SHML
Specific for Human Melanocyte Lineage
Dr. Hilary (Billy) Sheppard Dunbar Laboratory
• Originally identified as novel gene specifically expressed in melanoma
• Homologues in chimpanzee and rhesus monkey only
• Originally listed as hypothetical protein
• We identified homology to FRG2
SHML
ORF1 aligned to FRG2 – NLS? Putative P site? TF?
ORF2 aligned to FRG2 – targeted to exocytosis?
SHML expression in melanoma cell lines
SHML expression in skin
Skin 1 Skin 2 Skin 3
Where is SHML expressed intracellularly?
ORF1
punctate, cytoplasmic, near nucleus ORF2 (red = tubulin)
punctate, throughout cytoplasm HEK 293T
Where is SHML expressed intracellularly?
ME275
ORF2 - punctate, throughout cytoplasm
Can we detect endogenous SHML?
SHML ORF1 PAb
2 L ORF-1
56.9 kDa
ME275 DAGI SK-Mel29
Can we detect endogenous SHML?
2ndry alone ORF1 purified PAb DAPI merge
DAGI cells x 100
2ndry alone ORF1 purified PAb DAPI merge
Can we detect endogenous SHML?
ME275 cells x 100
SHML function?
• Knock down expression in DAGI (siRNA) microarray 54,000 probe sets
• 56 genes = greater than 2 fold change
• Dickkopf 1 (DKK1) most significant hit
* p<0.05 two tailed t test
Negative siRNA
SHML siRNA
100 X
SHML KD in p.1 melanocytes
**p<0.01 ***p<0.001 two tailed t test
DKK1
DKK1
DKK
SHML
SHML and the canonical Wnt signalling pathway
• Alterations associated with cancers
• Important in stem cell maintenance and proliferation
• Does SHML activate stem cell like properties in melanomas?
More functional studies Overexpression using AAV
AAV1 105 AAV1
104 AAV1
103 AAV2 105 AAV2
104 AAV2
103 AAV5 105 AAV5
104 AAV5
103 AAV6 105 AAV6
104 AAV6 103 0
2 4 6 8 10 12 14 16
4 best serotypes at a range of MOI
% EGFP pos cells
M03 M14 M23 M26 M34 M38 M47 M48 M49 M51 M38 1
10 100 1000
TILN derived lines normalised to HPRT
SHML MelanA Tyr
Identification of SHML reactive T cells
10/21 TILNS +ve SHML Expand to useable nos.
SHML peptides – IFN g assay
ORF1MGGFGNEGRWELCWRVSHSHASFAGICPGEEHSLMLKKKLKSSTLHSNKLKETCDAHHRGPF MTHTGCTKQHRSQTLGVQWLSFQKSLVTLQAVSRPFIKTYTGYRCSRSILH
5x29-mers
01-29 MGGFGNEGRWELCWRVSHSHASFAGICPG 22 - 50 SFAGICPGEEHSLMLKKKLKSSTLHSNKL 43 - 71 STLHSNKLKETCDAHHRGPFMTHTGCTKQ 64 - 92 THTGCTKQHRSQTLGVQWLSFQKSLVTLQ 85 - 113 QKSLVTLQAVSRPFIKTYTGYRCSRSILH
ORF2MAVISEKLGDFTSCVKAIYQDLYWVSVQQVHSPLTWEQLTQLARFWRPLCAQVQTFYFIAT QVGYVFPAKNWLVSATLPGPGDPALDREAHPLPGQEITEPVSGSDEA
5x28-mers
01-28 MAVISEKLGDFTSCVKAIYQDLYWVSVQ
21-49 DLYWVSVQQVHSPLTWEQLTQLARFWRPL
41-69 QLARFWRPLCAQVQTFYFIATQVGYVFPA
61-88 TQVGYVFPAKNWLVSATLPGPGDPALDR
81-108 PGDPALDREAHPLPGQEITEPVSGSDEA
IFN g assay suggests presence of SHML responsive T cells
Can we clone them?
Neg UL% UR%
34 0.66
ELA UL% UR%
40 1.57
ORF2 UL% UR%
41 0.26
ORF1 UL% UR%
40 1.54
Conclusions
• Identified and characterised a new gene, SHML,
expressed in human cells of melanocyte lineage
• SHML levels affects DKK1 expression,
and may be involved in the Wnt pathway
• We have identified putative SHML reactive CD8+ T cells
Future Work
• Clone the SHML reactive T cells/killing assays
• Further expression studies using MAbs
• Functional studies - overexpression
Acknowledgements
Lottery Health Research
Geoff Williams, Margaret Brimble
Vaughan Feisst Anna Brooks Jenni Chen
Rod Dunbar
Oliver Horlacher, James Ussher