Download Example 2. - the Gene Ontology Consortium Wiki

General information that should be discussed and agreed for
guidelines
For a long time scientists have agreed that the level of an mRNA does not directly
correlate with the level of a protein and this is, for the most part, reflected in the way
that curators annotate experimental data. Similarly, the level of mRNA expression
(which can be modulated e.g. by microRNAs) is not necessarily equivalent to the level of
transcription; however, curators sometimes do not capture this distinction, especially
when they take into consideration authors’ interpretation of results, i.e. the authors’
intent. With this in mind we would like to have an open discussion about how mRNA
expression data is annotated.
Objectives:
(1) To understand how curators annotate non-high-throughput mRNA expression data;
(2) To discuss how to improve consistency when annotating mRNA expression
experiments.
Example 1.
Re: PMID: 19805038, Figure 2.
Curator note: Human T-box protein 21 (T-bet) summary: Transcription factor that
controls the expression of the TH1 cytokine, interferon-gamma. Initiates TH1 lineage
development from naive TH precursor cells both by activating TH1 genetic programs and
by repressing the opposing TH2 programs. (http://www.uniprot.org/uniprot/Q9UL17).
In this example we are interested in how you would annotate the regulation of
expression of targets of T-box protein 21 (T-bet).
Abstract
Upon detection of antigen, CD4+ T helper (Th) cells can differentiate into a number of
effector types that tailor the immune response to different pathogens. Alternative Th1
and Th2 cell fates are specified by the transcription factors T-bet and GATA-3,
respectively. Only a handful of target genes are known for these two factors and
because of this, the mechanism through which T-bet and GATA-3 induce differentiation
toward alternative cell fates is not fully understood. Here, we provide a genomic map of
T-bet and GATA-3 binding in primary human T cells and identify their target genes, most
of which are previously unknown. In Th1 cells, T-bet associates with genes of diverse
function, including those with roles in transcriptional regulation, chemotaxis and
adhesion. GATA-3 occupies genes in both Th1 and Th2 cells and, unexpectedly, shares a
large proportion of targets with T-bet. Re-complementation of T-bet alters the
expression of these genes in a manner that mirrors their differential expression
between Th1 and Th2 lineages. These data show that the choice between Th1 and Th2
lineage commitment is the result of the opposing action of T-bet and GATA-3 at a shared
set of target genes and may provide a general paradigm for the interaction of lineagespecifying transcription factors.
Figure 2. T-bet activates the expression of its target genes. (A) Relative levels of
expression of T-bet target genes in primary stimulated Th1 cells compared with Th2 cells
(column 1) and in secondary stimulated Th1 compared with Th2 cells (column 2). Each
row represents a single gene. Genes are ordered by their relative expression level in Th1
cells versus Th2 cells and this average expression ratio is shown on the left hand side.
The colors represent relative gene expression levels between Th1 and Th2 cells, with
shades of red indicating higher expression in Th1 cells, black equal expression levels and
shades of green indicating higher expression in Th2 cells. (B and E) T-bet ChIP signals at
NKG7 and CCL4, in human Th1 cells (green) and Th2 cells (blue), and signal from an IgG
control IP in Th1 cells (black). Details as for Fig. 1B. (C and F) Real time PCR for NKG7 and
CCL4 in WT (open bars) and T-bet−/− (filled bars) murine T cells. Cells were unstimulated
(u/s), stimulated with anti-CD3 antibodies (αCD3), or stimulated with anti-CD3
antibodies and anti-CD28 antibodies (αCD3/28). RNA abundance is relative β-actin RNA.
(D and G) Real time PCR for NKG7 and CCL4 in CD4+ T cells from T-bet−/−× IFN-γ−/−
murine CD4+ T cells transduced with empty vector (open bars) or T-bet (filled bars)
expressing retrovirus. (H) Percentage of human Th1 genes whose orthologs are
upregulated (at least 1.5-fold, black bars) or downregulated (white bars) in murine Tbet−/−× IFN-γ−/− CD4+ cells upon expression of exogenous T-bet. Th1 genes were
defined as those expressed at least 2-fold or higher in Th1 cells than Th2 cells and
divided into those bound by T-bet or those not bound by T-bet.
Example 2.
Re: PMID: 24296617, Figure 2.
Curator note: miR-19b is predicted to target CTGF for silencing (Fig. 3a not shown
here).
Human AngII summary: A peptide hormone that is an essential component of the
renin-angiotensin system, a potent regulator of blood pressure, body fluid and
electrolyte homeostasis.
AngII acts directly on vascular smooth muscle as a potent vasoconstrictor (Full
UniProt description: http://www.uniprot.org/uniprot/P01019).
In this example we are interested in how you would annotate the regulation of CTGF
expression by AngII.
Abstract
Objectives: The present study was designed to decipher the molecular mechanisms
underlying angiotensin (Ang) II-induced overexpression of connective tissue growth
factor (CTGF) in cultured cardiomyocytes. Methods: Cardiomyocytes isolated from
1- to 3-day-old neonatal rats were cultured and treated with 100 nM [human] Ang II
with or without pretreatment with 10 nM telmisartan, an Ang II type 1 receptor
antagonist. The role of microRNA (miR)-19b in the regulation of Ang II-induced CTGF
expression was evaluated in cultured cardiomyocytes with quantitative real-time
reverse transcription polymerase chain reaction and Western blot
analysis. Results: We provide several lines of evidence to show that miR-19b
contributes to the Ang II-induced overexpression of CTGF in cultured
cardiomyocytes. Firstly, administration of Ang II decreased the level of miR-19b
dramatically (p < 0.05 vs. control), which was abolished by telmisartan. Secondly,
Ang II increased the level of CTGF significantly (p < 0.05 vs. control), which was also
prevented by pretreatment with telmisartan. Thirdly, overexpression of miR-19b
decreased CTGF levels (p < 0.05 vs. control). Finally, transfection of miR-19b into
cardiomyocytes prevented the upregulation of CTGF induced by Ang
II. Conclusion: Downregulation of miR-19b contributes to Ang II-induced
overexpression of CTGF in cultured cardiomyocytes.
“As shown in figure 2 a and b, CTGF mRNA expression was significantly increased after
Ang II stimulation (p < 0.05 vs. control group). When compared with the Ang II-treated
group, the induction of CTGF gene expression was prevented by preincubation with
telmisartan (p < 0.05). The result of Western blot showed that Ang [angiotensin] II
regulated the expression of CTGF [connective tissue growth factor] at protein level, and
preincubation with telmisartan prevented the effect of Ang II on CTGF expression (fig. 2
c, d). These results suggest a mechanism that mediates the regulation of expression of
CTGF genes by Ang II via an Ang II type 1 receptor-dependent (AT 1 R-dependent)
pathway.”
Example 3.
Re: PMID:20675382, Figure 2.
Curator note: CYR61 is known to bind the extracellular matrix, heparin and integrins.
CYR61-mediated gene regulation is dependent on heparin-binding. (Full UniProt
description: http://www.uniprot.org/uniprot/O00622).
In this example we are interested in how you would annotate the regulation of BMP-2
expression by CYR61
"CYR61 Induced BMP-2 Production in Osteoblast Cells.
Given the crucial role of BMPs in osteoblastic differentiation, we tested whether CYR61
mediated alteration of osteoblast proliferation and differentiation through regulation of
BMP expression. According to Cheng et al. (43), BMP-2, -6, and -9 may be the most
potent molecules that induce osteoblast lineage-specific differentiation. Therefore, we
explored possible target gene expression during rCYR61-induced osteoblastic
differentiation. We examined expression levels of members of the BMP family by RTPCR in MC3T3-E1 osteoblast cells in response to recombinant CYR61 (rCYR61)
treatment. Treatment with rCYR61 stimulated BMP-2 expression in a time-dependent
manner by RT-PCR and Western blot analysis (Fig. 2, A and D). A significant increase in
BMP-2 mRNA could be detected as early as 2 h after rCYR61 treatment. Elevation of
BMP-2 mRNA was maximal at 8 h and lasted until 24 h after treatment. Expression level
of other BMP mRNAs, such as BMP-4, -6, -7, and -9, were not significantly affected by
rCYR61 (Fig. 2A). In addition, the stimulatory effect of rCYR61 on BMP-2 expression was
dose-dependent in the range between 10 and 200 ng/ml (Fig. 2, B and C). The BMP-2
protein expression level in response to rCYR61 treatment was increased at 4~24 h after
treatment (Fig. 2D). Furthermore, we found rCYR61 induced BMP-2 secretion into
cultured medium of both MC3T3-E1 cells and primary osteoblasts by BMP-2 ELISA assay
(Fig. 2E). These data indicate that rCYR61 induces expression of BMP-2 in osteoblast
cells in a time- and dose-dependent manner."...
"we hypothesized that, as a positive extracellular signaling protein, CYR61 controls
osteoblast functions by regulating BMP2 gene expression in osteoblasts. This study was
designed to test this hypothesis and also determine the precise signaling mechanisms
involved in CYR61 regulation of BMP2 transcription in osteoblasts.”
Figure 2. Recombinant CYR61 induces BMP-2 expression. A and B, RT-PCR for mRNA of
different BMP family members is shown. The coding regions of cDNA were used as
probes as indicated under “Experimental Procedures.” A GAPDH probe was used as an
internal control for RNA quantity. C, MC3T3-E1 cells were incubated with rCYR61 for 8 h,
after which mRNA expression of BMP-2 was determined using qPCR. D, shown is
Western blot analysis of BMP-2 protein expression in MC3T3-E1 cells. Total proteins
were extracted from cells and probed with polyclonal antibody specific for BMP-2. Each
lane contains 80 μg of total protein. The internal loading control was β-actin. E, shown is
an ELISA assay of BMP-2 protein expression in MC3T3-E1 cells (left) and primary
cultured osteoblasts (right). MC3T3-E1 cells were incubated with the indicated dose of
rCYR61 for 24 h. The protein secreted into conditioned medium was determined by
BMP-2 ELISA assay kit. All the results except those in D are expressed as the means ±
S.E. Each assay was performed in three separate experiments. The asterisk indicates p <
0.05 between rCYR61 treatment and vehicle treatment cells.