Annotation Conf. Call, February 24, 2015
Annotation Consistency Exercise
Here is a link to the paper that I've chosen for the first annotation consistency exercise/discussion:
It describes studies on C. elegans bcl-7 (http://www.wormbase.org/species/c_elegans/gene/WBGene00016192) and human BCL7B (http://www.uniprot.org/uniprot/Q9BQE9).
The main thing I'd like to focus on for the annotation exercise is what Biological Process annotations curators make, noting that, at the moment, the all-encompassing Biological Process terms might not yet exist for what is described in the paper. In particular, I'd like to discuss annotating to a process vs. regulation of a process, and the use of annotation extensions to provide more context for the BP terms.
I've included some links to entries on the anatomy terms and phenotypes described in the paper, in case they're helpful:
- Seam cells
- During larval development, seam cells divide to generate new seam cells renew as well as a number of differentiated cell types (hypodermal/epidermal cell, neuron, glia). They stop renewing once the animals reach adulthood and do not appear to reside in a stem cell-like 'niche'.
- Somatic gonad development
- Egl phenotype
- Pvl phenotype
- Burst phenotype
- Alae morphology phenotype
Note that the paper starts with gross anatomical defects and then progresses to a more detailed characterization of the phenotype. This is fairly typical.
Summary of Phenotypes Observed
|Process||Phenotype - C. elegans bcl-7||Phenotype - Human BCL7B|
|Apoptosis||increased expression of anti-apoptotic factor in mutant animals; no organismal changes||increased apoptosis from overexpression; increased survival with knockdown and actinomycin D treatment|
|Cell Cycle||decreased number of mitotic germ cells||increased number of cells in G0/G1 phase|
|Cell Differentiation||increased expression of markers associated with undifferentiated state||increased expression of markers associated with undifferentiated state|
|Gene Expression||increased expression of a number of different genes - Wnt pathway, Notch pathway, apoptosis||increased expression of a number of different genes - pluripotency markers, Wnt pathway, apoptosis|
|Nuclear Organization||larger nuclei, irregularly shaped||larger nuclei, irregularly shaped, multinucleate cells|
|Tissue/Organ Development||epidermal defects, gonad defects||n/a|
|Wnt Signaling Pathway||increased expression of beta-catenins, mis-localization of WRM-1 beta-catenin, mis-localization of POP-1 TCF/LEF||increased expression of beta-catenin, HMGA1|
Summary of Molecular Markers Affected in bcl-7 Mutant Animals
|Gene/Marker Name||Molecular Identity||Anatomical Expression Pattern||Result|
|scm::gfp||not known||nuclei of seam cells, all larval stages||reduced number except for early L1 larval stage|
|cdh-3::gfp||cadherin||cytoplasm of seam cells||reduced number|
|col-19::gfp||collagen||hypodermal syncytium (hyp7) and seam cells in adult stage||decreased in hyp7 (not significant), decreased in seam cells (significant)|
|des-2::gfp||nicotinic acetylcholine receptor||PVD neurons||no extra neurons|
|dat-1::gfp||dopamine transporter||PDE neurons||no extra neurons|
|egl-27::mCherry||MTA1 homolog, NODE complex||strong in intestine, weak in epidermis in L4 larval stage||increased expression ubiquitously, but especially in seam cells and hyp7; increased expression - qRT-PCR|
|ceh-6||POU-homeodomain transcription factor, NODE complex||not reported in this paper, but head and tail region, excretory cell, neurons, vulva - dynamic expression pattern||increased expression - qRT-PCR|
|histone H3 (anti-phospho-histone, PH3)||chromatin||mitotic germ line||decreased number of mitotic cells, further from source of proliferative signal (distal tip cells, DTCs)|
|lag-2||Notch ligand DSL (Delta, Serrate, LAG-2)||2 distal tip cells (DTCs)||loss of expression in one distal tip cell|
|wrm-1||beta-catenin||seam cells, somatic gonad precursors (SGPs, distal tip cell parents)||increased expression (not significant) - qRT-PCR|
|bar-1||beta-catenin||not discussed in this paper||increased expression (significant) - qRT-PCR|
|sys-1||beta-catenin||not discussed in this paper||increased expression (significant) - qRT-PCR|
|WRM-1::GFP||beta-catenin||anterior cortex of mother seam cell at L2; higher in posterior daughter cell||anterior daughter cells higher or equal to posterior daughter cell (50% of animals)|
|POP-1::GFP||TCF/LEF transcription factor||high anterior seam cell daughter confers non-seam fate, low posterior seam cell daughter confers seam fate||increased in posterior seam daughter|
|POP-1::GFP||TCF/LEF transcription factor||higher in Z1.p and Z4.a (proximal, P)||proximal cells equal to, or lower, than distal (D) cells|
|GFP::HLH-2||basic helix-loop-helix transcription factor||present in both distal tip cells (DTCs)||decreased expression - seen in only one or no DTCs|
|ced-9||anti-apoptosis factor||not described in paper||increased expression (significant), qRT-PCR|
Summary of Molecular Markers Affected in BCL7B Knockdown in KATOIII cells
|Gene/Marker Name||Molecular Identity||Result|
|Nanog||homeodomain transcription factor - maintains pluripotency||increased - qRT-PCR|
|Oct3/4||POU domain transcription factor - expressed in ES cells||increased - qRT-PCR|
|Sox2||Sry-related transcription factor - marker for pluripotency||increased - qRT-PCR|
|NEAT1||lncRNA - core molecule of nuclear paraspeckles||increased - qRT-PCR|
|beta-catenin||Wnt signaling pathway||increased - qRT-PCR|
|HMGA1||high mobility group protein - Wnt signaling pathway target||increased - qRT-PCR|
|c-FLIP||apoptosis inhibitor||increased - qRT-PCR|
|Bcl2||apoptosis inhibitor||increased - qRT-PCR|
|PTEN||lipid phosphatase - pro-apoptotic factor||no significant change - qRT-PCR|
|Bax||pro-apoptotic factor||increased - qRT-PCR (not as great an increase as Bcl2)|
|Gene/Marker Name||GO term||Comment||Evidence Code||Annotation Extension||Comment||Annotation Extension||Comment||Annotation Extension||Comment|
|bcl-7||nucleus||IDA||part_of||range of cell types were filled in, including: seam cells, hyp7, neurons, intestine, distal tip cell, germ cell, gonadal sheath cell|
|egl-27||nucleus||IDA||part_of||intestine, epidermis - but immunofluorescence experiment was actually performed with the egl-27 promoter only||during||L4 stage|
|pop-1||nucleus||IDA||part_of||Z1 and Z4, hermaphrodite somatic gonad cell, Z1.p|
|wrm-1||cell cortex||IDA||part_of||seam cell||exists_during||L2 stage (note that wiki documentation states that GO:BP is allowed value for exists_during)|
|bcl-7||cell differentiation||regulation of cell differentiation? new term: seam cell differentiation?||IMP||results_in_acquisition_of_features_of (results_in_differentiation_of?)||seam cell, hyp7?||happens_during||L1 stage (but text said 'except L1 stage'?)||part_of||asymmetric cell division|
|bcl-7||cell differentiation||IMP||acts_on_population_of||seam cell|
|bcl-7||cell differentiation||regulation of cell differentiation?||IMP||results_in_acquisition_of_features_of (results_in_differentiation_of?)||distal tip cells, sister cells?||part_of||asymmetric cell division|
|bcl-7||positive regulation of cell differentiation||IMP||results_in_specification_of||distal tip cells||during||comma stage, early L1 stage|
|bcl-7||stem cell development||IMP|
|bcl-7||stem cell differentiation||IMP|
|bcl-7||asymmetric cell division||IMP||occurs_in||seam cell, hermaphrodite somatic gonad cell|
|bcl-7||asymmetric stem cell division||IMP||occurs_in||seam cell|
|bcl-7||somatic stem cell maintenance||IMP|
|bcl-7||regulation of epidermis development||IMP|
|bcl-7||regulation of epidermal cell differentiation||IMP|
|bcl-7||positive regulation of epidermal cell differentiation||IMP|
|bcl-7||positive regulation of epidermal cell fate specification||IMP|
|bcl-7||epithelial cell development||IMP||results_in_development_of||seam cell|
|bcl-7||external genitalia morphogenesis||IMP|
|bcl-7||hermaphrodite genitalia morphogenesis||IMP|
|bcl-7||regulation of vulval development||IMP|
|bcl-7||distal tip cell migration||placeholder - would prefer to have new term: 'distal tip cell differentiation'||IMP|
|bcl-7||multi-organism reproductive process||IMP|
|bcl-7||germ cell migration||IMP|
|bcl-7||germline cell cycle switching, mitotic to meiotic cell cycle||IMP||part_of||gonad development|
|bcl-7||oocyte fate determination||IMP|
|bcl-7||regulation of gene expression||IMP|
|bcl-7||negative regulation of gene expression||new term? negative regulation of Wnt signaling pathway by negative regulation of gene expression||IMP||has_regulation_target||bar-1, sys-1, wrm-1||part_of||negative regulation of Wnt signaling pathway|
|bcl-7||negative regulation of gene expression||IMP||has_input||bar-1, sys-1, wrm-1, egl-27, ceh-6|
|bcl-7||negative regulation of gene expression||IMP||has_regulation_target||ced-9|
|BCL7B||negative regulation of gene expression||IMP||has_regulation_target||beta-catenin, HMGA1|
|bcl-7||regulation of RNA metabolic process||IMP|
|bcl-7||regulation of Wnt signaling pathway||IMP|
|bcl-7||negative regulation of Wnt signaling pathway||IMP|
|BCL7B||negative regulation of Wnt signaling pathway||IMP|
|bcl-7||negative regulation of Wnt signaling pathway||With/From: wrm-1 or lsy-22 (suppressor screen)||IGI|
|bcl-7||negative regulation of Wnt signaling pathway||IEP||Are cell lines used for annnotation extensions?|
|bcl-7||negative regulation of canonical Wnt signaling pathway||With/From: negative regulation of gene expression||IC|
|BCL7B||negative regulation of canonical Wnt signaling pathway||IMP|
|bcl-7||cellular protein localization||IMP|
|bcl-7||asymmetric protein localization involved in cell fate determination||IMP||has_input||WRM-1|
|wrm-1||protein localization to nucleus||NAS||has_regulation_target||POP-1|
|bcl-7||regulation of protein localization to nucleus||IMP||has_regulation_target||wrm-1, pop-1||part_of||asymmetric cell division|
|bcl-7||regulation of nucleus organization||IMP|
|bcl-7||regulation of nucleus size||better to use regulation of nucleus size involved in G1 to G0 transition?||IMP||part_of||hyp7|
|BCL7B||regulation of nucleus organization||IMP|
|bcl-7||positive regulation of apoptotic process||IMP|
|BCL7B||positive regulation of apoptotic process||IMP|
|bcl-7||negative regulation of apoptotic process||IMP|
|BCL7B||regulation of cell cycle||IMP|
|BCL7B||regulation of G1/S transition of mitotic cell cycle||IMP|
|BCL7B||positive regulation of G1/S transition of mitotic cell cycle||IMP|
Possible New GO Terms
- seam cell development
- seam cell division
- seam cell asymmetric division
- seam cell differentiation
- seam cell terminal differentiation (note that 'terminal differentiation' is a related synonym of 'cell development')
- negative regulation of Wnt signaling pathway by negative regulation of gene expression
- negative regulation of Wnt signaling pathway by regulation of asymmetric protein localization
- regulation of epidermis development by regulation of seam cell differentiation (or terminal differentiation)
- regulation of gonad development by regulation of distal tip cell differentiation
- regulation of germ cell development by regulation of distal tip cell differentitation (may be overstating what is shown)
- positive regulation of apoptosis by negative regulation of gene expression
- Wnt/beta-catenin asymmetry signaling pathway (and appropriate regulation terms)
- When to use development vs. differentiation vs. asymmetric cell division?
- Is regulation of expression of genes known to be involved in a process sufficient evidence for regulation of that process? What more evidence might be needed?
- How much understanding of mechanism of action is needed for annotating to regulation of a process vs to the process? What about positive regulation of a process vs regulation of a process, especially at the level of differentiation, development, specification?
- How much understanding of mechanism is needed for assessing downstream effects, e.g. nucleus organization?
- For the cell differentiation which is the right relationship to indicate type of cell in col-16: results_in_acquistion_of is the right one. 'acts_on_population' should be used for behavior of a population of cells.
- Can we use stem cells for seam cells? Are they the same?
- DH: stem cells is fine to use here because terminal differentiation of stem cells is being described here.
- Suzi, David: we should make sure seam cells are defined to match CL ontology.
- Is differentiation part of division?
- aysmmetric cell division is not always part of development.
- How is asymmetric defined? Is spatial asymmetry? This needs to be clarified in GO.
- Annotating C. elegans bcl-7 to WNT signalling pathway. Evidence is not direct. But one can look at the human data and make that inference.
- Annotating to nuclear organization: nucleus changes under so many conditions. Hard to make a call that the gene is involved in nuclear organization. Do not make this annotation.
About the tool
- couldn't see the figures. This was a big drawback.
- couldn't edit parts of an annotation
- special characters
- should provide ability to suggest new GO terms
We will present an overview of various ontology files, file format, annotation file format pipeline
Follow-Up to Exercise
Annotating to Development Terms - Parent Term or a Regulation Term?
- For terms like 'cell differentiation' when is it appropriate to use the parent term, versus regulation or positive or negative regulation?
- In many (most?) cases, the experimental assays used to annotate to the different terms are the same, but the result, or outcome, is different. For example, to assay cell differentiation, the expression or activity of a gene product is manipulated and the resulting effect on cells is determined by examining morphological and/or molecular markers.
- From these experiments, the authors infer a role for a gene product in promoting and/or inhibiting cell differentiation.
- If the positive and negative terms in GO are meant to capture the promotion or inhibition of cell differentiation, respectively, then these are the terms that should be used for annotation.
- This would provide users with the information they probably really want to know: does the gene I'm interested in promote or inhibit differentiation of a particular cell type? If so, is it known through what mechanism?
- Annotation extensions could then be used to capture the specific cell types and markers affected by each gene product.
- The parent term and parent regulation term would get a 'do not manually annotate' flag.
Is it Development, Determination, Commitment, Specification, Differentiation, or Terminal Differentiation?
- See: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2830379/
- Classical embryology experiments defined various stages of cell development based upon how cells behaved when removed from their normal environment and placed in a new one.
- Experiments performed in model organisms or cell lines lead authors to draw conclusions about the role of gene products in these various developmental stages, even if the authors don't replicate the 'changed environment' types of experiments.
- How can curators decide what is the correct term to use?
- Differentiation - When the activity or expression of the gene product of interest is manipulated, does the cell lose expression of some or all cell type-specific markers? If the cell loses expression of some markers, but not all, this is likely an effect on differentiation.
- Terminal Differentiation - In GO, 'terminal differentiation' is a synonym of 'cell development' which is part_of 'cell differentiation'. In the literature, there is sometimes an association between 'terminal differentiation' and exit from the cell cycle.