Download Analysis of Histone Mutants (Cross-Talk between H3 and H4)

Analysis of Histone Mutants (Cross-Talk between H3 and H4)
Rehan S.M1 and Lehming N.2
Department of Microbiology, Faculty of Science, National University of Singapore
10 Kent Ridge Road, Singapore 117546
ABSTRACT
Core histones are an essential part of the nucleosome and undergo numerous post-translational
modifications, more frequently, on the first 20-40 amino acids located at the N-terminal tails.
Elucidating the functional role of specific patterns of these post-translational modifications across
histones (the histone code) has been the subject of intense research recently. For this project I
investigated the cross-talk between modifyable residues of histones H3 and H4. A yeast H3/H4
double deletion strain was generated and 220 combinations of histone H3 and H4 mutants (in which
the regular amino acid was substituted with alanine that does not undergoes covalent modification)
were tested for complementation and sensitivity to higher temperature, aminotriazole, methyl
methanesulfonate and antimycin A on galactose plates. The results of the phenotypic tests revealed
significant cross-talk between sixteen H3 and H4 residues. Moreover, by performing western blot
analysis, I also shed light on possible mechanisms by which two proteins, Sip5 and Hpa2, are able
to suppress the AT phenotype displayed by the H4Y98A mutant.
MATERIALS AND METHOD
1. Generation of the Histone H3 and H4 Double Knockout Yeast Strain
In Saccharomyces cerevisiae two identical histone H3 proteins are encoded by the genes HHT1
1. Student
2. Principal Investigator
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and HHT2 and similarly the genes HHF1 and HHF2 code for identical histone H4 proteins.
Therefore in order to study the cross-talk between different H3 and H4 mutants that were present on
plasmids, the HHT2/HHF2 locus was first knocked out using a NKY51 hisG-URA3-hisG gene
cassette, after which the HHT1/HHF1 locus was knocked out using a pUC8+his3+Thhtf1 deletion
cassette to generate the BY4742WHHTF+YCplac33-HHTF2 strain. Titration were performed in
a 96 well plate using a multi-channel pipette, scoop of cells was inoculated in 90µl of sterile water
in a well that was followed by serial dilution up to 10-5.
RESULTS AND DISCUSSION
1. Phenotype Tests
Phenotype tests revealed significant cross-talk between sixteen histones H3 and H4 residues.
Some of the results are shown in Figure 2.
Figure 2. Droplet assays show the suppression of the AT phenotype when the H4Y98A mutant was
combined with H3K4A, H3K9A, H3T32A and H3K37A. Ten-fold serial dilutions of the transformants were
titrated onto W-L- plate and histidine deficient (H-) plate containing 3-aminotriazole. The plates were then
incubated at 28oC. W-L- and AT plates were scanned after 6 days and 3 days of incubation, respectively.
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2.Analysis of the mechanism of suppression of H4Y98A AT phenotype by Sip5 and Hpa2.
From western blot analysis it was deduced that over-expression of Sip5 probably suppressed the
H4Y98A AT phenotype by restoring H3S10 phosphorylation levels (Figure 3) in H4Y98A mutant
while Hpa2 worked by restoring the wild type acetylation pattern of the N-terminal lysines of
histone H3, since over-expression of Hpa2 restored the reduced H3K14 acetylation levels (Figure 4)
and decreased the elevated H3K9 acetylation levels in H4Y98A mutant (not shown).
A)
B)
120
Percentage (%)
100
80
60
40
20
0
4WT,
316
4Y98A,
316
4Y98A,
316-SIP5
4Y98A,
316-Snf1
Figure 3. A) Western blot analysis revealed that that over-expression of Sip5 restores the H3S10
phosphorylation levels in H4Y98A mutant. BY4742WHHTF + Lane 1) YCplac111-H3WT+YCplac22H4WT+PactT316, Lane 2) YCplac111-H3WT+YCplac22-H4Y98A+PactT316, Lane 3) YCplac111-H3WT
+ YCplac22-H4Y98A+PactT316-Sip5 , Lane 4) YCplac111-H3WT + YCplac22-H4Y98A + PactT316-Snf1.
B) The bar graph shows the H3S10 phosphorylation levels relative to total histone H3 in the depicted strains
as a percentage of the relative H3S10 phosphorylation levels in H3WT, H4WT strain (Lane 1).
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120
Percentage (%)
100
80
60
40
20
0
H4WT,
181
H4Y98A,
181
H4WT,
181-HPA2
H4Y98A,
181-HPA2
Figure 4. Over-expression of Hpa2, restores the H3K14 acetylation levels. The bar graph shows the
H3K14 acetylation levels relative to total histone H3 in the depicted strains as a percentage of the
relative H3K14 acetylation levels H3WT, H4WT strain.
ACKNOWLEDGEMENTS
I would like to express my deep gratitude for Dr. Norbert Lehming for his guidance and patience,
fellow UROPS student Rachel with whom I collaborated on this project and everyone else in the
LAB.
REFERENCES
Angus-Hill, M.L., et al. 1999. Crystal structure of the histone acetyltransferase Hpa2: A tetrameric
member of the Gcn5-related N-acetyltransferase superfamily. J Mol Biol. 294(5), 1311-25.
Matsubara, K., Sano, N., Umehara, T., and Horikoshi, M. 2007. Global analysis of functional
surfaces of core histones with comprehensive point mutants. Genes to Cells 12, 13-33.
Peterson, C.L., Laniel, M.A. 2004. Histones and histone modifications. Current Biology 14, R546R551.
Sanz, P., et al. 2000. Sip5 interacts with both the Reg1/Glc7 protein phosphatase and the Snf1
protein kinase of Saccharomyces cerevisiae. Genetics 154(1), 99-107.
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