Download 2. Purification of WDR77

ABSTRACT
The WD40 repeat domain protein, WDR77
Post-translational modification of histone tails is one of the primary modes of epigenetic
regulation. The multiple types of post-translational modifications include acetylation,
methylation, phosphorylation, ubiquitylation, sumoylation, ADP-ribosylation, proline
isomerization, citrullination, butyrylation, propionylation and glycosylation. Methylation of
arginine residues in histone tails is a predominant type of modification and the marked
feature of this modification is that different methylated states i.e. monomethylated or
dimethylated can direct different transcriptional consequences.
Protein arginine methyltransferases (PRMTs) are a group of enzymes that attach a methyl
group to a guanidino nitrogen atom of arginine using S-adenosylmethionine (AdoMet) as the
source of the methyl group. PRMT5 has been shown to have an intracellular dependence on
the interacting WD40 repeat domain -containing protein WDR77 to determine specific
substrates. Furthermore, the WD40 domain containing protein WDR77 has been shown to
interact specifically with histone H2A. However, the dependence of the interaction between
WDR77 and modified histone H2A has not been clarified. Some evidence suggests that
H2AR3 methylation occurs in the cytosol and is deposited into nucleosomes by chaperones
as a dimer with H2B, suggesting a sophisticated mechanism of nucleosome assembly and
remodeling to ensure proper installation of H2AR3me2 throughout the genome.
Furthermore, the role between PRMT5 and WDR77 to identify specific substrates remains
unclear. In this study we begin to determine the criteria for WDR77 recognition of specific
substrates for PRMT5 and to determine the functions of both cytosolic and nuclear isoforms
of the WDR77/PRMT5 heterodimeric complex to enact specific genomic regulatory functions
with H2AR3me2 methylation.
Results
WD repeat containing protein 77 (WDR77) is a 342 aa WD repeat protein that is present in
the nucleus and cytoplasm. It has seven putative WD domains, three Nuclear Localization
Sequences (NLS) and two Nuclear Exclusion Sequences (NES). It has been separately
reported as MEP50 (Methylosome protein 50) and p44 (co-factor of androgen receptor).
Cytoplasm
1. Peptide pull down assay
Nuclear Extract – PC3 nuclear extract
Peptides used
1. No peptide – C
2. Unmethylated H3R2 peptide- UN
3. Assymmetrically dimethylated H3R2 peptide – 2A
4. Symmetrically dimethylated H3R2 peptide – 2S
20S Methylosome
pICln
Nuclear membrane
WDR77
Sm proteins
WDR77
Heat denatured
fractions
UN
C
Me
PRMT5
PRMT5
Acid elution
fractions
H2A
C
2A
2S
UN
2A
2S
Nuclear Pore complex
M
WDR5
Sm proteins
Nucleus
H2AR3me2s
M
WDR77
H2AR3me2s/
H2B dimer
PRMT7
Figure 4 - WDR77 interactions in the cytoplasm. WDR77/MEP50 is a part of the 20S methylosome
complex. PRMT5/WDR77 complex catalyze the symmetric dimethylation of H2AR3 and this is essential for
stem cell maintenance. Figure is not drawn to scale.
2. Purification of WDR77
M FT
W E
E E2
FT W E E2
250
150
100
75
PRMT5
50
P
CycD1
WDR77
37
SUZ12
WDR77
WDR77
Transcription
FCP1
WDR77
NuRD
complex
PRMT5
PRC-2
complex
PRMT7
MBD2
WDR77
M-Marker
FT- Flow through fraction
W-Wash fraction
E and E2- Elution fractions
25
WDR77
AR/ER
Densely packed heterochromatin
Conclusion
mRNA
P
Figure 1- Organization of the nucleosomal histone NH terminus (Fullgrabe
30:3391-3403).
et. al. (2011) Oncogene
Figure 2- Distinction between assymmetrical and symmetrical arginine dimethylation by protein arginine
methyltransferases (PRMTs) (Di Lorenzo A. and Bedford M.T. (2011) FEBS Letters 585(13):2024-31)
Figure 5- WDR77 interactions in the nucleus. WDR77 interacts with proteins in the actively
transcribing as well as repressed regions of the genome. AR/ER-androgen receptor or estrogen receptor, Cyc
D1- Cyclin D1. Figure is not drawn to scale.
WD40 proteins
Among the many protein domains that recognize histone modifications, the group of WD
repeat proteins constitute a large heterogenous family of proteins. The WD repeat proteins
get their name by carrying repeats of amino acid residues within a stretch of 40-60 amino
acids that begin with a Glycine-Histidine (GH) dipeptide and end in a Tryptophan-Aspartic
acid dipeptide (WD). WD repeat generally contain 7 repeats that are arranged in a circular
manner forming a β propeller structure (Figure 1). This structure enables WD repeat
domains to act as a scaffold to accommodate the interaction and assembly with a diverse
array of proteins and their structures.
Phosphate group
Methyl CpG
Although the peptide pull down assay showed WDR77 interaction with the
H3R2Me2s and H3R2Me2a peptides it remains to be confirmed if there is direct
binding. The assay should be repeated using the purified WDR77 protein. Future
experiments also include ChIP assays to determine WDR77 binding on a genome
wide scale and crystallization studies to determine the structure of WDR77.
Aims
1. To check if WDR77 recognizes and binds to specific histone modifications.
Peptide pull down assay
Biotinylated (bait) peptides
Immobilized on streptavidin beads
Incubated with nuclear extract
Collect beads
Remove bound proteins by acid elution/ denaturation by heat
References
1. Di Lorenzo, A. and M.T. Bedford, Histone arginine methylation. FEBS Letters, 2011. 585(13): p. 2024-2031.
2. Friesen, W.J., et al., A novel WD repeat protein component of the methylosome binds Sm proteins. J Biol Chem, 2002. 277(10): p.
8243-7.
3. Furuno, K., et al., Association of Polycomb group SUZ12 with WD-repeat protein MEP50 that binds to histone H2A selectively in vitro.
Biochem Biophys Res Commun, 2006. 345(3): p. 1051-8.
4. Hosohata, K., et al., Purification and Identification of a Novel Complex Which Is Involved in Androgen Receptor-Dependent
Transcription. Molecular and Cellular Biology, 2003. 23(19): p. 7019-7029.
5. Le Guezennec, X., et al., MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties.
Mol Cell Biol, 2006. 26(3): p. 843-51.
6. Licciardo, P., et al., The FCP1 phosphatase interacts with RNA polymerase II and with MEP50 a component of the methylosome
complex involved in the assembly of snRNP. Nucleic Acids Res, 2003. 31(3): p. 999-1005.
7. Margueron, R., et al., Role of the polycomb protein EED in the propagation of repressive histone marks. Nature, 2009. 461(7265): p.
762-7.
8. Peng, Y., et al., Androgen receptor coactivator p44/Mep50 in breast cancer growth and invasion. Journal of
Cellular and Molecular Medicine, 2010. 14(12): p. 2780-2789.
9. Tee, W.W., et al., Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency. Genes &
Development, 2010. 24(24): p. 2772-2777.
Western blot
2. Purification of WDR77 and check its binding with PRMT5 and PRMT7
Figure 3 - H3K27me3 binding by EED (left and middle) and H3R2me2s binding by WDR5 (right), as shown
from from crystal structures both at 1.9A. From Margureon et al. (2009) (left and middle) and Migliori et al.
(submitted) (right).
Constructs (Ernesto lab IMCB, Singapore)
1. WDR77-GST – pGEX6P1
2. PRMT7-GST – pGEX
3. PRMT5-His- pET32a
Acknowledgements
I would like to thank Dr. Martin Walsh for his invaluable guidance. I would also like to
thank all members of the Walsh lab for helpful discussions about experiments and
their unstinted support and encouragement.