research day poster final version
TRANSCRIPT
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.
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 steps References
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
2 1
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-stop 1b
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 1a 1b 1b 1b 1a
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 1b 1a 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
MLI
P C
M
LIP
1b
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 10 1c 1b 1a 11 (stop)
2 3 4 5 6 7 8 9 10 1c 1b 1a
2 3 4 5 6 7 8 9 10 1c 1b 1a
2 3 4 5 6 7 8 9 10 1c 1b 1a
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).