab
target antigens in exons 1a and 1b respectively.
1a 2 3 4 5 6 7 8 9 10 11-stop1b
Figure 4. (Left) Diagram demonstrating all the MLIP
exons and the locations of primers used for PCR
experiments. Red arrows indicate primer direction.
MLIPexon1a and MLIPexon1b are situated in the
translated region of the exon.
brain heart
1a 1b 1a 1b 1a 1b
+/+ +/+ +/+ +/+-/- -/- -/- -/-
brain heart
-/- -/- -/- -/-+/+ +/+ +/+ +/+
- ctrl
1a 1a1b 1b 1b1a
cDNA from mouse brain and heart was synthesized and PCR was
performed using the following primer combinations:
•MLIPexon1a/MLIPexon1b + MLIPexon9 (Figure 5)
•MLIPexon1a/MLIPexon1b + MLIPexon3(rvs) (Figure 6)
All reactions were done using both wild type (+/+) and MLIP K/O
(-/-) mice.
Figure 5 . Photograph of EtBr 2%
agarose gel with PCR on brain and
heart cDNA with a combination of
either MLIPexon1a + MLIPexon9
primers or MLIPexon1b +
MLIPexon9 primers.
Figure 6 . Photograph of
EtBr 2% agarose gel with
PCR on brain and heart
cDNA with a combination
of either MLIPexon1a +s
MLIPexon3(rvs) primers or
MLIPexon1b +
MLIPexon3(rvs) primers.
Exon 1a is expressed in the heart but not the brain,
whereas exon 1b expression is restricted to the
brain in this experiment.
Conclusion drawn from PCR:
-ctrl
liver sk. muscle
+/+ +/+ +/+ +/+-/- -/--/- -/-
1a 1b 1a 1b
+ctl
-ctl
1a 1b
-ctl
1a 1b exon3
1a 1b 1a 1b1a 1b
liver sk. muscle -ctl
+/+ +/+ +/+ +/+-/- -/--/- -/-
Figure 7 . Photograph of EtBr 2% agarose gel with PCR on liver
and skeletal muscle cDNA with a combination of either
MLIPexon1a +s MLIPexon9 primers or MLIPexon1b + MLIPexon9
primers.
Figure 8 . Photograph of EtBr 2% agarose gel with PCR on liver
and skeletal muscle cDNA with a combination of either
MLIPexon1a +s MLIPexon3(rvs) primers or MLIPexon1b +
MLIPexon3(rvs) primers.
Figure 9 . Photograph of EtBr 2%
agarose gel with PCR on
pancreas cDNA with a
combination of (i) MLIPexon1a
+s MLIPexon9; (ii) MLIPexon1b
+s MLIPexon9 or (iii)
MLIPexon3(fwd) +s MLIPexon9
primers
rvs
fwd
(1) Exon 1a is expressed in the skeletal muscle and may be expressed in the liver (2) Exon 1b is not expressed in any of these
tissues (3) Pancreas expresses MLIP variants that do not contain exon 1a or exon 1b
Conclusion drawn from PCR:
46 46 46
liver Sk. muscle lung kidney heart
MLIPCMLIP1b
Western blots were done on several mouse tissue lysates (Figure 10 and 11). Blots were probed with MLIPC (Figure 10a,
Figure 11) and MLIP1b (Figure 10b) antibodies.
brain
+/+ +/+ +/+ +/+ +/++/+-/- -/- -/--/- -/--/-
A)
B)
Figure 10. (Left) Image of Western
blot probed with: A) MLIPC or B)
MLIP1b as primary antibody. Various
tissues displayed, loaded 50ug of
protein per lane. Both +/+ and -/-
lysate samples shown.
(1) MLIP is expressed in liver, brain, lung and heart (skeletal muscle to be reassessed) as previously reported
(2) The heart expresses MLIP isoforms containing exon 1a only (deleted in -/- mice)
(3) Liver, brain and pancreas express MLIP isoforms containing other start sites than start site of exon 1a (expression
detected in -/- tissues)
(4) The specificity of the MLIP1b antibody is uncertain as bands are detected in tissues that do not expressed exon 1b
(liver, skeletal muscle and heart)
Conclusion drawn from Western Blot:
From both the PCR and the
Western blot results, the following
can be concluded:
In the heart and skeletal muscle
(mesoderm):
 Exon 1a is expressed; exon 1b is
silent (Figure 5, 6, 10A)
58
46
30
23
80
175
heart pancreas
+/+ -/-+/+ -/-
(kDa)
Figure 11. (Right) Image of Western
blot probed with MLIPC as primary
antibody. Loaded 60ug of protein per
lane. Both +/+ and -/- lysate samples
shown.
58 58 58
30 30 30
23 23 23
80 80 80
58 58 58
30 30 30
23 23 23
17 17 17
2 3 4 5 6 7 8 9 101c1b1a 11 (stop)
2 3 4 5 6 7 8 9 101c1b1a
2 3 4 5 6 7 8 9 101c1b1a
2 3 4 5 6 7 8 9 101c1b1a
MLIP gene
MLIP mRNA (mesoderm)
MLIP mRNA (ectoderm)
MLIP mRNA (endoderm)
Figure 12. Diagram of
proposed MLIP gene
structure, along with the
5’UTR/promoter regions
used for each germ layer.
Purple box: promoter for
exon 1a; pink box:
promoter for exon 1b, blue
box: promoter for exon 1c.
∴ MLIP appears to be ubiquitously expressed, but uses different promoters in each of the germ layers
Further work needs to be done on this project such as examining the specificity of the MLIP 1b antibody, sequencing 5’-3’
RACE and verifying PCR results in the liver. Other experiments include Immunofluorescence and immunohistochemistry in
different tissues to observe MLIP localization.
1. Stuurman, Nico, Susanne Heins, and Ueli Aebi. "Nuclear Lamins: Their Structure, Assembly, and Interactions." Journal of Structural
Biology 122.1-2 (1998): 42-66.
2. Ahmady, Elmira, Shelley A. Deeke, Seham Rabaa, Lara Kouri, Laura Kenney, Alexandre F. R. Stewart, and Patrick G. Burgon. "Identification
of a novel muscle A-type lamin-interacting protein (MLIP)."J Biol Chem. 286.22 (2011): 19702–19713.
Why is this finding important?
We already know MLIP is heavily spliced and ubiquitously expressed. Since the MLIP protein co-localizes with lamin A/C in
the nucleus, it likely has regulatory effects on gene expression. If different promoters are used in each of the germ layers, it
might signify that MLIP is significant in germ- specific gene regulation.
In the brain (ectoderm):
Exon 1b is expressed, exon 1a is silent (Figure 5, 6, 10B)
In the pancreas (endoderm):
Neither exon 1a or 1b are expressed (Figure 9), though MLIP is transcribed and translated (Figure 11)
In the liver (endoderm):
Neither exon 1a or 1b are expressed (Figure 7), but MLIP is transcribed and translated (Figure 10A)
Based on the above observations, a new model is proposed with 3 separate promoters, one for each
germ layer (Figure 12).