Chapter 5: GRIPE is a novel gene that may assume different roles during development and in adulthood Chapter 5: GRIPE is a novel gene that may assume different roles during development and in adulthood
5.1. Characterisation of GRIPE and E12 mRNA expression during development
As a first step towards describing the expression of GRIPE and E12 during neurodevelopment, Northern analysis was conducted to acquire a global pattern of mRNA expression. To do this, embryonic and adult tissues were harvested for RNA isolation as described (Section 2.2). Northern hybridisation was then performed using radiolabelled GRIPE and E12 cDNAs as probes, and these results are shown in Fig. 5.1.
A single band of approximately 8kb is detected in all samples; this was the only signal detected in all northern hybridisations performed with different GRIPE cDNA and cRNA probes (see Appendix 10.2). It would appear that GRIPE mRNA is abundant in the head and trunk at e11.5, and levels decrease during embryogenesis, and are undetectable by e17.5. In the adult, GRIPE is abundant in the brain, but is undetectable in the placenta and kidney. However, further RT-PCR experiments (see Figs 6.8 and 6.13) indicate that GRIPE is weakly detectable in these tissues, but not in the kidney.
Northern analysis of E12 mRNA shows high levels of expression at e11.5, with a gradual decrease during embryogenesis, and is almost undetectable by e17.5. Further, E12 is undetectable in adult brain, placenta and kidney. These observations demonstrate a coincidence in expression of GRIPE and E12 mRNA during embryogenesis, but not in the adult; where E12 mRNA is undetectable. In consideration of their protein- protein interaction described in the previous chapter, these observations bring up the possibility that the interaction between GRIPE and E12 may be important during neurogenesis, but not in the adult central nervous system.
5.2. GRIPE is an approximately 8kb polyadenylated mRNA
The size of the mature GRIPE transcript was further investigated by Northern analysis, performed with isolated polyadenylated mRNA. Messenger RNA was isolated using a standard oligo-d(T) cellulose method and Northern hybridisation performed as described (section 2.14). As shown in Fig. 5.2, the only detectable GRIPE mRNA signal is approximately 8 kb in size. In a pattern consistent with Northern analysis of total RNA (Fig. 5.1), the signal is high in e11.5 head and trunk (lanes 1 and 2 respectively), as well as in adult brain (lane 5), but is weakly detectable in e17.5 tissues (lanes 3 and 4). Further, the hybridised signal in total RNA derived from e11.5 trunk (lane 6) is identical to that detected in oligo-d(T)
65 e11.5 e12.5 e13.5 e14.5 e15.5 e17.5 h t h t h t h t h t h t ab pl k GRIPE
E12
18S rRNA
Figure 5.1. Characterisation of GRIPE and E12 mRNA expression. Northern blot of GRIPE transcript in total RNA isolated from mouse embryonic head (h) or trunk (t) at different stages of embryogenesis. Levels of GRIPE decrease during development, but remain high in adult brain (ab), and is low in placental (pl) and adult kidney (k) RNA. GRIPE expression correlates with the expression of E12 mRNA during embryogenesis.
1 2 3 4 5 6
28S
18S
5S
GAPDH
Figure 5.2. GRIPE is expressed as an approximately 8kb polyadenylated mRNA. Messenger RNA was isolated from 500µg of total RNA and electrophoresed on an agarose-formaldehyde gel before Northern blotting. GRIPE mRNA signal (arrow) is enriched after mRNA selection (lane 2), with respect to the same signal in total RNA from the same source of tissue (lane 6). Signals for GAPDH confirmed enrichment for mRNA after oligo-d(T) selection (compare lanes 2 and 6). Lane 1: 5µg e11.5 head poly A+ mRNA, Lane 2: 5µg e11.5 trunk poly A+ mRNA, Lane 3: 5µg e17.5 head poly A+ mRNA, Lane 4: 5µg e17.5 trunk poly A+ mRNA, Lane 5: 5µg adult brain poly A+ mRNA, Lane 6: 15µg total RNA from e11.5 trunk.
65i Chapter 5: GRIPE is a novel gene that may assume different roles during development and in adulthood purified mRNA preparations (compare lanes 2 and 6). Taken together, these data indicate that the mature
GRIPE transcript is a polyadenylated 8kb mRNA, and is the only signal detected by Northern analysis.
5.3. The interaction of GRIPE and E12 may be important for early neurogenetic events, but not in the adult brain.
To identify sites of GRIPE and E12 mRNA expression, in situ hybridisations were performed on 10µm sections of embryonic mouse tissue and adult brain (described in section 2.16). At e11.5, GRIPE and E12 are coincidentally detected in the ventricular zone of the developing forebrain, as well as in the mandibular arch (Figs. 5.3A,B; short and long arrows respectively). Parallel experiments conducted with corresponding sense control probes confirmed the detection of specific signals (Figs. 5.3C and D for
GRIPE and E12 respectively). In addition, tissue sections incubated without probe during hybridisation ruled out non-specific binding of DIG antibody and alkaline phosphatase activity (data not shown). In situ hybridisation experiments carried out with sections of e13.5 and e15.5 embryos were not successful.
In adult brain, GRIPE displays a widespread expression pattern, with prominent staining in the CA formation and dentate gyrus of the hippocampus (Fig. 5.4A, short arrow), as well as in deep layer neurons of the cerebral cortex (Fig. 5.4A, arrowhead), and in the frontal cortex (Fig. 5.4C, arrow). No specific staining was detected in the adult cerebellum (not shown). Experiments performed with coronal sections of adult mouse brain reveal staining in neurons of the medial habenula nuclei (Fig. 5.5A, arrowhead), piriform cortex and posterior basomedial amygdala (arrow and arrowhead in Fig.5.5B, respectively).
In contrast to GRIPE expression, E12 mRNA is undetectable in adult brain (Fig. 5.4B, arrowhead and arrow); an observation which is consistent with the results from Northern analysis. Taken together, these results suggest that the interaction of GRIPE and E12 may be functionally more significant during the neurogenetic period rather than in the adult. In particular, ventricular zone cells in the developing forebrain may require an intact GRIPE/E12 signalling pathway for proper migration and differentiation.
66 A B
C D
GRIPE E12
Figure 5.3. Localisation of GRIPE and E12 RNA. (A) In situ hybridisation reveals that GRIPE is expressed in most tissues of the e11.5 mouse embryo, including the ventricular zone of the neuroepithelium (short arrow) and mandibular arch (long arrow), but is absent in the heart (arrowhead). Similarly, E12 RNA is found in similar tissues (B). Panels C and D are sense probe controls for GRIPE and E12, showing the absence of specific staining. Scale bar represents 100 µm.
66i A GRIPE B E12
B C
D
C GRIPE
Figure 5.4. Localisation of GRIPE RNA in adult mouse brain. In situ hybridisation on sagittal sections of adult mouse brain show that GRIPE is expressed in the CA formation of the hippocampus (panel A, short arrow). In addition, staining is detected in deep layer neurons of the cerebral cortex (arrowhead in B), as well as in neurons of the frontal cortex (arrow in panel C). Conversely, in situ hybiridsation with an E12 probe reveals lack of expression in adult brain. Parasagittal section of rodent brain on Panel D as a guide for location of brain slices in A-C (from Swanson, 1992)
A
A GRIPE B
Figure 5.5. In situ hybridisation of coronal sections of adult brain. Coronal sections of adult brain indicate that GRIPE is expressed in neurons of the cerebral cortex, hippocampus and medial habenula nuclei (panel A, long arrow, short arrow and arrow head respectively).Furthermore, neurons of the priform cortex and posterior basomedial amygdala also express GRIPE (panel B, arrow and arrowhead respectively). Panel C depicts a coronal section of rodent brain for orientationof tissue slices in A and B (from Swanson, 1992).
B GRIPE 66ii Chapter 5: GRIPE is a novel gene that may assume different roles during development and in adulthood
5.4. GRIPE mRNA is upregulated in differentiating neurons.
To further investigate the association of GRIPE/E12 expression with neurogenesis, an in vitro approach was adopted. The mouse embryocarcinoma cell line P19 was induced to differentiate into neurons using retinoic acid, thereby producing an enriched population of neurons in culture (see Section 2.17). As shown in Fig. 5.6, bright field photomicrographs show undifferentiated cells (arrows in panel A) which are induced to aggregate in the presence of retinoic acid (panel B). These aggregates are finally plated, and clusters of differentiated cells have extended projections by Day 8 (panel C). Next, immunocytochemistry was performed with neuronal and glial specific markers to deduce the numbers of neurons and glia after 8 days in culture. As shown, neuronal nuclei are positive for NeuN (arrows in panel D), a known marker of neurogenesis (Mullen et al., 1992). Immunostaining for GFAP identified glial cells in the same field
(arrow in panel E; panel F). On the other hand, undifferentiated P19 cells, which do not exhibit a neuronal or glial phenotype (McBurney and Rogers, 1982), do not stain for NeuN (panel H) or GFAP(data not shown). Quantitation of the numbers of NeuN+ and GFAP+ cells revealed that greater than 98% of cells in culture were neurons by Day 8 (data not shown).
Following from these analyses, protein extracts were harvested and Western blot analysis performed to chart the progress of neurodifferentiation using established molecular markers. Shown in Fig. 5.6J,
Western analysis revealed an increase in expression of the neuronal protein NeuN; the 42 and 46 kDa doublet signal is markedly increased following 8 days differentiation in culture. Conversely, there is a corresponding decrease in levels of the neuronal progenitor cell marker Nestin, an intermediate filament protein. This indicates that there is an increase in the numbers of neurons in culture, concomitant with a decrease in the population of neuroprogenitor cells.
Next, Northern analysis was conducted to obtain a molecular profile of these cells upon neurodifferentiation with retinoic acid. As shown in Figure 5.7A, MASH1 and NSCL mRNAs were detected at Day 2, and is consistent with a neurogenetic role for these bHLH genes (Begley et al., 1992;
Johnson et al., 1992b; Uittenbogaard et al., 1999). The inhibitory HLH genes ID2 and BETA3 were also investigated. The levels of these two genes appear to peak at Day 2, then decrease as neurogenesis
67 NeuN Bis
A D G
GFAP NeuN
B E H
Merge Bis
C F I
J 0 2 4 6 8
Nestin
NeuN
Ponceau
Figure 5.6. Neuronal differentiation of P19 embryocarcinoma cells with retinoic acid. (A) P19 embryocarcinoma cells (arrows) exhibit a epithelial cell-like morphology, but aggregate (B) and finally differentiate into neurons after addition of retinoic acid (D) with visible cell projections. (D) Immunocytochemistry performed on fixed cells at Day 8 of culture shows neurons stained with NeuN in green (arrows), while glia are stained red with GFAP (arrowhead in E). Panel F represent merged images (D) and (E). (G) Cells were stained with bisbenzimide to reveal their nuclei. (H) Undifferentiated P19 cells do not express NeuN [arrow; bisbenzimide stained nucleus in (I)] or GFAP (not shown). Scale bar represents 40 µm. (J) Western blot analysis of Nestin and NeuN protein expression in P19 cells induced to differentiate with retinoic acid for 0, 2, 4, 6, and 8 days as described. The blot was stained with Ponceau S to assess sample loading.
67i A 0 2 4 6 8 GRIPE
E12
MASH1
NSCL1
BETA3
ID2
- 2.5 kb
mUBC9/UBCE2A
- 1.3 kb
18S rRNA
B 120.00
100.00
80.00
GRIPE 60.00 E12
40.00 Arbitrary Units Arbitrary Units 20.00
0.00 0 2 4 6 8 Days in culture
Figure 5.7. Expression of GRIPE and E12 mRNA during neurogenesis of P19 cells. (A) Northern analysis of GRIPE, E12, Mash1, NSCL1, BETA3, ID2 and mUBC9/UBCE2A transcripts during neurodifferentiation of P19 cells. Loading was determined using a probe for 18S rRNA. Total RNA was harvested from cells induced to differentiate for 0, 2, 4, 6 and 8 days, as described in Fig. 5.6. (B) Quantitation of GRIPE (white bars) and E12 (black bars) mRNA signals from triplicate experiments. Error bars represent s.e.m.. Single-factor ANOVA analysis of GRIPE (F(4, 10), 4.07, p <0.05) and E12 (F(4, 10), 13.05, p <0.01) signals indicate significant differences in mRNA expression during neurogenesis. Raw data is presented in Appendix 10.13.
67ii Chapter 5: GRIPE is a novel gene that may assume different roles during development and in adulthood progresses. While this observation is consistent with a postulated role for ID2 in cell proliferation (Alani et al., 1999; Loveys et al., 1996), it provides an important clue for the role of the novel HLH factor BETA3, since data here complements work by Peyton and colleagues (Peyton et al., 1996) which suggests that
BETA3 may behave as a negative regulator of HLH signalling.
Analysis of the expression of the ubiquitin-conjugating enzyme for E2A polypeptides, known as mUBC9/UBCE2A (Kho et al., 1997; Loveys et al., 1997), was also conducted. Northern analysis reveals developmental regulation of alternative splice forms of this gene during neurogenesis of P19 cells in vitro.
As shown, while levels of the 2.5kb transcript do not significantly change, there is a progressive decrease in levels of the 1.3kb splice form, indicating that the latter mRNA species may be more important during early stages of RA-induced neurogenesis of P19 cells.
Finally, the expression levels of GRIPE and E12 were assayed in these differentiating neurons. Results show that with increasing numbers of mature neurons in culture, there is a corresponding increase in levels of GRIPE mRNA, with the strongest signal detected at Day 8 of culture. Upon quantitation of the mature signals in triplicate experiments, a single-factor ANOVA test revealed significant (F(4, 10), 4.07, p <0.05) changes in GRIPE expression by Day 8 (Fig. 5.7B; raw data presented in Appendix 10.13). Conversely, the pattern of E12 gene expression mimics the behaviour of Id2 and BETA3; quantitation of E12 mRNA levels reveals a peak at Day 2, and follows a decrease upon the onset of neurodifferentiation (F(4, 10),
13.05, p <0.01). These observations suggest that the interaction of GRIPE and E12 may be an important event during the early phase of neurogenesis, perhaps prior to the neuroblast exiting from the cell cycle and before the expression of neuronal genes such as Mash1 and NSCL1. Furthermore, the continued expression of GRIPE in differentiated neurons suggests an ongoing role in mature neurons.
5.5. There are two novel GRIPE transcripts in P19 cells
Northern analysis of GRIPE expression in RNA isolated from P19 cells during 8 days of neurodifferentiation revealed two novel transcripts in some preparations. Shown on Fig. 5.8, these bands appear to migrate at approximately 12 kb (denoted as “a”) and 10 kb (denoted “b”), while the predicted 8
68 1 2 3 4 5 6 7 M
a GRIPE b c
4.40 kb
2.37 kb
1.35 kb
2.37 kb 18S rRNA 1.35 kb
Figure 5.8. Northern analysis showing alternative GRIPE RNA signals detected in total RNA isolates. Lane 1 = P19 Day 0; 2 = P19 Day 2; 3 = P19 Day 4; 4 = P19 Day 6; 5 = P19 Day 8; 6 = Adult brain; 7 = e11.5 trunk; M = RNA ladder. The signal for 18S rRNA is shown to account for differences in sample loading.
68i Chapter 5: GRIPE is a novel gene that may assume different roles during development and in adulthood kb signal is detected in all samples tested (denoted “c”). Signal “c” is identical to the mature transcript detected in Northern analysis for GRIPE mature mRNA in mouse tissue RNA (compare lane 5 with lanes 6 and 7; also see Fig. 5.2). Signal “a” increases to maximal levels at Day 8, while signal “b” is undetectable by Day 6. A comparison of P19 RNA samples with preparations from mouse e11.5 trunk and adult brain indicates that signals “a” and “b” are particular to P19 cell extracts (compare lanes 1 to 5 with lanes 6 and
7). Owing to repeated observations of these alternate signals in independent experiments, several explanations are presented here which could account for the detection of these novel transcripts in P19
RNA, and not in mouse tissues.
Firstly, it is possible that the novel transcripts may represent pre-spliced RNA species, which are processed to generate the mature GRIPE mRNA. While a valid explanation, the stoichiometric levels of each signal are inconsistent with this, as it would be expected that if signal “a” is the precursor to “b”, then the processed signal “b” should always increase as signal “a” increases. This is not seen during neurodifferentiation of P19 cells, since signal “b” decreases and is undetectable when signal “a” is highly expressed on Day 8 (lane 5). Further clarification could be sought by performing Northern analysis with isolated polyadenylated mRNA. To rule out the possibility that these signals are DNA contaminants, RNA samples should be pre-treated with deoxyribonucleases prior to Northern analysis.
Another explanation is that these transcripts may be a symptom of chromosomal abnormalities in the cell line. While it has been reported that the P19 cell line has a euploid karyotype of 40:XY (McBurney and
Rogers, 1982), it is possible that chromosomal translocations may have occurred which involve the GRIPE locus on chromosome 12 (see Chapter 6.1). This event could result in the creation of chimeric transcripts; one of which is up regulated during neurodifferentiation, and the other silenced. Consistent with this hypothesis is the observation that signal “a” is up-regulated upon neurodifferentiation of (Fig. 5.8), while signal “b” is down-regulated. Yet another possibility is that these transcripts are paralogous mRNA species which cross-hybridise to the radiolabelled cDNA probe. With these interpretations in mind, it is crucial that further experiments have to be conducted to reveal the true identity of these Northern signals.
69 Chapter 5: GRIPE is a novel gene that may assume different roles during development and in adulthood
5.6. Summary
5.6.1. An intact GRIPE/E12 signalling cascade may be important during early neurogenesis
Northern analysis reveals that GRIPE mRNA is a mature transcript of approximately 8kb, and expression coincides with E12 early in neurogenesis. GRIPE is coincidently expressed with E12 in the ventricular zone cells of the developing forebrain, and their interaction may be important during early neurogenesis. In adult brain, GRIPE is highly expressed in many regions of the cerebral cortex, as well as the hippocampus, but not in the cerebellum. Conversely, E12 is not detected in adult brain, and this leads to the possibility that the GRIPE/E12 interaction may not be important in mature neurons.
While Northern analysis indicates that GRIPE and E12 are expressed in embryonic tissues until e15.5, in situ hybridisation experiments must be conducted to identify sites of gene expression in e13.5 and e15.5 embryos. Furthermore, Roberts and coworkers (Roberts et al., 1993) had previously shown E12 expression in the ependymal cell layer lining the walls of the cerebral ventricles of the adult rat. The lack of specific signal in experiments conducted herein may be explained by differences in sensitivity between the colorimetric assay employed, compared to the approach used by Roberts and coworkers which utilised radiolabelled cRNA probes (Roberts et al., 1993). Nevertheless, GRIPE staining was not observed in the ependymal cell layer, hence, in the context of GRIPE and E12 signalling, their interaction is not important in ependymal cells of the adult mouse brain.
5.6.2. GRIPE may function independently of E12 in mature neurons.
An investigation of GRIPE expression in differentiating P19 embryocarcinoma cells indicates that GRIPE is expressed throughout neurogenesis, and levels are highest in mature neurons. Conversely, E12 levels peak on Day 2, but decrease as cells undergo neurodifferentiation. These studies recapitulate observations made with Northern analysis of mouse tissue RNAs: a coincidence of GRIPE/E12 expression early in neurogenesis, followed by a progressive reduction in E12 expression in mature neurons. Experiments carried out with P19 cells further implicate a role for GRIPE in mature neurons, and demonstrate the utility of this cell culture model in defining the role of GRIPE in neurogenesis. However, the detection of novel transcripts in this cell line must first be clarified with more experiments. To begin to address this, Northern
70 Chapter 5: GRIPE is a novel gene that may assume different roles during development and in adulthood analysis should be carried out with DNase-treated polyA mRNA. In addition, conventional approaches to isolate 5’ sequence information, such as primer extension or 5’RACE, should be informative. A proper description of GRIPE expression in these cells will then allow for further genetic analysis by employing over-expression or loss-of-function strategies to elucidate the contribution of GRIPE to neuronal development, as well as to mature cell function.
5.6.3. E12 function may be more important in the early stages of neurogenesis
Upon identification of interacting partners to E12 in developing mouse brain, and using a yeast 2-hybrid assay, further experiments were conducted to evaluate the timing of expression of these binding partners during in vitro differentiation of P19 cells. Importantly, these observations demonstrate a role for E12 in the early stages of neurogenesis, with a peak in mRNA expression at Day 2. Upon exposure to retinoic acid on Day 2, the neuronal Class II HLH factors Mash1 and NSCL1 are up-regulated for transactivation of downstream target genes, and may require heterodimerisation to E12 for proper function. Similarly, the negative regulators, ID2 and BETA3, are maximally expressed by Day 2, and, together with the Class II
HLH factors, constitute a highly regulated HLH signalling cascade which directs the early stages of neuron formation in postmitotic neuroblasts. Importantly, experiments conducted here are the first to report developmental regulation of mUBC9/UBCE2A transcripts in an in vitro model of neurogenesis, and suggest a role for the 1.3kb species in early, not late, neurogenesis. Indeed, since both the 2.5kb and 1.3kb transcripts for mUBC9/UBCE2A have been shown to encode identical polypeptides, and that cellular pools of mUBC9 protein remain unchanged (Loveys et al., 1997), data presented here (Fig. 5.7) shows the 1.3kb splice form may be important for maintaining steady-state levels of this ubiquitin-conjugating protein factor early in neurodifferentiation of P19 cells.
71 Chapter 6: The GRIPE gene encodes a 1485 amino acid protein, and its function may be conserved during evolution Chapter 6: The GRIPE gene encodes a 1485 amino acid protein, and its function may be conserved during evolution
6.1. Conservation of the GRIPE gene during evolution
A thorough investigation of Genbank and other nucleotide databases revealed that GRIPE is found in many organisms, including H. sapeins, Xenopus laevis, Mus musculus, Rattus norvegicus, Danio rerio,
Drosophila melanogaster and possibly in Caenorhabditis elegans. In the human, there appears to have been a duplication event that resulted in three copies of GRIPE, located on three different chromosomes. A summary of putative GRIPE orthologues in these organisms is provided in Table 6.1. The identification of expressed sequence tags (ESTs) from these organisms implies, at least, that these genes are transcribed.
Further, prevalence of GRIPE in these genomes may indicate some conservation of gene function during evolution. Evidence for a possible role for GRIPE in mature neurons is also discussed.
Organism Gene Identifier Comments Rattus Norvegicus Tulip-1 (AF041106) and Unpublished cDNA; no data available Tulip-2 (AF041107) Mus musculus Mouse Tulip (AB032400) Unpublished cDNA; no data available (Homologene Mm 35575)
Drosophila AE003758 (CG5521 gene Predicted transcript melanogaster product) Xenopus laevis BG159952 EST sequence from an unfertilized egg library
Danio rerio WZ16720 EST sequence from Washington University Zebrafish genome resources (http://zfish.wustl.edu/); sequence available through http://fisher.wustl.edu/fish_lab/cgi-bin/search.cgi; sequence is linked with homologene asignment Hs. 167031 Caenorhabditis elegans NM_061527 and Both hypothetical proteins show homology only to the GAP domain of NM_058371 GRIPE; these may represent undiscovered GAP proteins, and may be orthologues of GRIPE Homo sapiens KIAA0884; KIAA1272 Three unique cDNAs which may be orthologues of mouse GRIPE and DKFZp566D133 Table 6.1. A summary of cDNAs which show nucleotide and/or amino acid homology to mouse
GRIPE.
6.2. The GRIPE gene is encoded by at least 32 exons, and is located on mouse chromosome 12
With the observations from Northern analyses that GRIPE is an 8kb mRNA, it was noted that a significant portion of 5’ cDNA sequence of GRIPE still had to be cloned; only 3.8kb of cDNA sequence (including the polyA tail) was cloned using the yeast 2-hybrid library screen. Several attempts to clone the 5’ end using conventional methods were met with little success; conventional RACE and RNA ligase-mediated RACE
72 Chromosome 12 Band C1 GA_x5J8B7W899C (5’ to 3’ sequence) 12C1 (46.5- 49Mb)
1 500000 1) 2) 3) 4)
Input Sequence Homology to known Predicted exons and cDNAs polypeptide length 1) 343283 - 469607 Similarity to Tulip-1 28 exons, 1359 amino (126324 nucleotides) acids 2) 333823- 469607 Similarity to Tulip-1 28 exons, 1359 amino (136324 nucleotides) acids 3) 333823- 489607 Similarity to Tulip-1 31 exons, 1531 amino (156324 nucleotides) acids 4) 333823- 499607 Similarity to Tulip-1 32 exons, 1531 amino (166324 nucleotides) acids
Figure 6.1. A gene prediction approach to clone the 5’ sequence of GRIPE cDNA. Successive lengths of DNA sequence from mouse chromosome 12BandC1 were subjected to GRAIL analysis (see section 5.1) to predict the 5’ sequence of GRIPE. A hypothetical transcript comprising 32 exons, encoding 1531 amino acids, was then obtained for further analysis.
72i Chapter 6: The GRIPE gene encodes a 1485 amino acid protein, and its function may be conserved during evolution experiments yielded less than 1 kb of 5’ cDNA sequence (data not shown). While this was already informative, more than half of the putative transcript had yet to be identified.
Release of the annotated mouse genome database by Celera (Celera Genomics, Rockville; access available through http://www.celera.com) allowed a genomics approach to clone the 5’ sequence of GRIPE transcript, predicted from DNA sequence adjacent to its putative chromosomal location. Upon interrogation of the Celera mouse genome database, it was revealed that GRIPE was located on mouse chromosome 12, band C1. Using a predictive method outlined in Fig. 6.1, successively larger portions of genome sequence were evaluated for coding exons using the prediction program GRAIL version 3.1
(available on http://grail.lsd.ornl.gov/grailexp/). This approach resulted in the identification of a putative target “transcript” which comprised 32 exons, generating a mRNA encoding 1531 amino acids; the putative start codon was empirically determined as the first methionine which follows in-frame stop codons upstream of the cDNA sequence. Following from this information, oligonucleotide primers designed to clone these predicted 5’ sequences, known as “guessmers”, were employed for RT-PCR experiments using conditions described in appendix 10.10. Shown in Fig. 6.2., each predicted exon was targeted by two guessmers, to be used as forward primers for RT-PCR experiments. A reverse primer corresponding to previously known GRIPE sequence (nt 2152 to 2173), named “SP2”, was used to ensure faithful amplification of the target(s). This experiment was performed with RNA extracted from e11.5 trunk, a source of tissue rich in GRIPE transcripts (see Fig. 5.1). To validate this, RT-PCR was performed with primers previously known to detect GRIPE, and a predicted amplified product of 0.4kb was generated (Fig.
6.3. lane 1). Parallel experiments conducted without reverse transcriptase ruled out the possibility that this
PCR product was amplified from genomic DNA (lane 2). Next, RT-PCR was performed with the
“guessmer” forward primers, and with SP2 as the reverse primer in all preparations. As shown in Fig. 6.3, almost all primer pairs generated a RT-PCR product of predicted sizes (lanes 3, 5, 7, 9, 15 and 17). Parallel experiments performed with e11.5 trunk RNA and without reverse transcriptase indicate the absence of
DNA contamination (all even lanes). The lack of PCR product in lanes 11 and 13 may not indicate an absence of predicted sequence, as it could also be explained by sub-optimal PCR amplification conditions.
Regardless, the largest PCR fragments (isolated from lanes 3, 5, 7 and 9) were harvested and nested PCR
73 A CATTCTCATTGATGAAATAGTAAAAGCTTGATATTTTGCTTCAGGAAAATGGGACCA* * GTACTTCTGAACATGTTCGCAATTCTAGCTGGACAAAAAATGGTTCCTACCAAGAAG CTTTTCATGTCTGTGAAGAAGCCACAGAACAGAATATTCAAGCTGGCACTCAGGCCG TTTTGCAGGTGTTTATTATAAACTCATCGAACATATTTCTTCTTGAACCTGCAAATG AAATAAAGAATCTTCTGGATGAGCATACAGATATGTGTAAACGCATTCTTAATATTT ATCGATACATGGTTGTGCAAGTATCAATGGACAAAAAGACCTGGGAACAGATGCTGC TCGTGCTGCTCAGAGTCACGGAGTCCGTACTGAAGATGTCGTCACAAGCTTTTCTGC AGTTCCAGGGGAAAAAAAGCATGACCCTGGCGGGTCGACTTGCAGGACCACTTTTTC AGACTCTCATTGTTGCCTGGATCAAAGCAAATCTAAATGTATACATTTCTCGAGAAC TTTGGGATGACTTACTTTCGGTATTGTCATCATTGACCTATTGGGAAGAATTGGCCA CTGAGTGGTCACTGACCATGGAGACACTGACTAAAGTTTTGGCTAGAAATTTATATA GTTTGGATCTCAGTGATTTGCCATTGGATAAGCTCAGTGAACAGAAACAAAAAAAGC ACAAAGGGAAAGGAGTTGGACACGAATTTCAGAAAGTTTCAGTTGACAAGTCATTTT CTAGAGGATGGAGTCGCGATCAGCCTGGCCAGGCACCTATGAGACAGAGAAGTGCAA CGACCACTGGCTCCCCAGGAACTGAGAAGGCCAGGAGTATAGTACGGCAAAAAACTG TTGCCATGAGAAGCCGATCCATTGGTGAATGTGCTCTGCCATCGGCCTATATTCGCA GTGCTAAAAGTGCTCCTGTTCTGATCCATACTTCCAAACCCTTCTTGCCTGATATTG TTCTCACTCCCCTTTCTGATGAGCTTTCAGATATTGATGATGCTCAAATCCTTCCCC GCTCAACCAGAGTCAGACATTTTTCACAGAGTGAAGACACTGGAAATGAAGTTTTTG GTGCTTTGCATGAGGAACAGCCACTGCCTCGAAGTAGCAGCACTTCTGACATCTTGG AACCATTCACTGTTGAGCGAGCCAAAGTAAATAAAGAGGACACGAGCCCCAAACTGC CTCCTCTTAATAGTGAGACTGGTGGCAACAGCGCCAATGTTCCTGACCTGATGGATG AGTTTATCGCAGAGCGACTTCGAAGTGGCAATGCCTCGACTATGACAAGAAGGGGGA GTAGTCCAGGCAGCCTTGAAATCCCGAAAGACCTCCCTGATATTCTAAACAAGCAGA ACCAGATGCGGCCTGTTGATGACCCTGGTGTACCCTCCGAATGGACGTCTCCAGCCA GTGCAGGGAGCAGTGATCTCATGAGCTCAGATAGTCACTCGGATTCCTTCAGTGCCT TCCAGTGTGAGGGACGAAAATTTGACAACTTTGGCTTCGGGACTGACATTGGGATCC CATCTTCTGCTGATGTGGATTTAGGTTCTGGCCATCATCAAAGCACTGAGGAGCAGG AAGTGGCCAGCCTAACTACGCTTCATTTAGATTCAGAAACAAGCAGCCTTAATCAGC AAGCCTTCTCTGCAGAAGTTGCAACCGTTACTGGTTCAGAAAGTGCTTCCCCCGTTC ATTCAGCTCTGGGATCCAGGTCGCAGACCCCCTCCCCATCTACACTGAGCAGAGCTC ACATAGAACAGAAGGACCTACAGCTCGATGAGAAGCTCCACCACTCTGTTCTTCAGA CGCCGGATGACCTAGAAATCAGTGAATTTCCATCTGAATGTTGTAGCGTGATGGCAG GAGGTACTCTCACTGGGTGGCATGCTGATGTTGCTACTGTTATGTGGCGCAGAATGC TGGGCATTTTGGGAGATGTCAATGCTATTATGGATCCTGAAATACATGCTCAAGTTT TTGATTACCTCTGTGAACTTTGGCAGAATCTAGCCAAGATCAGAGATAACCTTGGCA TTTCAGCTGACAACCTGACTTCCCCTTCTCCACCCGTCCTGATTCCTCCACTAAGAA TTCTTACACCTTGGCTTTTTAAGGCAACCATGTTGACTGATAAGTATAAACAAGXXT ACAATATAATGCATTGTGGATTGCTTCATATTGACCAGGACATTGTCAATACGATCA TCAAGCACTGCTCACCTCAGTTTTTTTCACTCGGTTTGCCTGGGGCCACGATGCTTA TTATGGATTTTATTATAGCAGCTGGGAGAGTGGCTTCTTCGGCTTTTCTTAATGCAC CAAGAGTGGAAGCACAAGTTCTTCTGGGATCTTTAGTTTGCTTTCCCAACTTATATT GTGAATTGCCTGCTCTCCATCCCAACATTCCTGATATTGCTGTGTCTCAATTTACAG ATGTTAAGGAACTTATAATTAAAACTGTATTAAGCTCGGCAAGAGATGAGCCCTCTG GTCCTGCACGATGTGTAGCACTTTGCAGTTTAGGTATTTGGATTTGTGAAGA
R1: tgtgaagaagccacagaacag R5: cattgttgcctggatcaaagc R2: gcactcaggccgttttgcagg R6: tgccattggataagctcagtg R3: gcatacagatatgtgtaaacgc R7: atgagacagagaagtgcaacg R4: gttgtgcaagtatcaatggac R8: ctgagaaggccaggagtatag
B
R1 R2 R3 R4 R5 R6 R7 R8 (known sequence)
G2 G4 SP2 Hypothetical transcript - 2.5kb
Figure 6.2. Cloning the 5’ cDNA sequence of GRIPE. (A) The hypothetical transcript predicted from GRAIL analysis (see Fig. 6.1) was used as a template to design “guessmers” for RT-PCR cloning. In-frame stop codons upstream of the putative ATG start methionine codon are indicated with “*”. Blocks represent primer sequences targeted for cloning GRIPE cDNA using RT-PCR. Highlighted “ATG” sequences (in grey) denotes predicted start codons, while “XX” in bold indicates ambiguous sequence output from the prediction program. (B) The guessmers were arbitarily named R1 to R8, and target the hypothetical transcript (black bar). Reverse primers G4, and SP2 were designed from cDNA sequence already cloned (represented in grey). Primer G2 was employed for nested PCR experiments (see text).
73i M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Figure 6.3. Cloning the 5’ sequence of GRIPE by RT-PCR. Even numbered lanes represent RT-PCR products generated from reverse transcribed e11.5 trunk RNA. Parallel RT-PCR experiments were performed without reverse transcriptase to account for possible genomic DNA amplification (odd numbered lanes). PCR products from lanes 3, 5, 7, and 9 (named S3, S5, S7 and S9 respectively) were recovered for nested PCR experiments shown in Fig. 6.4. Primer pairs used: G2/SP2 (lanes 1, 2); R1/SP2 (lanes 3, 4); R2/SP2 (lanes 5, 6); R3/SP2 (lanes 7, 8); R4/SP2 (lanes 9, 10); R5/SP2 (lanes 11, 12); R6/SP2 (lanes 13, 14); R7/SP2 (lanes 15, 16); R8/SP2 (lanes 17, 18). Amplification products are visible in lanes 3, 5, 7, 9, 15 and 17.
M 1 2 3 4 5 6 7
~2400 bp ~1900 bp
387 bp
Figure 6.4. Verification of sequence composition of cloned 5’ fragments of GRIPE through nested PCR. Templates used for PCR analysis: the ~2.2kb fragment S3 was used as template for PCR using primer pairs G2/SP2 (lane 1), R4/G4 (lane 2) and G2/SP2 (lane 3). Amplification of predicted DNA fragments provide additional confirmation of faithful RT-PCR cloning of predicted 5’ fragments of GRIPE cDNA. Further, primers G2 and SP2 were used for PCR reactions using PCR fragments S5, S7 and S9 as templates (lanes 4-6). In all cases, a predicted nested PCR product was amplified. Representative reactions without template indicate absence of DNA contamination (lane 7). Predicted fragment lengths are: R2/SP2 (2428 bp); R4/G4 (1908 bp); G2/SP2 (387 bp).
73ii Chapter 6: The GRIPE gene encodes a 1485 amino acid protein, and its function may be conserved during evolution reactions conducted to verify the sequence composition of cloned GRIPE cDNAs. As shown in Fig. 6.4, nested primer pairs generated PCR products of predicted sizes, hence these experiments confirmed that genuine GRIPE cDNA fragments had been successfully isolated for subsequent sequencing.
6.3. GRIPE is a 1485 amino acid protein with distinct domains
The entire cloned cDNA sequence spans almost 6kb, and is displayed in Fig. 6.5. A 3’ polyadenylated tail is present, as well as a corresponding polyadenylation signal (nt 5833 to 5838; underlined) and a conserved sequence which promotes message stability (nt 5818 to 5827; underlined). This cDNA sequence has been submitted to Genbank as AY066011.
The translated protein sequence for GRIPE is shown in Fig. 6.6. Interrogation of public databases using resources of the ExPASy web site (Expert Protein Analysis System; a service provided by the Swiss
Institute of Bioinformatics; http://www.expasy.org) allowed for the detection of conserved protein domains within the GRIPE protein. As shown in Fig. 6.6, GRIPE contains a putative GAP (GTPase Activating
Domain; shaded grey) domain that could facilitate hydrolysis of GTP bound to small G-proteins such as
Rap or Ran. Within this domain is a conserved sequence motif which may be important for association with a guanine nucleotide triphosphate molecule (amino acids 1409 to 1416; underscored in Fig. 6.6A). In addition, a putative leucine zipper is predicted from amino acids 535 to 556. This motif may be important for mediating protein-protein dimerisation, and could indicate that GRIPE may interact with yet unknown proteins through which bind its N-terminus. Further, the prediction algorithm also detected a conserved prokaryotic membrane lipoprotein-lipid attachment site (amino acids 1004 to 1014; shaded yellow). This domain may be responsible for targeting EGFP-GRIPE701-1485 to the perinuclear region of transiently transfected HEK293T cells (see section 4.4). Together, these attributes provide clues that may explain the function and behaviour of GRIPE in vivo.
74 ATGTGTAAACGCATTCTTAATATTTATCGATACATGGTTGTGCAAGTATCAATGGACAAAAAGACCTGGGAACAGATGCTGCTCGTGCT GCTCAGAGTCACGGAGTCCGTACTGAAGATGTCGTCACAAGCTTTTCTGCAGTTCCAGGGGAAAAAAAGCATGACCCTGGCGGGTCGACTT GCAGGACCACTTTTTCAGACTCTCATTGTTGCCTGGATCAAAGCAAATCTAAATGTATACATTTCTCGAGAACTTTGGGATGACTTACTTT CGGTATTGTCATCATCGACCTATTGGGAAGAATTGGCCACTGAGTGGTCACTGACCATGGAGACACTGACTAAAGTTTTGGCTAGAAATTT ATATAGTTTGGATCTCAGTGATTTGCCATTGGATAAGCTCAGTGAACAGAAACAAAAAAAGCACAAAGGGAAAGGAGTTGGACACGAATTT CAGAAAGTTTCAGTTGACAAGTCATTTTCTAGAGGATGGAGTCGCGATCAGCTTGGCCAGGCACCTATGAGACAGAGAAGTGCAACGACCA CTGGCTCCCCAGGAACTGAGAAGGCCAGGAGTATAGTACGGCAAAAAACTGTTGATATTGATGATGCTCAAATCCTTCCCCGCTCAACCAG AGTCAGACATTTTTCACAGAGTGTAGACACTGGAAATGAAGTTTTTGGTGCTTTGCATGAGGAACAGCCACTGCCTCGAAGTAGCAGCACT TCTGACATCTTGGAACCATTCACTGTTGAGCGAGCCAAAGTAAATAAAGAGGACACGAGCCCCAAACTGCCTCCTCTTAATAGTGAGACTG GTGGCAACAGCGCCAATGTTCCTGACCTGATGGATGAGTTTATCGCAGAGCGACTTCGAAGTGGCAATGCCTCGACTATGACAAGAAGGGG GAGTAGTCCAGGCAGCCTTGAAATCCCGAAAGGCCTCCCTGATATTCTAAACAAGCAGAACCAGATGCGGCCTGTTGATGACCCTGGTGTA CCCTCCGAATGGACGTCTCCAGCCAGTGCAGGGAGCAGTGATCTCATGAGCTCAGATAGTCACTCGGATTCCTTCAGTGCCTTCCAGTGTG AGGGACGAAAATTTGACAACTTTGGCTTTGGGACTGACATTGGGATCCCATCTTCTGCTGATGTGGATTTAGGTTCTGGCCATCATCAAAG CACTGAGGAGCAGGAAGTGGCCAGCCTAACTACGCTTCATTTAGATTCAGAAACAAGCAGCCTTAATCAGCAAGCCTTCTCTGCAGAAGTT GCAACCGTTACTGGTTCAGAAAGTGCTTCCCCCGTTCATTCAGCTCTGGGATCCAGGTCGCAGACCCCCTCCCCATCTACACTGAGCAGAG CTCACATAGAACAGAAGGACCTACAGCTCGATGAGAAGCTCCACCACTCTGTTCTTCAGACGCCGGATGACCTAGAAATCAGTGAATTTCC ATCTGAATGTTGTAGCGTGATGGCAGGAGGTACTCTCACTGGGTGGCATGCTGATGTTGCTACTGTTATGTGGCGCAGAATGCTGGGCATT TTGGGAGATGTCAATGCTATTATGGATCCTGAAATACATGCTCAAGTTTTTGATTACCTCTGTGAACTTTGGCAGAATCTAGCCAAGATCA GAGATAACCTTGGCATTTCAGCTGACAACCTGACTTCCCCTTCTCCACCCGTCCTGATTCCTCCACTAAGAATTCTTACACCTTGGCTTTT TAAGGCAACCATGTTGACTGATAAGTATAAACAAGGTAAATTACATGCATATAAACTTATTTGTAATACAATGAAGAGAAGACAAGATGTA TCTCCAAATAGAAATTTTTTAACACATTTCTACAATATAATGCACTGTGGATTGCTTCATATTGACCAGGACATTGTCAATACGATCATCA AGCACTGCTCACCTCAGTTTTTTTCACTCGGTTTGCCTGGGGCCACGATGCTTATTATGGATTTTATTATAGCAGCTGGGAGAGTGGCTTC TTCGGCTTTTCTTAATGCACCAAGAGTGGAAGCACAAGTTCTTCTGGGATCTTTAGTTTGCTTTCCCAACTTATATTGTGAATTGCCTGCT CTCCATCCCAACATTCCTGATATTGCTGTGTCTCAATTTACAGATGTTAAGGAACTTATAATTAAAACTGTATTAAGCTCGGCAAGAGAT GAGCCCTCTGGTCCTGCACGATGTGTAGCACTTTGCAGTTTAGGTATTTGGATTTGTGAAGAATTAGTCCACGAATCTCATCATCCTCAAA TTAAAGAAGCACTGAATGTAATTTGTGTTTCATTAAAGTTTACTAATAAAACAGTTGCCCACGTCGCCTGTAACATGCTTCACATGCTGGT TCACTATGTCCCTAGACTTCAGATTCACCAGCCGCAGTCTCCCCTGAAAATTATTCAGATCCTAATAGCAACTATCACCCACCTTTTGCCA AGTACAGAAGCTTCATCATATGAAATGGACAAGAGGTTGGTTGTCTCATTACTTCTTTGCCTTCTGGACTGGATCATGGCCTTACCTTTAA AGACACTGCTCCAACCAGTCCATGCTACAGGAGCAGAAAATGATAAGACCGAAAAGTCTGTTCTCAACTGCATTTATAAGGTATTACATGG GTGTGTCTATGGAGCGCAAAGTTTTAGCAATCCCAAGTACTTTCCAATAAGCCTTTCTGATTTGGCATCTGTAGATTATGATCCGTTTATG CATTTGGAAAGTCTGAAAGAACCTGAACCTTTACACTCTCCAGATTCAGAGCGGTCTTCTAAACTGCAGCCTGTGACAGAAGTGAAAACTC AGATGCAACAAGGATTAATCTCTATAGCAGCCCGTACTGTTATTACCCATCTGGTGAATCACTTGGGCCATTATCCAATGAGTGGTGGTCC TGCTATGCTAACAAGTCAGGTGTGTGAAAATCATGACAATCATTACAGTGAAAGTACTGAACTTTCTCCTGAGCTCTTTGAAAGTCCAAAC ATCCAGTTCTTTGTGCTGAATAATACAACCTTAGTGTCCTGTATCCAGATAAGATCGGAAGAGAGTATGCCTGGAGGAGGTCTAGCTGCTG GCCTTGTGTCAGCCAACTCAAATGTTAGAATCATAGTACGTGATCTCTCTGGAAAGTACTCATGGGATTCTGCCATACTGTATGGACCACC CATTGTAAGTGGTTTGCCTGAACCTACATCTTTCATACTTTCAATGTCTGACCAAGAGAAACCGGAAGAGCCTCCTACATCTAACGAATGC TTAGAAGACATCGCAGTAAAAGATGGGCTTTCTCTCCAGCTTAGAAGATTTAGAGAAACTGTACCAACTTGGTCTACGATACGAGAGGAAG AAGATGTTCTTGACGAGCTCCTGCAGTATTTAGGCACTACAAGTCCTGAGTGTTTACAGAGAACGGGAATCTCCCTCAATGTTCCTGCTCC ACAGCCTCTGTGCATTTCTGAAAAACAGGAGAATGATGTCATTAATGCTATCCTTAAGCAATACACGGAAGAAAAAGAGTTTGTAGAGAAG CACTTTAATGACTTAAACATGAAAGCCTCAGAACAAGATGAGCCAACACCTCAGAAACCTCAGTCAGCTTTTTATTACTGCAGATTGCTTC TTAGTATATTGGGAATGAATTGGGCTGCAGGAATTCCTGGGACAAACGGAGGGAGCTTTCATCTCTTGAAGAAAAATGAAAAGCTGCTCAG AGAACTTAGGAACTTGGATTCAAGGCAATGCCGAGAGACACACAAGATTGCAGTATTTTACGTTGCTGAAGGACAAGAAGATAAATACTCC ATTCTCACCAATATAGGAGGAAGCCAAGCATATGAAGATTTTGTAGCTGGCCTTGGTTGGGAGGTAAACCTTACAAACCACTGTGGCTTCA TGGGAGGCCTTCAGAAGAACAGAAGCACTGGACTGACCACTCCCTATTTTGCTACCTCGACAGTAGAAGTAATATTTCATGTATCTACACG AATGCCTTCTGATTCTGATGACTCTCTGACCAAAAAATTGAGACATCTAGGAAATGATGAAGTGCACATTGTTTGGTCAGAACATACTAGA GACTACAGGAGAGGAATTATTCCTACTGAGTTTGGTGATGTCCTCATTGTAATATACCCAATGAAAAACCACATGTTCAGTATCCAGATCA TGAAAAAACCAGAGGTTCCCTTCTTTGGTCCACTTTTTGATGGTGCTATTGTGAATGGGAAAAGTTCTGCCATTATGGTTCGATCCACAGC AATAAATGCTAGCCGTGCTCTGAAATCACTGATTCCATTGTATCAAAATTTCTATGAGGAGAGAGCACGGTACCTGCAGACAATTGTCCAG CACCATTTAGAGCCAACAACATTTGAAGACTTTGCAGCACAGGTTTTCTCTCCAGCTCCCTACCATCATTTCCCTGCTGACGCAGATCATT AAGTGTCAGTTCTGTTTATCTGAAGGATCCTACCCAGAGATTCCACCCAGTGACGCTGCCCCAGCAGCACAGGTAGACGGGGCTGACCTGG CCTCTCCCATGTCTCCTCGAACTAGCAAAAGCCGCATGTCCATGAAGCTGCGTCGGTCCTCTGGCTCAGCCAATAAATCCTAAGGAGACAA CCAGCCTGGCAGTGAGCAGCAGTAGCCGCCCTGATCAGCAGTAGTGTTAACTCTTTCAGGCCTCGCGTGTTTGCCATAACACTCATGCTCC TTCCTGTGCGTTTATCTGAAAAAACACTGGACTTCAAAACTTACTATTTTAAGTTTGGACTATTTATTTAATTCAGGTTTTTTCCACACTC CAAGCTGCCATATTTTGAGGGTTGAGTTTTATTCCATACTTCACGTTCCTAGATAATCATGTCTAAGTGGTGTTACCTTCAAGTTCATGAT GAATGTTGAGACAAAATATAATTGGACAGATAGTCTTGTGGTTATTTATTGTGCCTCATTACAACTTTATGATTCAATAACTACATAGTTT TTACTAATTTTTTTGGCGGTGCTGGGAATAGAAGCCAGTATCCCAAGCATGCTAGGCAAGCACACTGTCCTTCACAGCCCAGTGCTACACA TTTAAATTAATGGAAGCATTTAGAGAGCTAGAGCAAAGCTGCACATCTGTTTGTCACATAGGCTGTACACAGAGTATTTGTGTTACAGAGA GAAACACTCCTAATGGCAGTGCAGCAGCTCAGATTCCAACTGAAATTACTTACATGTGGTAACATTTTCTTCCCAAGTTGGTATCTCATTT CTGGGCAGTGCAGGTGATAGCGGCACATACTCAGTACCCAGGAAGCCCACCACTGACCTTTGATTTCCGTTTAGCTTCCAGACAACTTTGT TCTGCCTTTTTAGGTGGCATCACTGCTAAATGCTATTTCTGTTTTGAGGCTGTGGTCTTTTATTATTGACCAGTTTATCCCGGTGTGTCTG TGTACTTTCTTAAGTACCAAAAAAGGTGATGGAGAAAAGGGGGAAAAGCTTGTTTTTGCTCAAATTATGTGTATTGTTAAAAAGCAATGGA ACATGACCCACAAGTCTAAGTTAAATATAATATTTTAGTACTAAACTTGGCTGCAGGTGCAGGTTGGTATGCACCCCACCTCCAGAAGTAT TTCCTGTAGTAAATAAGCAGTCTCCTTTTGTTTAACATGGGCATCTCCTAAGGAAATGTAGCATGATTGATACCAACTGCATGTGTAAATA TATCATCCTGACAAACCACAGAATATAAATTATGTATCATTCATGTAGTGTCTACAGATTTGTAATGGGACAAAAAGATGACATGGTATTT AATAAAAACAAAATAAAAATATTCTTCACAAAAAAAAAAAAAAAAA
Figure 6.5. Nucleotide sequence of GRIPE. The cloned 5867 nucleotide coding sequence for GRIPE is shown. Nucleotides 1 to 2100 were cloned by predictive RT-PCR (Fig. 6.2), while nucleotide 2101 (“A” in large font) indicates the start of cDNA sequence cloned using the yeast 2-hybrid assay. The polyadenylation (nt 5833 to 5838) and mRNA stability signals (nt 5818 to 5827) are indicated. This sequence was submitted to Genbank as AY066011. 74i Chapter 6: The GRIPE gene encodes a 1485 amino acid protein, and its function may be conserved during evolution
6.4. There is only one unique GRIPE mRNA in the mouse.
Sequence similarity searches using Blastn (Altschul et al., 1997) reveals that the 3’ end of GRIPE (nt 2101 to 5860) is similar to a supposedly full-length 2.2kb mouse cDNA named Tulip-1 (AB032400). A nucleotide-sequence alignment of GRIPE to mouse Tulip-1 indicated that these two cDNAs were almost identical (data not shown), bringing up the possibility that GRIPE may be expressed as a 2.2kb mRNA isoform. This hypothesis, however, is not substantiated in observations from Northern analysis of GRIPE mRNA (Section 5.1), since only a single 8kb species of GRIPE mRNA was ever observed. A 2.2kb transcript was never detected, even when cDNA probes complementary to GRIPE and Tulip-1 message were used (see section 2.14). In light of these findings, it is concluded that the authors of AB032400 have cloned a partial cDNA fragment of GRIPE.
Further sequence similarity searches revealed that GRIPE is homologous to two rat cDNAs, named Tulip-1
(AF041106) and Tulip-2 (AF041107). These cDNAs are 4.25kb and 3.34kb in size, respectively. An alignment of AF041106 and AF041107 with GRIPE cDNA reveals high sequence identity with the 3’ end of GRIPE cDNA (nt 2101 to 5860), and that GRIPE cDNA is more similar to Tulip-2 (alignment not shown). Closer inspection of these sequence alignments reveals rat Tulip-1 contains an additional 140 nucleotides adjacent to its stop codon, and is distinct from rat Tulip-2 and GRIPE, which brings up the possibility that multiple isoforms of GRIPE may have been overlooked in Northern analyses. To address this, an RT-PCR experiment was conducted which targeted this polymorphism (see Appendix 10.8 for protocols), and an outline of the strategy is shown in Fig. 6.7. Based on sequence dissimilarities between
GRIPE and Tulip-1, a unique PCR product would be amplified using primers that generated distinct products for target cDNAs; a predicted product of 201bp represents GRIPE cDNA (homologous to Rat
Tulip-2), while amplification of a 298bp product identifies an isoform of GRIPE which is homologous to
Rat Tulip-1. As shown in Fig. 6.8, a PCR product of 201bp was detected in all samples tested, while the
298bp product was not present in any of these tissues. Negative control experiments rule-out the possibility of genomic DNA contamination in any of the samples tested. These results indicate that there is only one isoform of GRIPE mRNA found in all tissue mRNAs tested.
75 A
MCKRILNIYRYMVVQVSMDKKTWEQMLLVLLRVTESVLKMSSQAFLQFQGKKSMTLAGRLAGPLFQTL IVAWIKANLNVYISRELWDDLLSVLSSSTYWEELATEWSLTMETLTKVLARNLYSLDLSDLPLDKLSE QKQKKHKGKGVGHEFQKVSVDKSFSRGWSRDQLGQAPMRQRSATTTGSPGTEKARSIVRQKTVDIDDA QILPRSTRVRHFSQSVDTGNEVFGALHEEQPLPRSSSTSDILEPFTVERAKVNKEDTSPKLPPLNSET GGNSANVPDLMDEFIAERLRSGNASTMTRRGSSPGSLEIPKGLPDILNKQNQMRPVDDPGVPSEWTSP ASAGSSDLMSSDSHSDSFSAFQCEGRKFDNFGFGTDIGIPSSADVDLGSGHHQSTEEQEVASLTTLHL DSETSSLNQQAFSAEVATVTGSESASPVHSALGSRSQTPSPSTLSRAHIEQKDLQLDEKLHHSVLQTP DDLEISEFPSECCSVMAGGTLTGWHADVATVMWRRMLGILGDVNAIMDPEIHAQVFDYLCELWQNLAK IRDNLGISADNLTSPSPPVLIPPLRILTPWLFKATMLTDKYKQGKLHAYKLICNTMKRRQDVSPNRNF LTHFYNIMHCGLLHIDQDIVNTIIKHCSPQFFSLGLPGATMLIMDFIIAAGRVASSAFLNAPRVEAQV LLGSLVCFPNLYCELPALHPNIPDIAVSQFTDVKELIIKTVLSSARDEPSGPARCVALCSLGIWICEE LVHESHHPQIKEALNVICVSLKFTNKTVAHVACNMLHMLVHYVPRLQIHQPQSPLKIIQILIATITHL LPSTEASSYEMDKRLVVSLLLCLLDWIMALPLKTLLQPVHATGAENDKTEKSVLNCIYKVLHGCVYGA QSFSNPKYFPISLSDLASVDYDPFMHLESLKEPEPLHSPDSERSSKLQPVTEVKTQMQQGLISIAART VITHLVNHLGHYPMSGGPAMLTSQVCENHDNHYSESTELSPELFESPNIQFFVLNNTTLVSCIQIRSE ESMPGGGLAAGLVSANSNVRIIVRDLSGKYSWDSAILYGPPIVSGLPEPTSFILSMSDQEKPEEPPTS NECLEDIAVKDGLSLQLRRFRETVPTWSTIREEEDVLDELLQYLGTTSPECLQRTGISLNVPAPQPLC ISEKQENDVINAILKQYTEEKEFVEKHFNDLNMKASEQDEPTPQKPQSAFYYCRLLLSILGMNWAAGI PGTNGGSFHLLKKNEKLLRELRNLDSRQCRETHKIAVFYVAEGQEDKYSILTNIGGSQAYEDFVAGLG WEVNLTNHCGFMGGLQKNRSTGLTTPYFATSTVEVIFHVSTRMPSDSDDSLTKKLRHLGNDEVHIVWS EHTRDYRRGIIPTEFGDVLIVIYPMKNHMFSIQIMKKPEVPFFGPLFDGAIVNGKSSAIMVRSTAINA SRALKSLIPLYQNFYEERARYLQTIVQHHLEPTTFEDFAAQVFSPAPYHHFPADADH
B GRIPE: 1295 LTNIGGSQAYEDFVAGLGWEVNLTNHCGFMGGL-QKNRSTGLTTPYFATSTVEVIFHVST 1353 pfam02145: 2 YSNEHGSPAFDEFLTGLGQRVRLKDFEPYRGGLDTKGDDTGTHSVYWHYKIMEIMFHVST 61
GRIPE: 1354 RMP-SDSDDSLTKKLRHLGNDEVHIVWSEHTRDYRRGIIPTEFGDVLIVIYPM----KNH 1408 pfam02145: 62 LLPYTEGDKQQLQRKRHIGNDIVTIVFQEPGTPFSPDTIRSHFQHVFVIVRPHDPCTNHV 121
GRIPE: 1409 MFSIQIMKKPEVPFFGPLFDGAIVNGKSSAIMVRSTA--INASRALKSLIPLYQNFYEER 1446 pfam02145: 122 TYLVSVTRRKDVPFFGPTLPTGAVFDKNLEFRAFLLAKLINAENAVHKSRKFAKMAYRTR 181
GRIPE: 1447 ARYLQTI 1453 pfam02145: 182 QKLLADL 188
Figure 6.6. Amino acid sequence of GRIPE. (A) GRIPE is a 1485 amino acid polypeptide which contains a putative leucine zipper (shaded blue), a conserved membrane anchor (yellow) and a GAP domain (grey). A conserved motif within the GAP domain may govern interaction with guanine nucleotide triphosphate (underlined). (B) Alignment of GRIPE with a consensus sequence for a Rap/Ran-GAP (Conserved Domain pfam02145). Residues in red indicate sequence correspondence, while boxed arginine residues (R1358 and R1446 of GRIPE) could be important for the GAP activity of GRIPE.
75i GRIPE GCTACCTCGACAGTAGAAGTAATATTTCATGTATCTACACGAATGCCTTCTGATTCTGATGACTCTCTGACCAAAAAA----- RatTulip1 GCTACCTCTACAGTAGAAGTAATATTTCATGTATCTACAAGAATGCCTTCTGAATCTGATGACTCTCTGACCAAAAAAATAAA RatTulip2 GCTACCTCTACAGTAGAAGTAATATTTCATGTATCTACAAGAATGCCTTCTGAATCTGATGACTCTCTGACCAAAAAA----- ******** ****************************** ************* ************************
GRIPE ------RatTulip1 TTGGAGAATTGCTCTTTCTTTCTCTATAAAGGATTCAGTTGGAATTTTGATGGGGATTCCATTGACTCTGTAGATTGCTTTAG RatTulip2 ------
GRIPE TTGAGACATCTAGGAAATGATGAAGTGCACATTGTTTGGTCAGAACATACTAGAGACTACAGGAGAGGAATTATTCCTACTG RatTulip1 TTGAGACATCTAGGAAATGATGAAGTACACATTGTTTGGTCTGAACATACTAGAGACTACAGGAGAGGAATTATTCCTACAG RatTulip2 TTGAGACATCTAGGAAATGATGAAGTACACATTGTTTGGTCTGAACATACTAGAGACTACAGGAGAGGAATTATTCCTACAG ************************** ************** ************************************** *
GRIPE AGTTTGGTGATGTCC RatTulip1 AGTTTGGTGATGTCC RatTulip2 AGTTTGGTGATGTCC ***************
Figure 6.7. GRIPE is homologous to Tulip-1 and Tulip-2. Sequence alignment of GRIPE nt 3961 to 4635 with Rat Tulip 1 (AAB97075) and Rat Tulip 2 (AAB97076) cDNAs shows an additional 97 nucleotide insert in Tulip-1, and not in Tulip-2 or GRIPE. Grey blocks highlight oligonucleotide primers sequences for RT-PCR experiments to detect predicted isoforms of GRIPE in mouse tissue.
A 1 2 3 4 5 6 7 8 9 10 11 12 - +
298bp 201bp
+RT enzyme
B 1 2 3 4 5 6 7 8 9 10 11 12 - +
-RT enzyme
Figure 6.8. There is only one isoform of GRIPE in the mouse. (A) RT-PCR experiment to detect potential isoforms of GRIPE cDNA in embryonic and adult RNA preparations. The predicted RT-PCR product is 201bp (arrowhead), while the PCR product homologous to Rat Tulip-1 is predicted to be 298bp (arrow). Due to poor image capture in (A), ghosting of the 201 bp signals appear as patches adjacent to lanes “12” and “+”, and are not genuine signals on the agarose gel. Two micrograms of DNase treated total RNA
was reverse transcribed with oligo-d(T)12-16 as a primer. Key : 1 = E11.5 head; 2 = E11.5 trunk; 3 = E13.5 head; 4 = E13.5 trunk; 5 = E15.5 head; 6 = E15.5 trunk; 7 = E17.5 head; 8 = E17.5 trunk; 9 = placental; 10 = adult liver; 11 = adult kidney; 12 = adult brain; - = no template; + = positive control. (B) RT-PCR performed on template preparations without reverse transcriptase indicates absence of DNA contamination.
75ii Chapter 6: The GRIPE gene encodes a 1485 amino acid protein, and its function may be conserved during evolution
As a final proof, an exhaustive search of public (http://www.ncbi.nlm.nih.gov/) and private
(http://www.celera.com) mouse genome databases failed to reveal any significant nucleotide homology of the GRIPE cDNA sequence to any other chromosomal locus, other than on chromosome 12C1. Taken together, it is concluded that there is only one copy of GRIPE gene in the mouse.
6.5. There may be two orthologues and one paralogue of GRIPE in the human
Database homology searches using the mouse cDNA sequence for GRIPE uncovered several human chromosomal loci with high sequence identities. These are located on human chromosomes 14q13.2 and
9q31.2. Further, homology searches performed with the amino acid sequence of mouse GRIPE identified yet another putative human GRIPE gene located on human chromosome 20p11.23. A summary of these genes, as well as the evidence for their relationship to mouse GRIPE, is provided in Table 6.2.
Gene Evidence for Chromosomal location Gene structure Comments identity to (h = human; m = mouse) GRIPE 1) GRIPE Located on mouse At least 30-40 exons; chromosome 12C1 spanning >200kb
2) DKFZp566D133 Nucleotide and Located on human At least 8-10 exons; Possible homologue, but its (partial amino acid amino acid chromosome 9q31.2, spanning <6kb PolyA tail is present in genomic sequence; 381 aa) sequence syntenic to mouse sequence – a duplication event matches chromosome 4 producing a pseudogene?
3) KIAA0884 Nucleotide and Located on human At least 30-40 exons; Most likely orthologue of mouse (partial amino acid amino acid chromosome 14q13.2, spanning >200kb GRIPE; available RT-PCR sequence; 943 aa) sequence syntenic to mouse expression data parallels work matches chromosome 12C1 done in this thesis
4) KIAA1272 Amino acid Located on human At least 28 exons; Most probable paralogue of (predicted amino acid match only, no chromosome 20p11.23, spanning >160kb mouse GRIPE – a result of either sequence; 1023 aa) identity at the syntenic to mouse convergent or divergent nucleotide level chromosome 2 evolution. RT-PCR data suggests different pattern of expression to mouse GRIPE
Table 6.2. Comparison of the three putative human orthologues of mouse GRIPE.
76 Chapter 6: The GRIPE gene encodes a 1485 amino acid protein, and its function may be conserved during evolution
6.6. The human homologue of GRIPE, named hGRIPE, is located on chromosome 14q13.2.
A close examination of synteny maps between mouse and human genomes indicate that mouse chromosome 12C1 is syntenic to human chromosome 14q13.2. As a corollary, neighbouring genes which flank this chromosomal location (such as Pax9 and Foxa1) in the human show an identical arrangement in the mouse, providing further evidence for synteny (for a useful graphical representation of synteny maps, see http://www.ncbi.nlm.nih.gov/Homology/). Furthermore, this region of human chromosome 14 encodes a partial cDNA that bears extremely high nucleotide and protein sequence homology to mouse GRIPE.
Assigned with the identifier KIAA0884, this cDNA encodes a human homologue of GRIPE, but lacks a stop codon, 3’UTR and polyA tail (not shown), hence this cDNA will hereafter be named hGRIPE.
Certainly, there is good correspondence in amino acid sequence between these two polypeptides (see Fig.
6.9). A close investigation of alignments of mouse GRIPE compared with human chromosome 14q13.2 sequence reveals that hGRIPE is organised into 30 to 40 exons, and spans at least 20kb (not shown). This chromosomal organisation of hGRIPE is consistent with mouse GRIPE. Finally, semi-quantitative RT-
PCR experiments conducted by Kikuno and colleagues (Kikuno et al 2001; http://www.kazusa.or.jp/huge/) show a pattern of gene expression which parallels that of mouse GRIPE; hGRIPE is detected in adult brain and kidney, as well as in fetal brain. Since the authors who conducted these RT-PCR experiments had targeted KIAA0884 cDNA sequence that was identical to mouse GRIPE (nt 1050 to 1155), their expression data provides corroborating evidence demonstrating that KIAA0884 is indeed the putative cDNA for hGRIPE.
6.7. Human GRIPE, or hGRIPE, may have been duplicated to human chromosome 9q31.2 during evolution.
Homology searches of the published human genome sequence with mouse GRIPE cDNA sequence revealed a second chromosomal locus: human chromosomes 9q31.2. Further, the amino acid sequence of mouse GRIPE displayed a near-perfect match with a human EST localised to this locus on human 9q31.2, and named DKFZp566D133 (see Fig. 6.10). With the consideration that ESTs may represent putative transcripts that may or may not be correctly processed and translated in vivo, several lines of evidence indicate that DKFZp566D133 is not the human orthologue of GRIPE, but rather an EST which could be a
77 GRIPE: 1 MCKRILNIYRYMVVQVSMDKKTWEQMLLVLLRVTESVLKMSSQAFLQFQGKKSMTLAGRL 60 MCKRILNIYRYMVVQVSMDKKTWEQMLLVLLRVTESVLKM SQAFLQFQGKK+MTLAGRL KIAA0884: 396 MCKRILNIYRYMVVQVSMDKKTWEQMLLVLLRVTESVLKMPSQAFLQFQGKKNMTLAGRL 455
GRIPE: 61 AGPLFQTLIVAWIKANLNVYISREXXXXXXXXXXXXTYWEELATEWSLTMETLTKVLARN 120 AGPLFQTLIVAWIKANLNVYISRE TYWEELATEWSLTMETLTKVLARN KIAA0884: 456 AGPLFQTLIVAWIKANLNVYISRELWDDLLSVLSSLTYWEELATEWSLTMETLTKVLARN 515
GRIPE: 121 XXXXXXXXXXXXXXXEXXXXXXXXXXXXXEFQKVSVDKSFSRGWSRDQLGQAPMRQRSAT 180 E EFQKVSVDKSFSRGWSRDQ GQAPMRQRSAT KIAA0884: 516 LYSLDLSDLPLDKLSEQKQKKHKGKGVGHEFQKVSVDKSFSRGWSRDQPGQAPMRQRSAT 575
GRIPE: 181 TTGSPGTEKARSIVRQKTV------199 TTGSPGTEKARSIVRQKTV KIAA0884: 576 TTGSPGTEKARSIVRQKTVAMRSRSIGECALPSAYIRSAKSAPVLIHTSKPFLPDIVLTP 635
GRIPE: 200 ------DIDDAQILPRSTRVRHFSQSVDTGNEVFGALHEEQPLPRSSSTSDILEPFTVER 253 DIDDAQILPRSTRVRHFSQS +T NEVFGAL+EEQPLPRSSSTSDILEPFTVER KIAA0884: 636 LSDELSDIDDAQILPRSTRVRHFSQSEETANEVFGALNEEQPLPRSSSTSDILEPFTVER 695
GRIPE: 254 AKVNKEDTSPKLPPLNSETGGNSANVPDLMDEFIAERLRSGNASTMTRRGSSPGSLEIPK 313 AKVNKED S KLPPLNS+ GG+SANVPDLMDEFIAERLRSGNASTMTRRGSSPGSLEIPK KIAA0884: 696 AKVNKEDMSQKLPPLNSDIGGSSANVPDLMDEFIAERLRSGNASTMTRRGSSPGSLEIPK 755
GRIPE: 314 GLPDILNKQNQMRPVDDPGVPSEWTXXXXXXXXXXXXXXXXXXXXXXFQCEGRKFDNFGF 373 LPDILNKQNQMRP+DDPGVPSEWT FQ +GRKFDNFGF KIAA0884: 756 DLPDILNKQNQMRPIDDPGVPSEWTSPASAGSSDLISSDSHSDSFSAFQYDGRKFDNFGF 815
GRIPE: 374 GTDIGIPSSADVDLGSGHHQSTEEQEVASLTTLHLDSETSSLNQQAFSAEVATVTGSESA 433 GTD G+ SSADVD GSGHHQS EEQEVASLTTLH+DSETSSLNQQAFSAEVAT+TGSESA KIAA0884: 816 GTDTGVTSSADVDSGSGHHQSAEEQEVASLTTLHIDSETSSLNQQAFSAEVATITGSESA 875
GRIPE: 434 SPVHSALGSRSQTPSPSTLSRAHIEQKDLQLDEKLHHSVLQTPDDL 479 SPVHS LGSRSQTPSPSTL+ H+EQKDLQLDEKLHHSVLQTPDDL KIAA0884: 876 SPVHSPLGSRSQTPSPSTLNIDHMEQKDLQLDEKLHHSVLQTPDDL 921
Figure 6.9. Amino acid sequence alignment of GRIPE and KIAA0884 polypeptides. Stretches of “XXX” represent regions of absolute sequence conservation which were ignored by the alignment tool (Wootton and Federhen 1996)
GRIPE: 1102 SLQLRRFRETVPTWSTIREEEDVLDELLQYLGTTSPECLQRTGISLNVPAPQPLCISEKQ 1161 + Q +RFRETVPTW TIR+EED LDELLQYLG TSPECLQRTGISLN+PAPQP+CISEKQ DKFZp: 3 TFQFKRFRETVPTWDTIRDEEDALDELLQYLGVTSPECLQRTGISLNIPAPQPVCISEKQ 62
GRIPE: 1162 ENDVINAILKQYTEEKEFVEKHFNDLNMKASEQDEPTPQKPQSAFYYCRLLLSILGMN-W 1220 E+DVINAILKQ+TEEKEFVEKHFNDLNMKA EQDEP PQKPQSAFYYCRLLLSILGMN W DKFZp: 63 ESDVINAILKQHTEEKEFVEKHFNDLNMKAVEQDEPIPQKPQSAFYYCRLLLSILGMNSW 122
GRIPE: 1221 AAGIPGTNGGSFHXXXXXXXXXXXXXXXDSRQCRETHKIAVFYVAEGQEDKYSILTNIGG 1280 SFH DSRQCRETHKIAVFYVAEGQEDK+SILTN GG DKFZp: 123 ------DKRRSFHLLKKNEKLLRELRNLDSRQCRETHKIAVFYVAEGQEDKHSILTNTGG 176
GRIPE: 1281 SQAYEDFVAGLGWEVNLTNHCGFMGGLQKNRSTGLTTPYFATSTVEVIFHVSTRMPSDSD 1340 SQAYEDFVAGLGWEVNLTNHCGFMGGLQKN+STGLTTPYFATSTVEVIFH+STRMPSDSD DKFZp: 177 SQAYEDFVAGLGWEVNLTNHCGFMGGLQKNKSTGLTTPYFATSTVEVIFHMSTRMPSDSD 236
GRIPE: 1341 DSLTKKLRHLGNDEVHIVWSEHTRDYRRGIIPTEFGDVLIVIYPMKNHMFSIQIMKKPEV 1400 DSLTKKLRHLGNDEVHIVWSEHTRDYRRGIIPTEFGDVLIVIYPMKNHMFSIQIM+KPEV DKFZp: 237 DSLTKKLRHLGNDEVHIVWSEHTRDYRRGIIPTEFGDVLIVIYPMKNHMFSIQIMRKPEV 296
GRIPE: 1401 PFFGPLFDGAIVNGKSSAIMVRSTAINASRALKSLIPLYQNFYEERARYLQTIVQHHLEP 1460 PFFGPLFDGAIVNGK IMVR+TAINASRALKSLIPLYQNFYEERARYLQTIVQHHLEP DKFZp: 297 PFFGPLFDGAIVNGKVLPIMVRATAINASRALKSLIPLYQNFYEERARYLQTIVQHHLEP 356
GRIPE: 1461 TTFEDFAAQVFSPAPYHHFPADADH 1485 TTFEDFAAQVFSPAPYHH P+ ADH DKFZp: 357 TTFEDFAAQVFSPAPYHHLPSGADH 381
Figure 6.10. Sequence alignment of GRIPE and DKFZp566D133.1 polypeptides.
77i Chapter 6: The GRIPE gene encodes a 1485 amino acid protein, and its function may be conserved during evolution processed pseudogene. Firstly, human chromosome 9q31.2 is syntenic to mouse chromosome 4, and not mouse chromosome 12C1, where mouse GRIPE is located. If human GRIPE is located on 9q31.2, two explanations could account for this: either (i) a unique shuffling event has occurred in mice and not in humans, or (ii) there has been, rather improbably, an incorrect chromosomal assignment during the collation of these independently verified mouse-human synteny maps.
A closer examination of the putative GRIPE locus on human 9q31.2 uncovered more evidence of a possible pseudogene. Analysis of mouse GRIPE cDNA alignment to human chromosome 9q31.2 sequence reveals that the putative human transcript is organised into 8-10 exons, and spans no more than 6kb of genomic sequence. This gene structure is not consistent with its mouse counterpart, which is organised into 30 to 40 exons and spans over 200kb. Curiously, the entire 3’ sequence of mouse GRIPE cDNA, including the polyadenylation sequence and polyA tail, is encoded in the last exon of the putative human transcript (Fig.
6.11). In light of these observations, it is suggested here that 9q31.2 harbours a pseudogene which may have been duplicated from a canonical GRIPE homologue, and was inserted into chromosome 9q31 during the course of evolution.
6.8. A human paralogue of GRIPE, named GRIPE Related-1 (hGR-1), is located on human chromosome 20p11.23.
While nucleotide homology searches with mouse GRIPE cDNA sequence uncovered putative human homologues on human chromosomes 9q31.2 and 14q13.2, database searches using the amino acid sequence of mouse GRIPE uncovered yet another homologous human GRIPE protein, assigned KIAA1272 (Kikuno et al., 2002). This 1023 amino acid polypeptide is translated from a gene localised to human chromosome
20p11.23. An analysis of the amino acid sequence of KIAA1272 with mouse GRIPE reveals significant identity in their C-termini; shown in Fig. 6.12, KIAA1272 encodes a putative Ran/Rap GAP (panel A) which is nearly identical to the GRIPE polypeptide (panel B). In consideration of the lack of nucleotide sequence identity with putative human GRIPE homologues located on 9q31.2 and 14q13.2, this protein is possibly a paralogue of GRIPE that could have arisen during human evolution. This event may be explained either as a consequence of: (i) a GRIPE homologue that was duplicated and subsequently
78 A GRIPE: ttttgtttaacatgggcatctcctaaggaaatgtagcatgat 00001706 |||||||| ||||||||||||| ||||||||||||||||| <<<<<<<< h9q31.2: ttttgttttgaatgggcatctcctgaggaaatgtagcatgat 99090076
GRIPE: aactgcatgtgtaaatatatcatcctgacaaaccacagaatataaattat 00001763 ||||||||||||||||| ||||| ||| ||| || | |||||||||||| <<<<<<<< h9q31.2: aactgcatgtgtaaatacatcatactggcaagccgtaaaatataaattat 99090135
GRIPE: gtatcattcatgtagtgtctacagatttgtaa 00001795 |||||||||| ||||| |||| | |||||||| <<<<<<<< h9q31.2: gtatcattcacgtagtatctata.atttgtaa 99090166
GRIPE: aaaagatgacatggtatttaataa 00001826 |||||||||||||||||||||||| <<<<<<<< h9q31.2: aaaagatgacatggtatttaataa 99090200
GRIPE: aataaaaatattcttcacaaaaaaaaaaaaaaaaa 00001867 ||||||||||||||| ||||||||||||| |||| <<<<<<<< h9q31.2: aataaaaatattcttatcaaaaaaaaaaaagaaaa 99090238
gctcacaTTT TGTTTtgaAT GGGCATCTCC TgAGGAAATG TAGCATGATa ttggtactAA B CTGCATGTGT AAATAcATCA TaCTGgCAAg CCgtAaAATA TAAATTATGT ATCATTCAcG TAGTaTCTAt AATTTGTAAc agtggggggA AAAGATGACA TGGTATTTAA TAAtacAATA AAAATATTCT TatCAAAAAA AAAAAAgAAA Agaaaaccag cacaagacaa ggatgccctc tctcaccact cctactcaac atagtattga aagttctgac cagggcaatc aggcaagaga aagaaataaa g
Figure 6.11. Alignment of mouse GRIPE with human chromosome sequence (nt 99090028 to 99090338 of human chromosome 9q31.2) in block format (panel A), and as an overlay with human chromosome sequence (panel B). Capital letters indicate matching bases between mouse GRIPE sequence and human genome sequence. The polyadenylation signal and polyA tail of GRIPE maps to genomic sequence of human chromosome 9q31.2 (underlined).
A KIAA1272: 852 ILSNERGSQAYEDFVAGLGWEVDLSTHCGFMGGL-QRNGSTGQTAPYYATSTVEVIFHVS 910 Pfam02145: 1 MYSNEEGSPAFDEFLTGLGQRVRLKDFEPYRGGLDTKGDDTGTHSYYWHYDIMEIMFHVS 60
KIAA1272: 911 TRMP-SDSDDSLTKKLRHLGNDEVHIVWSEHSRDYRRGIIPTAFGDVSIIIYPM----KN 965 Pfam02145: 61 TLLPYTESDKQQLQRKRHIGNDIVTIVFQEPGEPFSPGTIRSHFQHVFVIVRPHDPCTNH 120
KIAA1272: 966 HMFFIAITKKPEVPFFGPLFDGAIVSGKLLPSLV--CATCINASRAV 1010 Pfam02145: 121 TTYLVSVTRRKDVPGFGPTLPAGAVFDKNLEFRAFLLAKHINAENAV 167
B KIAA1272: 805 DRRKNFHLLKKNSKLLRELKNLDSRQCRETHKIAVFYIAEGQEDKCSILSNERGSQAYED 864 +FH DSRQCRETHKIAVFY+AEGQEDK SIL+N GSQAYED GRIPE: 1227 TNGGSFHXXXXXXXXXXXXXXXDSRQCRETHKIAVFYVAEGQEDKYSILTNIGGSQAYED 1286
KIAA1272: 865 FVAGLGWEVDLSTHCGFMGGLQRNGSTGQTAPYYATSTVEVIFHVSTRMPSDSDDSLTKK 924 FVAGLGWEV+L+ HCGFMGGLQ+N STG T PY+ATSTVEVIFHVSTRMPSDSDDSLTKK GRIPE: 1287 FVAGLGWEVNLTNHCGFMGGLQKNRSTGLTTPYFATSTVEVIFHVSTRMPSDSDDSLTKK 1346
KIAA1272: 925 LRHLGNDEVHIVWSEHSRDYRRGIIPTAFGDVSIIIYPMKNHMFFIAITKKPEVPFFGPL 984 LRHLGNDEVHIVWSEH+RDYRRGIIPT FGDV I+IYPMKNHMF I I KKPEVPFFGPL GRIPE: 1347 LRHLGNDEVHIVWSEHTRDYRRGIIPTEFGDVLIVIYPMKNHMFSIQIMKKPEVPFFGPL 1406
KIAA1272: 985 FDGAIVSGKLLPSLVCATCINASRAVKCLIPLYQS 1019 FDGAIV+GK +V +T INASRA+K LIPLYQ+ GRIPE: 1407 FDGAIVNGKSSAIMVRSTAINASRALKSLIPLYQN 1441
Figure 6.12. KIAA1272, also known as hGR-1, may be a novel GAP protein. (A) Protein sequence alignment of hGR-1 with the conserved GAP domain pfam02145. Red residues indicate sequence conservation, while arginines (bold text and boxed) may be important for GAP activity. (B) Alignment of hGR-1 with GRIPE, demonstrating extremely high sequence conservation in their C- termini. 78i Chapter 6: The GRIPE gene encodes a 1485 amino acid protein, and its function may be conserved during evolution shuffled and mutated; or (ii) a convergent evolutionary event involving a non-homologous gene which acquired then eventually maintained a protein sequence similar to GRIPE. Semi-quantitative RT-PCR experiments conducted by the authors of KIAA1272 and KIAA0884 (Kikuno et al., 2002) indicate that these genes show non-complementary patterns of gene expression (Fig. 6.13). For example, KIAA0884
(hGRIPE) transcripts are detected in adult liver and spinal cord, while KIAA1272 is absent. These observations lead to speculation that KIAA1272 is a GRIPE paralogue which is located on human chromosome 20p11.23.
Further surveys of the sequence databases reveal the mouse homologue of KIAA1272 is represented by a predicted cDNA, assigned XM_141404. Located on mouse chromosome 2G2 (which is syntenic to human chromosome 20p11.23), this transcript encodes a predicted 2095 amino acid protein with a partial Ran/rap
GAP domain in its C-terminus. While protein alignments reveal that only an amino-terminal portion of a putative GAP domain is encoded in XM_141404 (Fig. 6.14), cloning experiments may clarify possible inaccuracies in gene prediction, and uncover an entire GAP domain encoded in this gene. Furthermore, although these two proteins differ in size by over 1000 amino acids (KIAA1272 is a 1023 amino acid polypeptide), their amino termini shows remarkable sequence similarity (not shown). Based on these observations, it is concluded that XM_141404 is the mouse homologue of KIAA1272, and both cDNAs are hereby-named mGR-1 (murine Gripe Related-1) and hGR-1 (human Gripe Related-1), respectively.
6.9. A role for GRIPE in controlling motor axon guidance and synaptogenesis in Drosophila.
Mouse GRIPE bears high nucleotide and protein sequence identity to a cDNA predicted from Drosophila melanogaster. Designated AE003758 (predicted gene product CG5521), this gene is localised to chromosome 3 and encodes a 1958 amino acid protein containing a GAP domain. Curiously, CG5521 was identified by Kraut and coworkers as a potential genetic determinant which orchestrates proper motor axon guidance and synaptogenesis in Drosophila (Kraut et al., 2001). This discovery was made through a P- element based gain-of-function screen which identified genes that, when misexpressed in motor neurons of the peripheral nervous system, caused alterations in the neuromuscular system of developing fly larvae. By tracking the reproducible innervation patterns of type I neuromuscular junction synapses, mutant larvae
79 KIAA0884 (hGRIPE)
Experimental conditions for KIAA0884 Primer_f : CTCAGATAGTCATTCGGATTC Primer_r : CACATCAGCAGAGGACGTAAC PCR conditions : 95 °C30 sec55 °C30 sec72 °C60 sec30 cycles
KIAA1272 (hGR-1)
Experimental conditions for KIAA1272 Primer_f : GCCCTAGCATTTATTGGACCA Primer_r : CAGTAATGTGTTGGCAGTGCG PCR conditions : 95 °C30 sec55 °C30 sec72 °C60 sec30 cycles
Figure 6.13. KIAA0884 (hGRIPE) and KIAA1272 (hGR-1) are expressed in non-complementary domains. Semiquantitative RT-PCR experiments performed by Kikuno and coworkers at RIKEN (Kikuno et al., 2002) indicate that hGRIPE and hGR-1 may assume analogous but non-identical roles in humans.
79i Chapter 6: The GRIPE gene encodes a 1485 amino acid protein, and its function may be conserved during evolution which exhibited aberrant innervation by motor neurons were identified for closer examination. From these experiments, it was reported that overexpression of Drosophila GRIPE (fly line designated as EP(3)0327 or
EP3527; http://flybase.bio.indiana.edu/) manifested as a defect in axonal pathfinding. While specific details were not elaborated, the authors had defined “axonal pathfinding” defects as flies which exhibited
“truncations and defasiculation failures of entire nerve branches, as well as missing branchpoints and individual axons with abnormal trajectories” (Kraut et al., 2001). In light of the apparent similarities in nucleotide and protein sequence between GRIPE and CG5521, it is conceivable that GRIPE may assume analogous roles in mature neurons of the mammalian central nervous system. Thus, these functional experiments carried out by Kraut and colleagues have provided valuable insight towards elucidating a role for GRIPE in neurodevelopment and axon guidance.
Amino acid alignments of CG5521 with hGRIPE and hGR-1 indicate that both human polypeptides may be orthologues of the Drosophila gene. In light of the non-complementary expression patterns of hGRIPE and hGR-1 (shown in Fig. 6.13), it is noteworthy to consider that GRIPE may be a genetic marker for an evolutionary expansion of CG5521 gene function in higher order organisms. That is, it is postulated here that the contribution of one gene product (CG5521) to the development of an ancestral organism is expanded to two genes (hGRIPE and hGR-1) in a more complex organism.
6.10. The SAGE Tag for GRIPE is not a unique identifier
Serial Analysis of Gene Expression (SAGE) is an approach to gene discovery which permits a global survey of the target transcriptome, and complements existing approaches which survey global gene expression, such as microarray analysis. Unlike microarray analysis, however, which surveys expression of a set of cloned cDNAs by hybridisation, SAGE additionally permits for identification of novel genes otherwise precluded from discovery in the former analysis (Tan et al., 2002). The approach involves generating unique identifying “tags” which comprise 3’UTR sequence adjacent to the polyA tail of target messenger RNAs. These identifying tags are concatenated and then sequenced to deduce the relative abundance of each identifying tag, regardless of identity. Since its invention in 1995 (Velculescu et al.,
1995), some researchers have made their data publicly available through SAGEmap, a web site which
80 XM_141404: 1997 ILANERGSQAYEDFVAGLGWEVDLSTHCGFMGGL-QRNGSTGQTAPYYATSTVEVIFHVS 2055 KIAA1272: 852 ILSNERGSQAYEDFVAGLGWEVDLSTHCGFMGGL-QRNGSTGQTAPYYATSTVEVIFHVS 910 pfam02145: 1 MYSNEEGSPAFDEFLTGLGQRVRLKDFEPYRGGLDTKGDDTGTHSYYWHYDIMEIMFHVS 60
XM_141404: 2056 TRMP-SDSDDSLTKK-VQEGVSFV 2077 KIAA1272: 911 TRMP-SDSDDSLTKKLRHLGNDEVHIVWSEHSRDYRRGIIPTAFGDVSIIIYPM----KN 965 pfam02145: 61 TLLPYTESDKQQLQRKRHIGNDIVTIVFQEPGEPFSPGTIRSHFQHVFVIVRPHDPCTNH 120
KIAA1272: 966 HMFFIAITKKPEVPFFGPLFDGAIVSGKLLPSLV--CATCINASRAV 1010 pfam02145: 121 TTYLVSVTRRKDVPGFGPTLPAGAVFDKNLEFRAFLLAKHINAENAV 167
Figure 6.14. XM_141404, also known as mGR-1 is the mouse homologue of hGR-1, and may lack a functional GAP domain. Homology searches reveal XM_141404, or mGR-1, contains a partial GAP domain, and lacks conserved arginine fingers found in other GAP proteins.
UniGene cluster ids UniGene cluster title Number of sequences Hs. 30148 homeodomain 6/46 interacting protein kinase 3
Hs. 167031 DKFZp566D133 protein 33/46
Hs. 15903 ESTs 5/46 N/A No UniGene cluster for 2/46 this sequence
Figure 6.15. The SAGE tag for GRIPE is not a unique identifier. The predicted NlaIII- prepared SAGE tag, defined as “GTATTTAATA” was found in 46 mRNA-source sequences. Of these, 44 clustered in 3 UniGene clusters, while 2 remained unclustered. This information is available through the following web link: http://www.ncbi.nlm.nih.gov/SAGE/index.cgi?tag=GTATTTAATA&org=Hs&anchor=NLAIII &Go=submit&cmd=tagsearch
80i Chapter 6: The GRIPE gene encodes a 1485 amino acid protein, and its function may be conserved during evolution serves as a user-group for SAGE biologists, as well as a depository for SAGE information
(http://www.ncbi.nlm.nih.gov/). Libraries of SAGE tag data can be interrogated for estimates of the relative abundance of a target gene in various transcriptomes. With respect to criteria for SAGE tag preparation, the tag for GRIPE in mice and humans is defined as “GTATTTAATA”, and represents sequence resolved following NlaIII cleavage at the extreme 3’ site of the mRNA (adjacent to the polyA tail). Information pertaining to this tag in published human libraries reveals that it is not a unique identifier for GRIPE. Rather, this SAGE tag may represent at least 3 human genes, grouped into 3 UniGene clusters
(http://www.ncbi.nlm.nih.gov/UniGene/). As shown in Fig. 6.15, this SAGE tag represents UniGene clusters Hs.15903, Hs.167031 and Hs.30148. While cDNA sequences clustered to Hs.15903 have yet to be better defined, it is clear that, upon inspection of curated cDNA sequences, “GTATTTAATA” is the genuine SAGE tag for GRIPE (ostensibly clustered to Hs.167031) and HIPK3 (Hs. 30148). In a further complication, the Hs.167031 UniGene cluster includes the possible pseudogene sequence DKFZp566D133
(evidence presented in section 6.6.), which further clouds interpretation of the relative abundance of this tag as an identifier for GRIPE. Therefore, the SAGE tag data collection for GRIPE is uninformative since it is not a unique identifier.
An explanation for the ambiguity of the SAGE tag as an identifier lies in its sequence composition; the internal “ATTTAA” is a conserved sequence adjacent to the polyA site of mRNAs, and has been demonstrated to increase the stability of these mRNAs (Shaw and Kamen, 1986). Therefore, this sequence may be conserved in more than one gene transcript that demonstrates message stability in vivo.
6.11. Summary
6.11.1. GRIPE is a 1485 amino acid protein with conserved domains that provide clues to its function in vivo.
An integrative approach, combining classical molecular biology techniques with Bioinformatics, led to successful cloning of the full-length coding sequence for GRIPE. Analysis of the translated polypeptide sequence revealed that GRIPE is a 1485 amino acid protein with several conserved domains. A predicted leucine zipper in the amino terminus of GRIPE may govern interaction with yet unknown binding partners
81 Chapter 6: The GRIPE gene encodes a 1485 amino acid protein, and its function may be conserved during evolution in vivo, while the GAP domain may be important for facilitating hydrolysis of GTP bound to small G proteins such as Rap and Ran. Further investigations of sequence databases revealed that GRIPE is found in many organisms, an observation which could suggest conservation of gene function throughout evolution. Ectopic expression studies in Drosophila demonstrate defects in axonal pathfinding by motor neurons which misexpress GRIPE, and provide the first clue to its role in mature neurons of the adult nervous system.
6.11.2. GRIPE is present in many genomes, suggesting conservation of gene function during evolution.
The prevalence of GRIPE in such diverse organisms suggests an evolutionary conservation of gene function. In the human, GRIPE appears to have even been duplicated to two chromosomal loci, although the second locus on 9q31.2 could harbour a pseudogene. Furthermore, a paralogous gene, hGR-1, may complement GRIPE function in humans; their over-lapping but non-identical patterns of gene expression suggests a division of labour for roles otherwise assumed by a single genetic determinant in their ancestral orthologue. This hypothesis is substantiated by observation that amino acid sequences of hGRIPE and hGR-1 are highly homologous to Drosophila GRIPE; an analagous role for hGRIPE and hGR-1 in peripheral nervous system development could be observed with ectopic expression assays using cultured motor neurons. In the rodent, GRIPE and mGR-1 may exhibit analogous expression profiles to their respective human homologues, and targeted deletion studies or transgenesis may reveal the contribution of these genes to neuronal function.
Database information for GRIPE-like sequences remain poorly documented. Since Northern analyses presented here (Chapter 4) documents a single 8kb mRNA species in all mouse tissues tested, it is conceivable that the authors of rat Tulip-1 and Tulip-2 have cloned partial cDNAs to GRIPE. It is also unfortunate that SAGE data is uninformative for GRIPE, since the tag is not a unique identifier.
82 Chapter 7: General discussion Chapter 7: General discussion
7.0. GENERAL DISCUSSION
Members of the helix-loop-helix (HLH) family of transcription factors are key contributors to a wide variety of developmental processes, including neurogenesis, myogenesis and lymphopoiesis. While the role of neurogenic Class II HLH factors is clearly demonstrated through loss-of-function (LOF) and gain- of-function approaches (GOF) (for a review see Bertrand et al., 2002), the role of the ubiquitously expressed Class I HLH factor, E12, in neurogenesis remains ambiguous. Originally discovered as an important transcriptional regulator of B cell development, expression studies subsequently revealed widespread expression of E12 in proliferative zones of multiple nascent organs of the embryo, including the developing cerebral cortex (Perez-Moreno et al., 2001; Roberts et al., 1993); implying a role for E12 in development of the nervous system. Numerous biochemical studies have implied a role for E12 in neurogenesis, but a definitive schema has not been formulated.
It is the focus of this discussion to clarify the role of E12 in brain development. A survey of the literature reveals distinct gradients of HLH gene expression during corticogenesis (see Figure 1.13), and suggests combinatorial signalling by these transcription factors in a temporally regulated fashion. Importantly, E12 expression is restricted to the early steps of neuron formation, with all available evidence indicating a role for this transcription factor in signalling cell cycle exit, and to function as a “co-neurogenic” factor during the early steps of lineage commitment in neuroblasts. Since the function of E12 is, in part, coded by its capacity for protein dimerisation through its HLH domain, a search was undertaken for HLH-binding partners in the developing mouse brain.
7.1. A neurogenic role for E12 through heterodimerisation with HLH and non-HLH factors during brain development
A yeast 2-hybrid interaction screen was employed to identify interacting partners to the HLH domain of
E12 during early (e11.5) and peak (e15.5) neurogenesis in the developing cerebral cortex. This resulted in cloning of the Class II HLH factors Mash1 and NSCL, as well as the Class V HLH factors Id2 and Id3. In addition to these, non-HLH genes were also cloned, such as Ubc9/UBCE2A which encodes the ubiquitin- conjugating enzyme for E12 and E47; as well as a novel binding partner which we name GRIPE (see below
83 Chapter 7: General discussion for discussion). Importantly, deletion analysis confirm that cloned binding partners require an intact HLH domain of E12 for binding, indicating these compete for dimerisation to E12 polypeptide.
Following from observations of their biochemical interaction, Northern analysis was conducted to evaluate the extent of coincident gene expression between E12 and its binding partners during neuron formation, and using a cell culture model of in vitro neurogenesis. During neurodifferentiation of P19 embryocarcinoma cells with retinoic acid, discrete waves of gene expression were observed for E12 and its functionally related counterparts, consistent with their postulated combinatorial functions at distinct phases of neurogenesis. For example, E12 and its inhibitory dimerisation partners Id2, and possibly Id3, are highly expressed at the early stages of RA-induced neurodifferentiation of P19 cells, with a progressive down regulation as neurogenesis proceeds. On the other hand, transcripts encoding the neurogenic HLH binding partners to E12, namely Mash1 and NSCL, are detected only as cells begin to differentiate in the presence of retinoic acid, consistent with their roles in programming neurogenesis. While NSCL is down- regulated, Mash1 expression persists throughout neurogenesis, suggesting a furhter role for this transcription factor in programming terminal differentiation. Importantly, expression of the ubiquitin- conjugating enzyme for E2A, mUBC9/UBCE2A, persisted throughout RA-induced neurodifferentiation, but the 1.3kb splice form was down-regulated as P19 cells began to differentiate into neurons, and may reflect a mode of gene regulation which maintains steady-state levels of this protein in vivo. Taken together, these data are invaluable for establishing correspondence between expression of E12 and its cloned binding partners, allowing for formation of theoretical models to define its function during neurodevelopment.
The positive identification of binding partners to E12, as well as elucidation of their patterns of gene expression during neurogenesis, now permits a summary of their presumed combinatorial functions with
E12 in the ventricular zone neuroblast, depicted in Figure 7.1. As shown, data presented in this thesis supports the role for E12 as a nuclear chaperone for cytoplasmic proteins, as well as a transcriptional modulator of downstream genes, signalling either as an HLH-homodimer or heterodimer. In the proliferating neuroblast, E12 heterodimerises with the Class II HLH factors Mash1, NSCL or Math2 to up- regulate expression of neuron-specific genes, thereby committing cells to adopt a neuronal fate.
84 β 1 β α 1- integrin mediated cell adhesion
C3G GDP GTP
Rap1 Rap1 “OFF” ? “ON”
GRIPE
ID3
ID2
Ubc9/ Id2 UBCE2A (Neuman et al., 1995)
Neuronal genes (Miyachi et al., 1999) heterodimer OR p21 E12 (Peverali et al., 1994; Pagliuca et al., 2000)
OR OR P# MCK (Markus et al., 2002) Mash1 NSCL homodimer OR E-cadherin Math2 (Perez-Moreno et al., 2001)
HAT
(Qiu et al., 1998; Massari et al., 1999)
Cytoplasm Nucleus
Figure 7.1. A proposed “co-neurogenic” role for the Class I HLH factor, E12, in developing neurons. E12 heterodimerises with neurogenic Class II HLH factors (such as Mash1, NSCL and Math2) to activate transcription of downstream genes (such as NeuroD) for proper terminal differentiation, as well as to regulate chromatin decompaction through recruitment of histone acetyltransferase (HAT) complexes to target DNA. Concomitantly, E12 may actively repress alternative cell fates through silencing of target genes, such as muscle creatine kinase. E12-expressing neuroblasts loose their adhesiveness through repression of E-cadherin, a function which may be important for endowing a migratory phenotype. Additionally, E12 inhibits cell cycle re-entry through upregulation of the CDKI protein, p21. Finally, E12 upregulates ID2 expression to establish a negative feedback loop for regulating dimerisation to Class II HLH factors. The available pool of E12 monomers in vivo is directly regulated by proteasome degradation (P#) through a ubiquitin-mediated pathway which involves Ubc9/UBCE2A. GRIPE is a novel interacting partner to E12 that contains a putative GTPase Activating Protein (GAP) domain. In addition to a role for GRIPE in negative regulation of E12-dependent gene transcription, this novel gene may regulate β1- integrin mediated adhesion through a novel signalling pathway that involves the cytosolic G-protein, Rap1.
84i Chapter 7: General discussion
Concomitantly, homodimers or heterodimers of E12 directly program cells to exit the cell cycle, through transactivation of CDKIs, such as p21. Functional dimeric complexes comprising E12 may additionally facilitate accessibility of the basal transcriptional apparatus to neuronal promoters, through recruitment of histone acetyltransferase (HAT) complexes for target-chromatin decompaction. Further, neuronal lineage commitment is reinforced through suppression of alternate cell fates, by direct transcriptional repression of non-neuronal genes such as muscle creatine kinase (Markus et al., 2002). As neuroblasts leave the ventricular zone of the developing cortex, E12 may be required for negatively regulating adhesiveness to the germinal matrix, through direct transcriptional repression of E-cadherin. Finally, E12 homo- or heterodimers positively regulate expression of the inhibitory Class V HLH factor Id2, thereby establishing a feedback loop which titrates the available pool of E12 monomers for dimerisation to the neurogenic Class
II HLH factors. Currently, there is no evidence for direct regulation of Id3 expression by E12, since E- boxes were not reported in a recent description of the mouse Id3 promoter (Yeh and Lim, 2000).
In addition to its presumed role as a transcriptional regulator of downstream genes, E12 also functions as an important nuclear chaperone for cytoplasmic proteins that signal in the nucleus. The Class V HLH proteins
ID2 and ID3 lack a nuclear localisation signal, but is imported into the nucleus upon E12-binding (Deed et al., 1996). Similarly, the cytoplasmic protein GRIPE is also imported into the nucleus, and can negatively regulate E12-dependent target gene transcription (see below). Thus, antagonism of HLH signalling cascades by HLH- and non-HLH dimerisation partners involves nuclear import through E12 binding, thereby demonstrating an active recruitment of negative regulators into the nucleus by this transcription factor . Importantly, the pool of available E12 protein is regulated through a ubiquitin-mediated, proteasome degradation pathway involving Ubc9/UBCE2A (Kho et al., 1997; Loveys et al., 1997). Work by Deed and colleagues demonstrate that the half-life of E12 protein is reduced upon heterodimerisation, a function which could involve this ubiquitin-conjugating enzyme as a dynamic sensor for regulating in vivo pools of multi-protein HLH complexes comprising E12. Taken together, the definitive role of E12 in neurodevelopment may be contingent upon its capacity to activate target gene transcription, as well as to facilitate intracellular signalling, through its role as a molecular chaperone.
85 Chapter 7: General discussion
While results presented in this thesis highlights a role for E12 in regulating a variety of intranuclear signalling events during the early stages of neurogenesis, analysis of E2A-deficient mice suggest this transcription factor signals in a redundant manner. Certainly, this genetic redundancy, observed as a lack of overt neurological phenotype in E2A null mutant mice, could underscore the importance of the contribution by Class I HLH factors to neurodevelopment. Nevertheless, the model presented in Figure 7.1 is consistent with presumed roles for the Drosophila homologue daughterless in neuronal-precursor differentiation (Hassan and Vaessin, 1997; Vaessin et al., 1994), reinforcing the concept of functional conservation within paralogues of Class I HLH factors in mammals. Taken together, elucidation of the role of E12 in neurogenesis may allow for clarification of general mechanisms by which this transcription factor programs cell cycle exit and lineage commitment in multiple cell types.
7.2. Class I HLH factors were not isolated from yeast 2-hybrid screens for E12 binding partners
Comprehensive screens for E12 binding partners failed to isolate Class I HLH factors from embryonic mouse brain 2-hybrid libraries, even when previous studies have demonstrated their widespread expression in the developing cortex (see Chapter 1.4.5.6.). Since library screening conditions employed in this thesis focused on maximising detection of interacting preys, this failure to isolate Class I HLH factors could thus be interpreted as an inability for E12 to heterodimerise with E47, HEB and E2-2. To rule out an absence of these genes in the prey libraries screened, multiple Class I HLH prey cDNAs, including isoforms of E2A
(E12 and E47), E2-2 (known as MITF2A and MITF2B) and ME1 were isolated using a Xenopus NeuroD bait construct (data not shown). Furthermore, there is currently no evidence describing heterodimerisation of E12 to these aforementioned Class I HLH factors, and homodimers represent only a small proportion of functional units in vivo (Sloan et al., 1996). Furthermore, the inability for E12 to efficiently homodimerise, coded in its inhibitory domain (Shirakata and Paterson, 1995; Sun and Baltimore, 1991), may also preclude heterodimerisation to related Class I HLH proteins. Taken together, observations here are consistent with a role for E12 to signal in a redundant manner with related Class I HLH factors, but not as heterodimers with these.
86 Chapter 7: General discussion
These observations warrant an explanation for the evolutionary expansion of Class I HLH factors seen in mammals, such as in rodents and humans. In their respective genomes, both mammals encode three Class I factors: E2A, E2-2 and HEB (also known as ME1). On the other hand, flies and nematodes contain only one Class I HLH factor, namely daughterless and CE/Da, respectively. This genetic expansion in mammals may be explained as a parsimonious route to genomic evolution, which broadens the role of
Class I HLH factors to multiple, overlapping signalling cascades in a multitude of progenitor cell types.
Certainly, gene duplication may overcome limitations seen with a single locus, since transcriptional regulation may be more readily saturable at the enhancer/insulator regulatory locus of a genetic determinant. Over time, paralogues of the common ancestral gene adopt dedicated roles, as their regulatory loci accumulate genomic mutations, leading to progressive restriction, or even silencing, of gene function.
Certainly, in the mouse, the Class I HLH factors E2A, HEB (ME1) and E2-2 (ME2) exhibit overlapping, but non-identical expression patterns during brain development, indicating that these three Class I HLH factors, could have evolved from a common ancestral gene, and are orthologous species of Drosophila daughterless.
7.3. A small subset of HLH genes identified as binding partners to E12
This thesis reports cloning of Mash1, NSCL, Id2 and Id3 as interacting partners to the HLH domain of E12.
However, other HLH factors which are known binding partners to E12, such as MASH2 and ID1 (see
Figure 1.12), demonstrate coincident expression patterns to E12, but were not isolated from the screens.
Similarly, members of the Class VI HLH factors, such as Hes1 and Hes5, are known to bind E2A proteins, but were not identified as interacting partners to E12. In consideration of this, several explanations may clarify their lack of representation in yeast 2-hybrid screens conducted herein. Firstly, the strategy for prey library construction may preclude some cDNAs from assessment for E12-binding by the encoded polypeptide. For example, HLH genes encoding E12-binding proteins may harbour long 3’ UTRs, hence the probability of isolating 5’ sequences which encode their amino-termini HLH domains may be detrimentally affected by the 3’ bias imposed through the use of an oligo-d(T) for generating cDNAs.
Furthermore, complications with the yeast 2-hybrid approach may confound results as well, since prey fusion proteins may not be properly expressed in yeast, or prey plasmid-transformed yeast cells may not be
87 Chapter 7: General discussion viable. Similarly, cDNAs encoding HLH genes may be fused out-of-frame to the GAL4 TAD during library construction, hence a nonsense fusion polypeptide is generated. Alternatively, the mouse E12505-651 bait may be incompatible for binding to cognate HLH protein targets, as additional N-terminal sequences not present on mouse E12505-651 may be required for interaction to the entire gamut of HLH-dimerisation partners in the tissue milieu. Certainly, the conserved C-terminal domain adjacent to the HLH domain of
E12 is demonstrated here (see Chapter 4) and by others (Goldfarb et al., 1998) to be crucial for dimerisation. Nevertheless, the cloning of interacting partners to E12 validates this approach, and further work must be carried out to clarify their combinatorial roles with E12 during neurogenesis.
It is reported here that the human and mouse E12 baits used in yeast 2-hybrid interaction screens are dissimilar in their capacities for protein dimerisation. For example, cDNAs encoding NFI/x,a nuclear transcription factor (Kruse et al., 1991), and GRIP1, a glutamate receptor interacting protein (Dong et al.,
1997), were cloned as binding partners to human E12, but could not bind the mouse E12 bait. While the yeast 2-hybrid assay indicates mouse E12501-651 does not bind NFI/x, it is curious that this transcription factor may function co-operatively to regulate expression of the myelin basic protein promoter in oligodendrocytes (Aoyama et al., 1990). Analysis of the distal promoter of this gene reveals NFI/x consensus sequences directly adjoining an E-box site. While by no means convincing, this observation indicates that these proteins could signal in tandem to regulate target gene transcription. This issue warrants clarification, thus alternative approaches to the re-assessment of protein-protein interaction between human E12-derived preys to mouse E12, such as co-immunoprecipitation or reporter assays, should be considered.
7.4. GRIPE is a novel interacting partner of E12, and may regulate its dimerisation state in the nucleus
A novel interacting partner to E12, named GRIPE, has been cloned from developing mouse brain. The association of GRIPE to E12 was mapped to the HLH domain of E12, suggesting that GRIPE may share and compete with other HLH proteins for dimerisation with E12. Furthermore, E12 may be important for the delivery of GRIPE to the nucleus, indicating that one facet of GRIPE function is dependent upon its
88 Chapter 7: General discussion dimerisation to E12, and subsequent nuclear import of the dimer. Attenuation of E12-dependent target gene transcription by GRIPE suggests a function akin to inhibitory HLH proteins, such as ID2, that are known to negatively regulate HLH signalling cascades by forming inactive heterodimers with E12. Furthermore, striking similarities between the HLH and adjacent C-terminal domains of E proteins, such as HEB and E2-
2 (Goldfarb et al., 1998), suggest that GRIPE may bind these as well, and influence multiple HLH signalling cascades involving these transcription factors. Taken together, these results indicate that GRIPE may be a transcriptional regulator in association with E12 but by itself has other cytoplasmic functions.
A parallel may be seen with the interaction of Tbr-1 (a T-box transcription factor) with CASK. The activity of CASK is dependent on subcellular localisation, as well as its interaction with other proteins in the cell; in the cytoplasm, CASK functions as a membrane-associated scaffold protein, involved in the assembly of specific protein complexes at sites of cell contact. However, in the presence of Tbr-1, CASK binds to its C terminus of Tbr-1 and translocates to the nucleus, where it behaves as a coactivator of Tbr-1 to induce transcription of T-element containing genes such as reelin (Hsueh et al., 2000). An analogous situation may also be seen with ß-catenin, which normally resides in the cytoplasm. Following Wnt signalling, ß- catenin gradually appears in the nucleus in a concentration-dependent manner, and interacts with members of the Tcf family of DNA-binding proteins to promote expression of Wnt-responsive genes (for a review see Sharpe et al., 2001).
While these examples demonstrate the importance of contextual function through specific genetic interactions, alternative explanations of GRIPE function abound. As GRIPE is only one of many possible interacting partners of E12 cloned from the prey library, it may function as a competitive binding partner with other HLH proteins found in this tissue milieu. Binding of GRIPE with E12 may reduce the available
E12 substrate binding to other HLH proteins such as Mash1 and Id2. Another explanation is that GRIPE has other bona fide functions in the cytoplasm but binding to E12 silences these activities, upon nuclear entry. Conversely, GRIPE may elicit its signalling activities in the nucleus, upon nuclear import by E12.
In any case, results presented in this thesis demonstrate biochemical interaction of E12 with this novel
89 Chapter 7: General discussion gene, and further work must be carried out to elucidate their true physiological roles in neuroblasts, as well as in mature neurons.
7.5. GRIPE may be a novel GAP protein
Members of the Ras-family of GTP-binding proteins function as molecular switches in growth (Altschuler and Ribeiro-Neto, 1998), differentiation (Asha et al., 1999; Dugan et al., 1999), survival and adhesion of eukaryotic cells (for reviews see Bos, 1997; Bos et al., 2001; Bos et al., 1997). These switches cycle between GDP-bound “inactive” and GTP-bound “active” forms, allowing for modulation of signalling by these cellular factors in vivo. Importantly, this cycling is mediated by guanine nucleotide exchange factors
(GEFs) and GTPase activating proteins (GAPs) which activate, or inactivate, G-protein signalling respectively. That is, GEFs exchange GDP for GTP on cognate G-protein targets (such as Ras, Rap and
Ran), while GAPs facilitate GTP hydrolysis of G-proteins. An analysis of the polypeptide sequence reveals
GRIPE may be a novel GAP involved in regulating GTP hydrolysis of small cytosolic G proteins such as
Rap and Ran (see Figure 6.6). Although the GAP function of GRIPE remains to be tested, amino acid residues comprising putative arginine fingers within the GAP domain are in good correspondence with those observed in other known GAP proteins such as RapGAP (P47736), mouse Tuberin (AAA86901) and
SPA-1 (BAA22197). This suggests that GRIPE may be a GTPase activating protein for Rap1a, Rap1b and
Rap2 in vivo. Therefore, further work must be conducted to verify functionality of this predicted domain, since there is considerable heterogeneity in GAP protein motifs that facilitate GTP hydrolysis (Scheffzek et al., 1998). A recent report has demonstrated that GAP proteins for the nuclear G-protein Ran, known as
RanGAPs, do not to require the canonical arginine finger motif for hydrolysis of GTP-bound Ran (Seewald et al., 2002). Therefore, an evaluation of the GAP function of GRIPE should allow identification of cognate G-protein targets which reside in the cytoplasm (through interaction with Rap and Ras), or the nucleus (through interaction with Ran). In addition, it remains to be tested if the GAP function of GRIPE is abrogated upon binding to E12 (see below).
The importance of GAP proteins in normal development is exemplified in the autosomal dominant disorder known as tuberous sclerosis. The condition is characterised by the development of hamartomas in the
90 Chapter 7: General discussion brain, skin, kidneys, heart and other organs. In familial cases the disease segregates as an autosomal dominant trait, linked either to chromosome 9q34 (TSC1) or to chromosome 16p13.3 (TSC2). Some patients carry missense mutations in the GAP domain of the TSC2 gene, indicating that this domain is critical for normal cellular function (Maheshwar et al., 1997). Studies of the Drosophila homologue of
TSC2, known as gigas, indicate that a deletion of the C-terminus of gigas protein, which includes the GAP domain, results in mutant cells that exhibit defects in cell cycling (Ito and Rubin, 1999). Analogously,
GRIPE may be an important regulator of cell cycling through a pathway that involves E12. Strikingly, these LOF analyses mentioned herein reveal non-compensatory function of related GAPs in mutant animals, indicating that these serve dedicate roles as conditional modulators of GTP-bound proteins (such as Ras, Rap and Ran), even though their biochemical targets may be identical (see Luo, 2000) for a review). Together, these observations provide immutable evidence that the GAP activity of GRIPE must be evaluated using various biochemical approaches, or functional assays (see below).
7.6.0. GRIPE may assume different functions during development and in adulthood
7.6.1. A role for GRIPE in negatively regulating cell adhesion in neuroblasts?
The coincidental expression of GRIPE and E12 mRNA during embryogenesis suggests that GRIPE function may be tightly linked to E12 signalling in the embryo. In the developing forebrain, GRIPE mRNA is localised to cells of the ventricular zone, similar to the distribution of E12 transcripts. This suggests that
GRIPE may have an important role in maintaining the dimerisation state of E12 to other HLH proteins, such as Mash1 or Id2, and may even behave as a negative regulatory protein for E12, thus contributing indirectly to neurogenesis. This is corroborated by observations of P19 cells induced to differentiate into neurons with retinoic acid; E12 mRNA levels peaked when neurogenic bHLH genes are activated, while
GRIPE levels continued to increase. This suggests that, in the presence of E12-binding proteins such as
ID2 and GRIPE, an up-regulation of E12 may be required to produce sufficient substrates for dimerisation with Mash1 and NSCL1, thus increasing the proportion of functional heterodimers required for the proper co-ordination of neurodifferentiation, while facilitating necessary entry of cytosolic proteins into the nucleus.
91 Chapter 7: General discussion
In addition to postulated roles in antagonism of E12-dependent gene transcription in the nucleus, the binding and subsequent nuclear delivery of GRIPE by E12 may silence the cytoplasmic activities of
GRIPE. Recently, accumulating evidence has implicated a role for Rap1, a potential G-protein target of
GRIPE, in regulating cell adhesion through an integrin-mediated pathway (for a review see Bos et al.,
2001). For example, following GTP-exchange through interaction with its guanine nucleotide exchange factor, C3G, the “active” GTP-bound Rap can stimulate β1-integrin mediated cell adhesion of haematopoietic cells (Arai et al., 2001). In a complementary approach, mice lacking C3G exhibit severe defects in cell adhesion (Ohba et al., 2001), and studies of hypomorphic alleles of C3G mice demonstrate a loss in migratory behaviour of cortical neurons, revealed through explant assays (Voss et al, manuscript in preparation). Importantly, overexpression of dominant negative Rap1 (Arai et al., 2001), or forced expression of a GTPase activating protein for Rap1, RapGAP (de Bruyn et al., 2002), resulted in loss of integrin-mediated adhesion of transfected cells. While it remains to be determined if GRIPE inactivates
Rap1 through its GAP domain, nuclear localisation of GRIPE may negatively regulate β1-integrin mediated adhesion in ventricular zone neuroblasts of the developing cortex.
Integrins are ubiquitous cell surface glycoproteins that mediate cell-to-cell, and cell-to-extracellular matrix
(ECM) interactions (Hynes et al., 1992; Hynes and Lander, 1992; Lilien et al., 1999; Milner and Campbell,
2002a; Milner and Campbell, 2002b). These extracellular glycoproteins provide a physical transmembrane link between the ECM and the cytoskeleton, and can transduce bi-directional signals across the cell membrane. Importantly, integrins have been shown to be crucial for proper development of the cerebral cortex, and can mediate neuronal migration along radial glia in vitro (Anton et al., 1999; Magdaleno and
Curran, 2001; Milner and Campbell, 2002a; Milner and Campbell, 2002b). Studies of Cre-lox mice which produce glial and neuronal precursors deficient in β1-integrin demonstrate defects in corticogenesis, resulting typically in a convoluted cerebral cortex marked by inappropriate invasion of neurons into a normally cell sparse Layer I (Graus-Porta et al., 2001). Further analysis of these mice reveals a gross disruption of the marginal zone, seen as a perturbation of the basement membrane and Cajal-Retzius cell heterotopia (Graus-Porta et al., 2001). Therefore, observations in these mutant mice reveal that proper co-
92 Chapter 7: General discussion ordination of integrin-mediated adhesiveness is indispensable for migration of cortical cells from the ventricular zone of the developing cortex.
With the observations that: (i) Rap1 can mediate β1-integrin dependent neuronal cell adhesion, and (ii) β1- integrin is crucial for proper development of the cerebral cortex, a physiological correlate may be drawn with the nuclear import of GRIPE by E12, and seen as a coincidence in gene expression seen in neuroblasts of the ventricular zone. Shown in Fig. 7.1, GRIPE may be important for abrogating the potential for Rap1 to signal integrin-mediated adhesion in neuroblasts of the ventricular zone. That is, the GAP function of
GRIPE behaves as a dynamic regulator of Rap1-GTP/Rap1-GDP levels, in conjunction with the guanine nucleotide exchange factor, C3G. Upon nuclear sequestration by E12, the GAP activity of GRIPE is silenced, allowing for accumulation of GTP-loaded Rap1, thereby facilitating integrin-mediated adhesion of the postmitotic neuroblast to radial glia, and leading to proper layer formation in the cortex. Thus, the combinatorial function of GRIPE and E12 may lead to modulation of nuclear activity, as well as abrogation of cytoplasmic signalling in vivo.
7.6.2. A role for GRIPE in axonal pathfinding in mature neurons?
In adult neurons, GRIPE is expressed in many regions of the adult brain, including the hippocampus and neocortex. The absence of E12 transcripts in adult hippocampus suggests that the activity of GRIPE in adult brain does not involve an interaction with E12. However, one cannot rule out the possibility that paralogues of E12 expressed in the adult brain, such as ME2 (Soosaar et al., 1994) or ME1a (Chiaramello et al., 1995), could also interact with GRIPE. Certainly, their C termini shows remarkably high protein sequence conservation (Goldfarb et al., 1998), hence paralogous Class I HLH proteins with similar C- termini to E12 may bind to GRIPE in cells of the adult brain.
In the absence of E12, GRIPE may serve a different function in the brain. Studies of GAP proteins such as
SPAR have demonstrated their importance in regulating dendritic spine morphology of hippocampal neurons, a role that requires a functional GAP domain (Pak et al., 2001). This brings up the possibility that
GRIPE may also have important roles for maintaining structural integrity of mature granule cells of the
93 Chapter 7: General discussion adult hippocampus, possibly through its GAP function. Furthermore, this function may be evolutionarily conserved, since the Drosophila homologue of GRIPE, known currently as CG5521, was identified as a potential regulator of axon guidance and synaptogenesis in motor neurons of the peripheral nervous system
(Kraut et al., 2001). In consideration of the conservation of Class I HLH factors, and of GRIPE cDNA sequences in both Drosophila and mammalian genomes, their biochemical interaction reported in this thesis could lead to identification of key signalling pathways for proper neuron formation and mature cell function that is conserved during evolution.
7.6.3. Multiple isoforms of GRIPE may code for overlapping function during development and in adult brains of mammals
An analysis of the mouse and human genomes reveals multiple species of GRIPE cDNAs in these mammals. Conversely, the Drosophila genome only encodes a single orthologous GRIPE species, which brings up the possibility that the function of this gene product has been duplicated (or triplicated) to accommodate function in higher order organisms. Certainly, sequence alignments reveal extremely high sequence conservation between GRIPE and GR-1 (see Chapter 6.7 and 6.8), indicating they arose from a common ancestor. Furthermore, experiments by Kikuno and colleagues (Kikuno et al., 2002; presented in
Figure 6.13) demonstrate overlapping but non-identical expression of these paralogues, suggesting a progressive restriction of gene function upon duplication in the host genome.
In addition to GRIPE and GR-1 in these organisms, humans also encode a putative pseudogene on chromosome 9q31.2 (see Chapter 6.7). While this pseudogene may not encode a functional protein product, it may be involved in post-transcriptional regulation of related sequences, such as the canonical
GRIPE and GR-1 genes, through a mechanism of RNA silencing known as RNA interference (for reviews see Mattick and Gagen, 2001; Moss, 2001; Nishikura, 2001; Tuschl, 2001). This process of post transcriptional gene silencing involves production of a 21 to 25 nucleotide double-stranded RNA molecule that targets specific mRNA sequences for degradation by RNAse III (Moss, 2001; Nishikura, 2001; Tuschl,
2001). Thus, transcriptional activation of the GRIPE pseudogene from chromosome 9q31.2 may result in post-transcriptional regulation of cellular pools of GRIPE and GR-1 mRNA.
94 Chapter 8: Future directions Chapter 8: Future directions
8.1. A re -examination of mice lacking Class I HLH factors for neurological defects.
Evidence presented in this thesis warrants a re-evaluation of the nervous systems of mice which lack Class
I HLH factors, such as E2A (Bain et al., 1994; Zhuang et al., 1994), HEB (Zhuang et al., 1996) and E2-2
(Bergqvist et al., 2000; Zhuang et al., 1996). However, since it is postulated herein that Class I HLH factors signal cell cycle exit and lineage commitment albeit in a redundant manner, a neurological phenotype may not be easily uncovered in single mutant mice. Nevertheless, this genetic redundancy underscores an indispensable role for these transcription factors in neurodevelopment, a contribution that should be revealed through breeding of double mutant mice. In anticipation of defects in cell cycle exit and lineage commitment, double homozygous mutant mice which lack multiple Class I HLH factors should exhibit dysregulated cell proliferation in multiple nascent organs, including the ventricular zone of the developing forebrain. In addition, these mutant mice should also present with reduced expression of
CDKIs, as well as possible tumorigenesis. Thus, these LOF approaches should clarify the contribution of
Class I factors to neurodevelopment. Certainly, a similar approach was employed to describe a neurogenic role for the inhibitory Class V HLH factors, ID1 and ID3 (Lyden et al., 1999). While single mutant mice lacking ID1 or ID3 did not produce any overt phenotype, breeding of double homozygous mutant mice uncovered their role in proper timing of neuroprogenitor differentiation and cell cycle exit (Lyden et al.,
1999).
During mammalian evolution, an expansion in Class I and V HLH gene compartments has lead to multiple redundant signalling mechanisms by these. In contrast, the genome of Drosophila melanogaster only harbours one species of Class I (daughterless) and Class V (extramacrochaete) HLH factor. Analysis of mutant flies that lack either gene has been invaluable in establishing a role for these in neuronal fate determination and terminal differentiation. Perhaps the extent of conservation in function between mammalian and Drosophila Class I HLH proteins may be evaluated through functional replacement of mammalian E12 gene at the daughterless gene locus. This approach has been successful in uncovering a partial compensatory function for the mammalian homologue of Drosophila atonal, Math1, in flies that encode Math1 at the atonal locus (Ben-Arie et al., 2000). Functional replacement of daughterless with E12 may allow for an evaluation of the potential for the mammalian homologue to rescue defects in neuronal
95 Chapter 8: Future directions differentiation seen in da-mutant flies. In consideration of the high sequence homology between these two polypeptides, it is suggested here that a partial rescue will be observed in these animals.
8.2. Gain-of-function approaches to elucidating E12 function in neuroprogenitors
Loss-of-function approaches to genetic dissection may be uninformative if the gene in question is functionally related to paralogous species which, additionally, exhibit coincidental spatial and temporal expression patterns. This certainly holds true for E12, since related family members, such as HEB, can functionally replace E2A in mice (Zhuang et al., 1998). Thus, gain-of-function strategies may be more effective in deducing the role of E12 in programming cell cycle exit and lineage commitment in target cells that ectopically express this transcription factor. Previous work by Peverali and coworkers has demonstrated that forced expression of E2A signals cell cycle arrest in fibroblasts (Peverali et al., 1994).
To investigate the possibility that E12 may also signal neuroprogenitors to exit the cell cycle, replication- defective retroviruses may be utilised for delivery of E12 expression constructs to ventricular zone cells of the developing e11.5 mouse forebrain. Ectopic expression of E12 should result in premature cell-cycle exit and misexpression of the cyclin-dependent kinase inhibitor p21. Additionally, virally-infected cells will possibly display a down regulation of E-cadherin mediated cell adhesion, as well as premature expression of Hes5, a Class VI HLH factor which is a marker for corticogenesis (Akazawa et al., 1992; Casarosa et al.,
1999). In anticipation of premature neurogenesis in cells which overexpress E12, virally-infected cells may additionally express neuronal HLH factors, such as members of the neurogenic Class II HLH factors,
Ngn1/2 and NeuroD. These effects, however, should not be inferred as an intrinsic neuronal programming potential encoded by E12, since GOF experiments using undifferentiated P19 embryocarcinoma cells do not demonstrate a capacity for E12 to signal neurogenesis (Farah et al., 2000). Rather, the extrinsic cues within the ventricular zone may promote neurophenotype-programming in cells which have exit the cell cycle, a scenario akin to premature neurogenesis in mice which lack the negative HLH regulators ID1 and
ID3 (Lyden et al., 1999).
In a similar approach, forced expression of E12 in neuroprogenitors may also be important for identifying downstream target genes regulated by this transcription factor. A chromatin-immunoprecipitation (ChIP)
96 Chapter 8: Future directions assay (Cohen-Kaminsky et al., 1998; Weinmann et al., 2001) will be invaluable for cloning promoter DNA sequences which are bound by homo- or heterodimers of E12. Currently, this assay system for uncovering
E12-responsive neural promoters is in want of a suitable antibody for co-immunoprecipitation of protein-
DNA complexes, since available antibodies to E2A polypeptides recognise and bind to the HLH domain of their C-termini; access to the HLH domain may be occluded upon dimerisation by E12, and subsequent glutaraldehyde cross-linking during ChIP analysis. Thus, antibodies raised to the N-terminus of E12 may be more useful for these assays.
8.3. Evaluation of protein-protein dimerisation between mammalian HLH factors important for neurogenesis
From the available evidence, the most important subclasses for neural programming in mammals are members of Class II (Mash1 and Neurogenins), Class V (the inhibitory ID proteins) and Class VI (hairy and enhancer-of-split related HLH genes). Their roles are readily revealed in loss- and gain-of-function experiments in vitro and in vivo. In addition, the Class I HLH factors bind to members of Class II for proper neuro-phenotype programming as heterodimers, since the Class II factors homodimerise poorly.
Significantly, cross-specificities for protein-protein dimerisation between members of Class I, II, V and VI have not been comprehensively surveyed (see Figure 8.1). For example, while EMSAs have shown that ID proteins prevent formation of heterodimers between Class I and II (Cabrera et al., 1994), it is not clear if
Class V proteins bind members of Class II HLH factors. While members of Class V bind Class I (Loveys et al., 1996; Sun et al., 1991), there is differential capacity for Class V factors to bind members of Class II
(Langlands et al., 1997). Work by Langlands and colleagues demonstrated a preference for ID1 and ID3 to bind myogenic Class II HLH factors, such as Myf-5 and MyoD (Langlands et al., 1997). Conversely, ID1 and ID3 did not bind members of the Class II which program haematopoiesis, such as Tal-2 and Scl
(Langlands et al., 1997). These observations underscore different modes of negative regulation by members of Class V in various differentiation programs: by direct or indirect antagonism of myogenic, or haematopoietic Class II factors, respectively. Thus, these binding studies must be extended to include neurogenic Class II HLH factors, in order to elucidate their mechanisms of signalling cross-talk.
97 Chapter 8: Future directions
? ü da atonal achaete CE/Da III E12 lin-32 HEB Mash1 ü Neurogenins ? NeuroD ü ?
emc hairy ID1 enhancer-of-split ID2 V VI lin-22 ID3 ? Hes1 ID4 Hes5 ? ?
Figure 8.1. Assessment of functional inter-relationships between members of Class I, II, V and VI factors relevant to neurodevelopment. Unknown binding preferences are marked “?”, while “ü” represents known rules for protein-protein interaction. Lack of homo- or heterodimerisation is marked with “X”.
In addition to specificities between Classes of HLH factors, within-Class preferences should also be evaluated (Figure 8.1). These biochemical data will allow for a precise understanding of functional inter- relationships between all HLH proteins present in a given cell. Ultimately, knowledge of precise signalling mechanisms by HLH factors during neurogenesis will permit development of predictive models that reconcile combinatorial function by members of Class I, II, V and VI.
An understanding of cross-specificities for dimerisation is also important for an appreciation of HLH gene complements in different organisms, with respect to the complexity of their nervous systems. For example, while the role of Class II factors in neurophenotype programming is evident across model organisms, such as D. melanogaster (Jan and Jan, 1993; Jarman et al., 1993), C. elegans (Hallam et al., 2000; Portman and
Emmons, 2000) and M. musculus (reviewed in Bertrand et al., 2002), the role of Class I factors is less clear. Loss of the Class I factor daughterless in male mutant flies results in defects in neuronal precursor differentiation (Vaessin et al., 1994), but a neuronal phenotype is not reported in nematodes which lack the homologous gene, CE/Da (Krause et al., 1997). Similarly, there are three Class I HLH genes in the genome
98 Chapter 8: Future directions of M. musculus, and single-mutant mice which lack one of these do exhibit any overt neurological phenotype (Bain et al., 1994; Bergqvist et al., 2000; Zhuang et al., 1996; Zhuang et al., 1994).
Other observations suggest divergence in the biochemical properties of HLH factors between these model organisms during evolution. For example, the genome of C. elegans does not encode Class V HLH proteins (Ruvkun and Hobert, 1998), while M. musculus encodes four members of this subfamily.
Ostensibly, a comprehensive evaluation of binding specificities between all HLH proteins relevant to neurogenesis in these organisms should reveal their idiosyncrasies in regulation of downstream genes in vivo.
8.4. Further biochemical analysis of GRIPE
8.4.1. Colocalisation experiments with full-length GRIPE polypeptide
The cloning of GRIPE as a novel interacting partner to E12 is a novel finding arising out of the current project. Evidence presented here constitutes an inaugural description of their biochemical interaction, and may lead to an integration of cytoplasmic G-protein signalling cascades with nuclear transcriptional regulation under the control of HLH factors. Hence, further biochemical analyses of the GRIPE polypeptide must be conducted in order to reveal the true signalling potential of this novel gene product.
Following from observations that GRIPE701-1485 resides in the cytoplasm of transiently transfected cells, it remains to be tested if full-length GRIPE1-1485 protein exhibits a similar subcellular distribution. In anticipation of this observation, however, available direct and indirect evidence indicates that the full- length protein should be distributed in identical domains to GRIPE701-1485. For example, analysis of the amino acid sequence of full-length GRIPE reveals a conserved membrane anchor motif (encoded in amino acids 1004-1014) that targets this polypeptide to the perinuclear membrane (see Figure 6.6). Furthermore,
GTPase activating proteins for Rap1, such as SPA-1 (Tsukamoto et al., 1999) and Tuberin (Nellist et al.,
1999) are localised to the perinuclear region of host cells. In addition, the putative G-protein target of
GRIPE, Rap1, demonstrates coincidental subcellular localisation for proper activation by GAP proteins
99 Chapter 8: Future directions
(Mochizuki et al., 2000). Thus, further experiments with full-length GRIPE and E12 polypeptides should yield similar results as those described herein.
While heterologous expression systems have been invaluable for describing biochemical interaction between GRIPE and E12, these observations must be reconciled through approaches that more closely reflect the nature of their combinatorial signalling in vivo. To this end, reciprocal coimmunoprecipitation experiments may be performed using whole cell extracts from embryonic brain, and with specific antibodies to GRIPE and E12. Thus, these experiments serve to demonstrate that, indeed, these two proteins interact in cells of the embryonic brain. Since an antibody is currently not available, it is of the utmost importance to produce this invaluable reagent for further biochemical analyses of the novel polypeptide, GRIPE.
In addition to whole protein coimmunoprecipitations to reveal protein-protein interaction in vivo, immunocytochemistry may also be carried out to determine the subcellular distribution patterns of GRIPE and E12 during neurodevelopment, and in adult brain. Since the data presented in this thesis postulates a combinatorial role for GRIPE and E12 in embryonic brain, but not in the adult central nervous system, then it stands to reason that GRIPE may be localised to the nucleus of neuroblasts which express E12 protein.
Similarly, GRIPE may be predominantly localised in the cytoplasm of mature hippocampal neurons that lack E12. Certainly, antibodies to GRIPE will be useful to track its subcellular localisation in vivo.
8.4.2. Does GRIPE encode a functional GAP domain?
It is well documented that proteins that contain a GAP domain have demonstrable roles in signalling proliferation, morphogenesis and cell adhesion in eukaryotic cells. With such demonstrable signalling potential, the GAP domain of GRIPE must be evaluated for its cognate G-protein targets. To do this, several bacterial expression constructs have been generated which encode GRIPE polypeptides (which comprise the GAP domain) as C-terminal fusions to glutathione S-transferase, and named GST-GRIPE proteins (see Appendix 10.12). Initial characterisation of the fusion constructs confirm expression of the designated polypeptides in the E. coli strain DH5α (data not shown). These fusion proteins may be
100 Chapter 8: Future directions purified for use in GTPase activation assays, which evaluate the potential for accelerating hydrolysis of
GTP-bound small G-proteins such as Rap, Ras and Ran (Der et al., 1986; Kurachi et al., 1997; Pak et al.,
2001).
In an alternate approach, a yeast 2-hybrid assay could be utilised to screen for binding partners to the GAP domain of GRIPE. While this indirect approach for assessing function may be useful, it lends to the assumption that the GAP domain binds G-proteins, which is only implied through database homology searches. Secondly, the GAP domain may only exhibit a transient interaction to its G-protein target, and may not bind the GDP-loaded form. Finally, this approach relies on endogenous GEF systems in the yeast cell host to correctly recognise and load GTP onto murine G-proteins. Regardless, yeast 2-hybrid constructs were generated (see Appendix 10.11), and these have yet to be employed in 2-hybrid library screens. While this strategy for evaluating GAP function has so far not been reported, several research groups have successfully isolated GAP and GEF proteins using G-proteins as yeast 2-hybrid baits (Cantor et al., 1995; Nancy et al., 1999) demonstrating, at least, that the reciprocal approach is informative.
8.4.3. Is GRIPE a direct or indirect transcriptional repressor?
Evidence presented in this thesis implicates GRIPE as a potent negative regulator of E12-dependent target gene transcription. This function is likely attributable to the capacity for GRIPE to heterodimerise with
E12 to form inactive dimers that do not bind E-box promoter DNA. Therefore, electrophoretic mobility shift assays (EMSAs) should be carried out to confirm that heterodimers of E12 and GRIPE, indeed, fail to bind a target E-box sequence. This approach will verify that the function of GRIPE is akin to the Class V
ID proteins, and sequester available E12 monomers to negatively regulate E12-dependent target gene transcription. In addition, the use of EMSAs will be valuable for quantifying the affinity of GRIPE for
E12; competitive EMSAs should also reveal the potential for GRIPE to compete for E12 binding in the presence of other HLH binding partners. These experiments will involve the use of the aforementioned
GST-GRIPE fusion proteins.
101 Chapter 8: Future directions
Alternatively, the potential for GRIPE to interfere with the accessory roles for neurophenotype programming may be assessed using in vitro models of neurogenesis, and signalled through Class II HLH factors. For example, forced expression of Mash1 results in neurodifferentiation of P19 cells in transient transfection assays (Farah et al., 2000). Owing to their low efficiency for homomerisation, Mash1 signals neurogenesis through formation of heterodimers with E12 or E47. Therefore, this system may be useful in deducing the potential for GRIPE to antagonise heterodimers of E12 and Mash1; since it is anticipated that a repressor role for GRIPE will manifest as reduced potential for Mash1 to generate neurons upon co- transfection.
While the above experiments focus on the potential for GRIPE to indirectly regulate transcription of downstream genes, its large molecular mass (at least 140 kDa), as well as its possible nuclear localisation in the presence of E12, lends to speculation that this polypeptide may additionally code for intrinsic transactivation potential; or function as a direct transcriptional repressor. For example, the predicted N- terminal leucine zipper (located on amino acids 535 to 556) may be important for mediating protein-protein interaction to yet undescribed binding partners, of which some may be nuclear transcription factors.
Alternatively, this leucine zipper may constitute a transcriptional activation domain, akin to the ADII domain of E2A proteins (see Figure 1.11). To entertain these possibilities, yeast 2-hybrid interaction screens could be conducted to search for binding partners to ancillary domains predicted on the GRIPE polypeptide. Remarkably, a GRIPE701-1485 bait construct activates yeast reporter genes in the presence of a spurious bait, SV40 (see Table 10.1 of Appendix 10.11) or in the absence of prey plasmid (not shown).
Since the bait constructs for GRIPE727-1485 and GRIPE1280-1485 do not activate yeast reporter genes, it is possible that the C-terminal portion of GRIPE may encode a transcriptional activation domain which warrants further investigation. Failing the use of yeast 2-hybrid assays, coimmunoprecipitations may also be employed to isolate protein factors which bind GRIPE. These could involve GRIPE antibodies, or epitope-tagged fusion constructs through heterologous expression systems, such as EGFP-GRIPE constructs for use with eukaryotic cells.
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8.5. Biochemical analysis of homologues of GRIPE and GR-1 in model organisms
With the knowledge that GRIPE function may be conserved during evolution, the task remains to clone and characterise full length cDNAs for both GRIPE and GR-1 in Mus musculus, Homo sapiens and Drosophila melanogaster. The availability of published genome sequence information for all three organisms will accelerate molecular cloning approaches, including 5’RACE, predictive RT-PCR (similar to the strategy described in Chapter 6.1) and conventional cDNA library panning.
The paralogue of GRIPE, named GR-1, may encode a partial GAP domain in mice and not humans (see
Chapter 6.7). Since the murine mGR-1 cDNA sequence represents a predicted transcript, 3’RACE should determine if an erroneous gene-prediction assignment has been made to the coding sequence at its 3’ end.
Further, semi-quantitative data performed by Kikuno and colleagues (Kikuno et al., 2002) demonstrate overlapping expression patterns of human GRIPE and hGR-1 in the adult. To clarify this, Northern blot analyses should be conducted to provide clarification of transcript sizes, while in situ hybridisation be performed to describe tissue distribution of these novel genes. These data will complement immunohistochemical approaches that reveal coincidence in expression of these paralogous genes during development and in adult tissues.
8.6. Functional analyses of GRIPE using Drosophila and mouse models
Work by Kraut and colleagues have uncovered a role for GRIPE in regulating proper axonal guidance and synaptogenesis of motor neurons of the peripheral nervous system in Drosophila (Kraut et al., 2001). With the observation that GRIPE negatively regulates E12-dependent gene transcription, the role of daughterless may be functionally linked with GRIPE in Drosophila. While biochemical approaches, such as yeast 2- hybrid assays, coimmunoprecipitations and EMSAs should be informative in describing their possible interaction, existing mutant lines of flies may be useful, as a first step, in describing their combinatorial signalling roles. For example, male flies lacking daughterless may exhibit a predominantly cytoplasmic distribution of GRIPE, consistent with their biochemical interaction. Similarly, the mutant fly line which overexpresses GRIPE, designated EP(3)0327 or EP3527 (Kraut et al., 2001), will be invaluable in establishing a functional relationship with daughterless. Embryos derived from EP3527 should be
103 Chapter 8: Future directions evaluated for expression of genes normally regulated by daughterless, such as couch potato, deadpan and asense (Hassan and Vaessin, 1997; Vaessin et al., 1994). If GRIPE antagonises the transcriptional activity of daughterless, then ostensibly these abovementioned downstream genes should demonstrate a concomitant decrease in the fly line EP3527.
In light of the postulated roles for GRIPE in neuronal development and function, a definitive description of its function in brain development may be elucidated through production of mutant mice that harbour null alleles for this gene. Certainly, the importance of “activators” (GEFs) and “silencers” (GAPs) is demonstrated in their non-complementary roles for programming growth and differentiation through regulation of GTP-exchange or hydrolysis; even though their biochemical targets may be identical. Mice which lack GRIPE may exhibit increased Rap1-dependent adhesion through hyperstimulation of the β1- integrin signalling pathway. In another scenario, lack of GRIPE may result in increased signalling by E12, which may lead to early cell cycle withdrawal in mutant mice. Furthermore, mature hippocampal neurons in these mice may exhibit defects in axonal pathfinding and synaptogenesis, since RapGAPs have demonstrable roles in regulating dendritic spine morphologies (Kraut et al., 2001; Pak et al., 2001). Studies of mutant mice harbouring hypomorphic alleles for the guanine nucleotide exchange factor C3G demonstrate reduced cell adhesion, excessive proliferation of neuroblasts of the telencephalon, as well as defects in development of the ganglionic eminences (Voss, manuscript in preparation). These data may be interpreted as an accumulation of the GDP-bound “inactive” form of Rap1 (Ohba et al., 2001), thereby serving as an analogous model for GAP function during neurodevelopment.
8.7. The role of GRIPE in mature neurons evidenced through in vitro models
In anticipation of the tremendous technical challenge faced when producing homozygous null mutant mice, the use of transformed cell lines for analysis LOF and GOF phenotypes in vitro may also be informative for evaluating the role of GRIPE in β1-integrin mediated neuronal adhesion. Studies of the embryocarcinoma cell line P19, induced to differentiate into neurons using retinoic acid, demonstrated an increase in GRIPE mRNA expression as neurogenesis progressed (see Chapter 5.3). In addition, previous work by Dedhar and colleagues (Dedhar et al., 1991) demonstrated increases in expression of integrin V and integrin β1 mRNA
104 Chapter 8: Future directions as these cells are induced to differentiate with retinoic acid. Therefore, dominant negative approaches to elucidating a role between GRIPE and β1-integrin function may be tested, and using this cell line. To do this, the relatively recent technology of RNA interference (RNAi) may be employed to selectively silence
GRIPE mRNA expression in mature neurons (for reviews see Moss, 2001; Nishikura, 2001; Tuschl, 2001).
Firstly, P19 cells are transfected with an expression construct which programs RNAi for GRIPE mRNA, and is under the control of an inducible promoter, such as the tetracycline-induction system (Gossen and
Bujard, 1992; Gossen et al., 1995). Following selection of stable transformants harbouring this expression construct, P19 cells are induced to differentiate with retinoic acid, before activating RNAi for GRIPE in these cells through addition of an activator (such as tetracycline). It is anticipated that these cells may exhibit symptoms of excessive β1-integrin mediated adhesion, such as aberrant axonal pathfinding and synaptogenesis. Conversely, targeting the guanine nucleotide exchange factor, C3G, for gene silencing through RNAi should produce an opposite effect; with decreased adhesion through down-regulation of β1- integrin. In addition to defects in adhesion, loss of GRIPE function in precursor cells may result in derepression of E12-dependent signalling pathways, and may signal premature withdrawal from the cell cycle, as well as reduced adhesion through transcriptional repression of E-cadherin by E12. Thus, the P19 cell line may be an important tool for deducing the functions of GRIPE and E12 in vitro.
In an alternative approach, GOF assays using primary cultures of hippocampal neurons may uncover roles for GRIPE in regulating dendritic spine morphology. It was recently reported that expression of a dominant negative form of SPAR, a GTPase activating protein for Rap1, causes narrowing and elongation of dendritic spines of transiently transfected hippocampal neurons; this modulation of spine morphology was dependent on its GAP (Pak et al., 2001). In light of high homology between the GAP domains of
GRIPE and SPAR, it is likely that GRIPE may also elicit roles in spine elaboration. Certainly, transcripts for GRIPE are readily detected in granule cells of the dentate gyrus, bringing up the possibility that GRIPE may be the endogenous effector for Rap1 in these cells.
105 Chapter 8: Future directions
8.8. The role of GRIPE/E12 signalling in the development of multiple cell lineages
The widespread expression of E12 transcripts in multiple nascent organs imply a role for this transcription factor in co-ordination of a diverse range of cell differentiation programs (Perez-Moreno et al., 2001;
Roberts et al., 1993). Ostensibly, the role for GRIPE/E12 signalling should be extended to understand their possible function during development of the stomach, kidney, lung, as well as lymphoid and myeloid cells.
Importantly, analysis of mice lacking functional E2A reveals severe defects in B-cell (Bain et al., 1994;
Zhuang et al., 1994) and T-cell (Bain et al., 1999; Yan et al., 1997) development, hence further work should additionally focus on a potential for GRIPE to modulate E12 function during lymphopoiesis.
Available EST information sourced from the National Centre for Biotechnology Information
(http://www.ncbi.nlm.nih.gov) reveals GRIPE transcripts are detected in cDNA libraries constructed from lymphoid and myeloid cells, though the reliability of the data is confounded by potential pseudogenes of identical sequence composition (see Chapter 6.7). Regardless, the GAP function of GRIPE should be evaluated with respect to lymphocyte function, since other GAP proteins, such as SPA-1 (Kurachi et al.,
1997) and RapGap (de Bruyn et al., 2002) negatively regulate β1-integrin mediated adhesion in lymphocytes through modulation of Rap1 signalling. To do this, Northern blot analysis of multiple organs will reveal tissue-specific patterns of gene expression, while transient expression assays with lymphoid or myeloid cell lines should yield significant observations of a gain-of-function phenotype. Significantly, understanding the role of GRIPE and E12 in programming lineage commitment of haematopoietic stem cells (HSCs) could lead to elucidation of novel signalling roles by these which direct neurogenesis of
HSCs. Thus, the description of GRIPE and E12 function may be extended to formulate general principles for their combinatorial roles in signalling growth and differentiation in multiple cell lineages.
8.9. Concluding remarks
Work presented in this dissertation detail a yeast 2-hybrid interaction screen for dimerisation partners to the
HLH domain of E12, a Class I HLH transcription factor that is highly expressed in proliferative cells of the developing forebrain. This led to the cloning of known binding partners, including members of Class II and V HLH factors, and the ubiquitin conjugating enzyme Ubc9/UBCE2A. Most importantly, the search for dimerisation partners to E12 has led to cloning of GRIPE, a novel GAP-Related Interacting Protein to
106 Chapter 8: Future directions
E12. GRIPE binds to the HLH region of E12, and may require E12 for nuclear import. Furthermore,
GRIPE may negatively regulate E12-dependent target gene transcription. High levels of GRIPE and E12 mRNA were coincidently detected during embryogenesis, but only GRIPE mRNA levels remained high in adult brain, particularly in neurons of the cortex and hippocampus. These observations were recapitulated through an in vitro model of neurogenesis. Taken together, these results indicate that GRIPE is a novel protein whose dimerization with E12 has important consequences for cells undergoing neuronal differentiation. Furthermore, positive identification of binding partners to E12 in embryonic brain permit synthesis of a unifying model for neurogenic HLH signalling cascades that involve this transcription factor.
Thus, further assessment of the functional inter-relationships between E12 and its binding partners will complement existing LOF approaches to reveal its proposed role as a “co-neurogenic” factor in cortical development.
107 Chapter 9: Bibliography Chapter 9: Bibliography
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10.0. Appendix
10.1. Enzymes used: The following enzymes were purchased from Promega (Promega, Madison, WI) T4 DNA polymerase RQ1 DNAse T4 DNA ligase AMV reverse transcriptase T3 RNA polymerase Klenow DNA polymerase T7 RNA polymerase T4 polynucleotide kinase RNAse inhibitor All restriction endonucleases
Taq DNA polymerase was purchased from Perkin Elmer Life Science (Perkin Elmer Life Science, Boston, MA)
10.2. Plasmid constructions used for generating cDNA probes for Northern analysis Plasmid constructs which contain GRIPE cDNAs (with reference to Genbank Acc. No. AY066011): Vector Plasmid stock # Sequence of GRIPE encoded in cDNA insert* 1 pBSIIKS 58 2101 to 3668 2 pBSIIKS 60 3669 to 5867 3 pBSIIKS 79 3669 to 3917 4 pBSIIKS 80 4282 to 4698 * all four constructs harbouring GRIPE cDNAs were employed as probes for Northern analysis.
Plasmid constructions which contain E12 cDNAs (with reference to Genbank Acc. No. AK017617): Vector Plasmid stock # Sequence of E12 encoded in cDNA insert 1 pBSIIKS 42 1452 to 1960 2 pBSIIKS 122 1 to 2838
10.3. PCR amplification of prey cDNAs. Template DNA (either as phage eluate or purified DNA) was added to reaction mix containing 1 x AmpliTaq buffer, 2.5mM dNTPs, 5µM of each primer (5’AD primer sequence AGGGATGTTTAATACCACTAC; and 3’AD primer sequence GCACAGTTGAAGTGAACTTGC), 1% Tween 20, and 1 unit of Taq polymerase. The final volume was 40µl, and PCR cycling conditions were as follows: 1 cycle (93°C for 5 min, 48°C for 5 min), followed by 30 cycles (72°C for 3 min, 93°C for 1 min, 48°C for 1 min), with a final extension at 72°C for 10 min, followed by storage at 4°C before analysis.
131 Chapter 10: Appendices
10.4. Description of prey cDNAs cloned through yeast 2-hybrid library screens HLH genes isolated as preys to human and/or mouse E12 baits.
Name of cDNA Accession number Coding sequence cloned Comments Id2 M69293 1 to 134* HLH domain encoded in aa 36 to 76 Id3 M60523 1 to 119* HLH domain encoded in aa 41 to 81 Math2 D44480 103 to 337 HLH domain encoded in aa 107 to 147 Mash1 X53725 1 to 231* HLH domain encoded in aa 127 to 166 NSCL-1 M82874 1 to 133* HLH domain encoded in aa 73 to 133 *denotes entire polypeptide sequence cloned
Non-HLH genes isolated as preys to human and/or mouse E12 baits.
Name of cDNA Accession number Coding sequence cloned Comments GRIP1 U88572 414 to 1102 HGCP U61837 28 to 154 NFI/x U57636 97 to 441 PBP U43206 168 to 186 the last 18 amino acids of the polypeptide Ubc9 X99739 1 to 158* *denotes entire polypeptide sequence cloned
10.5. Cloning of mouse E12 bait plasmids. The pBSIIKS-A1/A7 plasmid was generously provided by Dr. Yoshitaka Kajimoto (Osaka University School of Medicine, Japan). This construct was digested with BamHI and SalI to excise the insert cDNA (accession number D29919). The cDNA fragment was further digested with Sau3AI then directionally cloned into pBD2.4.1 which was predigested with EcoRI and blunted. The construct, named pBD- muE12505-651 was sequence verified, and multiple clones evaluated for utility in the yeast 2-hybrid assay.
Cloning of the C-terminal mutant pBD-muE12505-600 was carried out as follows: coding cDNA for E12505-600 was generated by PCR and the resulting target phosphorylated using T4 polynucleotide kinase; pBD2.4.1 was digested with EcoRI and SalI and end-filled with T4 DNA polymerase before ligation to E12505-600 PCR product.
The construct pBD-muE12505-571 lacks helix 2 and adjacent loop sequence, and was prepared accordingly: the bait plasmid pBD-muE12505-651 was digested with StuI and SmaI to excise coding cDNA for E12572-651 before an end-filling reaction with T4 DNA polymerase followed by self-ligation of the remaining linear vector.
132 Chapter 10: Appendices
To prepare the N-terminal mutant pBD-muE12534-651, coding cDNA for E12534-651 was generated by PCR and the resulting target phosphorylated using T4 polynucleotide kinase; pBD2.4.1 was digested with EcoRI and SalI and end-filled with T4 DNA polymerase before ligation to E12534-651 PCR product
10.6. Cloning of deletion prey constructs of GRIPE. The construct pAD-GRIPE701-932 encodes a C-terminal deletion and was prepared as follows: the 0.7kb EcoRI fragment of pAD-GRIPE701-1485 was ligated to EcoRI digested pAD2.4.1 prey vector.
The N-terminal deletion mutant, pAD-GRIPE933-1485, was prepared by isolating the 3kb EcoRI fragment of pAD-GRIPE701-1485. This insert cDNA was end-filled with T4 DNA polymerase before ligation to EcoRI digested, T4 DNA polymerase end-filled, pAD2.4.1.
The third deletion prey construct, pAD-GRIPE701-1458, was prepared by inserting the 2.3kb NheI fragment of pAD-GRIPE701-1485 to NheI digested pAD2.4.1.
10.7. Cloning of mammalian expression plasmids pEGFP-GRIPE701-1485, pcDNAF-E12505-651 and pcDNAF-E121-651. The construct pEGFP-GRIPE701-1485 was prepared as follows: a BamHI fragment of GRIPE cDNA was excised from the prey plasmid pAD-GRIPE701-1485 and cloned into pEGFP-C1 vector (Clontech, Palo Alto, California, USA ) pretreated with BamHI and dephosphorylated with T4 polynucleotide kinase.
For expression of an N-terminal FLAG epitope, an oligonucleotide duplex was cloned into KpnI and BamHI digested sites of mammalian expression vector pcDNA3 using two primers: 5’- GCCACCATGGCGCGCCAGGACTACAAGGACGACGATGACAAG-3’ and 5’- GATCCTTGTCATCGTCGTCCTTGTAGTCCTGGCGCGCCATGGTGGCGTAC-3’. This plasmid was defined as “pcDNAFlag”.
To prepare pcDNAF-E12505-651, coding sequence for E12505-651 was amplified from A1/A7 template cDNA (D29919) using primers which generated a PCR product containing a 5’ BamHI restriction site. This fragment was then phosphorylated with T4 polynucleotide kinase, digested and directionally cloned into pcDNAF, which was predigested with BamHI and EcoRV (and dephosphorylated).
Full-length cDNA for E12 was encoded in AK017617, and was provided by RIKEN (Yokohama Institute, Japan). The construct pcDNAF-E121-651 was prepared as follows: the entire coding sequence of E12 was amplified by PCR with primers encoding BamHI restriction sites on either end. This amplified insert cDNA was digested with BamHI then ligated to BamHI/dephosphorylated pcDNAF vector.
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All constructs were sequence verified and fusion proteins evaluated using standard Western blotting techniques.
10.8. Reverse Transcriptase-Polymerase Chain Reaction. Total RNA was prepared for RT-PCR according to the following precodure: 15µg of total RNA was pretreated with 2 units of RQ1 DNase in reaction buffer containing 10-20 units of RNase inhibitor before acid phenol extraction, followed by ethanol precipitation. A representative aliquot (2µg) of RNA was electrophoresed on a denaturing agarose gel to assess integrity of each sample.
Reverse transcriptase reactions were performed as follows: Each 50µl RT reaction contained the following: 2µg of pretreated RNA, 50µM oligo-d(T) primer, 500µM dNTPs and 5-10 units of AMV reverse transcriptase. The reaction was incubated at 42°C for 50 min and stored at -20°C before use.
PCR reactions were prepared as follows: 25µl reactions in buffer containing 1.2µM of each primer, 100µM dNTPs, 1mM MgCl2, 1 unit of Taq (Perkin Elmer Life Science, Boston, MA) and 200ng (5µl of a 50µl reaction) template DNA from reverse transcription reaction. The cycling conditions are: pre-PCR incubation at 94°C for 2 minutes, 30 cycles (94°C for 30secs, 55°C for 45 secs, 72°C for 45 secs) followed by a final incubation at 72°C for 7 mins. Reactions were stored at 4°C until further analysis.
10.9. Preparation of cRNA probes Radioactively labelled cRNA fragments were prepared as follows: plasmid (1µg) encoding GRIPE cDNA (nt 3669 to 3917) was linearised then incubated with cRNA reaction buffer which contained 1 mM of each rNTP (ATP, CTP, GTP), and 5 µM rUTP. Following a brief incubation on ice, the following components were added: 25µCi of α32P-UTP (with a specific activity of 3000 Ci/mmol), 8 units of T3 RNA polymerase and 20-40 units of ribonuclease inhibitor. The reaction was incubated at 37 °C for 1 hour, then excess EDTA was added to stop the enzyme reaction. Probe solution was heated at 65 °C for 15 minutes then chilled on ice before adding to hybridisation bottles.
Preparation of DIG-labelled cRNA probes for in situ hybridisation experiments was as follows: 1µg of linearised vector was incubated with cRNA synthesis buffer containing rNTP mix (2mM of ATP, CTP, GTP, 0.7µM DIG-11-UTP, 1.35mM UTP; purchased from Roche (Roche Biochemicals Gmbh, Germany)), 10-20 units of RNAsin, 10-20 units of T3 or T7 RNA polymerase in a final volume of 20µl. The reaction was incubated at 37°C for 90 mins, then 1 unit of RQ1 DNase added and incubated 15 mins at 37°C. Finally, the preparation was phenol extracted and ethanol precipitated. A representative aliquot was analysed on a denaturing agarose gel to assess quality of transcribed cRNA.
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10.10. Cloning 5’ cDNA sequences for GRIPE by RT-PCR. Reverse transcription of DNase-treated e11.5 total RNA was carried out as dsscribed (Section 10.8). PCR reaction conditions are as follows: reaction buffer contained 1mM dNTPs, 1mM MgCl2, 1.2µM of each primer, 1 unit Taq polymerase, 80 ng (2µl of a 50µl reaction) of template from reverse transcription reaction. PCR cycling conditions are: pre-PCR incubation at 94°C for 2 minutes, 30 cycles (94°C for 30secs, 57°C for 45 secs, 72°C for 45 secs) followed by a final incubation at 72°C for 7 mins. Completed reactions were stored at 4°C until further analysis.
10.11. Cloning and evaluation of yeast 2-hybrid bait constructs encoding GRIPE polypeptide. To prepare GRIPE bait constructs, additional cloning sites were first introduced to the multiple cloning site of pBDGal2.4.1 through ligation of a phosphorylated oligonucleotide. Primers “AATTCAGAGGATCCTCTGCTAGCAATCCCGGGAGATCT” and “TCGACAGATCTCCCGGGATTGCTAGCAGAGGATCCTCTG” were phosphorylated with T4 polynucleotide kinase then annealed before the resulting duplex oligo ligated to pBDGal2.4.1 (pre-digested with EcoRI and SalI). This ligation produced the vector “pBDMCS”.
The bait construct pBD-GRIPE701-1485 was prepared by cloning the BamHI fragment of pAD-GRIPE701-1485 into pBDMCS. The bait construct pBD-GRIPE727-1485 was prepared as follows: the insert cDNA was amplified by PCR using primers which encoded BamHI restriction sites on both ends. The cDNA was then digested and cloned into pBDMCS. To prepare the third bait construct, pBD-GRIPE1280-1485, the EcoRI/BamHI GRIPE cDNA fragment was first end-filled with T4 DNA polymerase, then ligated to a EcoRI digested pBDMCS vector which was also end-filled with T4 DNA polymerase. These bait constructs were evaluated for spurious activation of yeast reporter genes and shown in Table 10.1:
Bait Prey Growth in Growth in media Growth in media media –tl? –tlh (+ 3AT)? –tlha (+ 3AT)? 1pBD-GRIPE701-1485 SV40 +++ +++ +++ 2pBD-GRIPE727-1485 SV40 +++ - - 3pBD-GRIPE1280-1485 SV40 +++ - - 4 p53 SV40 +++ +++ +++ Table 10.1. Evaluation of GRIPE bait constructs. GRIPE bait constructs were evaluated for spurious activation of yeast reporter genes. While all bait-prey transformations produced cells which could grow in media selecting for these plasmids, introduction of pBD-GRIPE701-1485 with a spurious prey resulted in activation of reporter genes, allowing for survival of these cells in media lacking histidine and/or adenine (row 1). The bait constructs pBD-GRIPE727-1485 and pBD-GRIPE1280-1485 did not spuriously activate reporter genes in the presence of SV40 (rows 2 and 3). The SV40 prey binds a known bait, p53 (row 4). 10.12. Preparation of pGEX-GRIPE constructs for expression of GST-GRIPE fusion proteins.
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The construct pGEX-GRIPE701-1485 encodes a GRIPE polypeptide fused to bacterial gluthathione S- transferase (GST), and was cloned by ligating the BamHI fragment of pAD-GRIPE to BamHI digested vector pGEX-2T (Amersham plc, Buckinghamshire, UK). The construct pGEX-GRIPE701-1144 was prepared by digesting the parent vector pGEX-GRIPE701-1485 with SmaI and AccI, then the remaining fragment was end-filled with T4 DNA polymerase and self-ligated. In a similar approach, pGEX- GRIPE701-1334 was cloned by digesting the parent vector pGEX-GRIPE701-1485 with SmaI and StuI, then end- filling the remaining vector with T4 DNA polymerase before self-ligation.
Following sequence verification of cloned constructs, these plasmids were introduced into BL21 strain of E. coli. Transformed cells were maintained in media containing ampicillin, and expression of the fusion protein was stimulated through addition of 0.1mM isopropylthiogalactoside (IPTG) with incubation for 1-2 hours. Cells were then harvested by centrifugation at 20 800 x g for 10 secs. Upon removal of the supernatant, the pellet was resuspended in 300µl of PBS, and 10µl was saved for further analysis. The remaining cell suspension was sonicated before cold-centrifugation for 5 mins at top speed. The resulting supernatant is then incubated for 3 mins at room temperature with 50µl of a 50% slurry of glutathione- sepharose CL-4B (Amersham plc, Buckinghamshire, UK). The sepharose is pelleted with centrifugation at top speed and washed twice with PBS. Following the final wash, all traces of supernatant were removed before resuspending the sepharose in loading dye for denaturing SDS-PAGE gel electrophoresis.
10.13. Northern Analysis Northern analysis was performed as described (Section 2.14). Non-saturating radioactive signals for target RNA species (GRIPE, E12 and 18S rRNA) are shown in Table 10.2. All signals are normalised to 18S rRNA then multiplied by 100. A one-way analysis of variance was conducted on data sets for GRIPE and E12, and shown in Table 10.3 and 10.4 respectively.
GRIPE 1 GRIPE 2 GRIPE 3 Average Av x 100 STDEV STDEV x 100 STD Error RA0 0.554 0.496 0.586 0.545 54.528 0.045 4.539 2.621 RA2 0.510 0.536 0.695 0.580 58.029 0.100 9.979 5.762 RA4 0.487 0.664 0.509 0.553 55.328 0.096 9.640 5.565 RA6 0.550 0.670 0.641 0.620 62.024 0.062 6.215 3.588 RA8 0.637 0.959 1.058 0.884 88.439 0.220 22.016 12.711
E12 1 E12 2 E12 3 Average Av x 100 STDEV STDEV x 100 STD Error RA0 0.300 0.270 0.346 0.305 30.532 0.038 3.838 2.216 RA2 0.364 0.427 0.529 0.440 44.003 0.083 8.309 4.797 RA4 0.275 0.330 0.334 0.313 31.273 0.033 3.309 1.911 RA6 0.190 0.238 0.215 0.214 21.418 0.024 2.409 1.391 RA8 0.154 0.212 0.209 0.192 19.162 0.032 3.244 1.873 Table 10.2. Quantitation of GRIPE and E12 mRNA signals relative to 18S rRNA.
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GRIPE Groups Count Sum Average Variance Row 1 (RA0) 3 1.636 0.545 0.002 Row 2 (RA2) 3 1.741 0.580 0.010 Row 3 (RA4) 3 1.660 0.553 0.009 Row 4 (RA6) 3 1.861 0.620 0.004 Row 5 (RA8) 3 2.653 0.884 0.048 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.240 4 0.060 4.080 0.03247 3.478 Within Groups 0.147 10 0.015
Total 0.388 14 Table 10.3. One-way analysis of variance performed on data set of normalised measurements of GRIPE mRNA signals.
E12 Groups Count Sum Average Variance Row 1 (RA0) 3 0.916 0.305 0.001 Row 2 (RA2) 3 1.320 0.440 0.007 Row 3 (RA4) 3 0.938 0.313 0.001 Row 4 (RA6) 3 0.643 0.214 0.001 Row 5 (RA8) 3 0.575 0.192 0.001
ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.116 4 0.029 13.05 0.0006 3.478 Within Groups 0.022 10 0.002
Total 0.138 14 Table 10.4. One-way analysis of variance performed on data set of normalised measurements of E12 mRNA signals.
10.14. Data for Luciferase assay Luciferase assays were performed as described (Section 2.13). Values in Table 10.5 represent luciferase activity units relative to β-galactosidase activity.
Expt 1 Expt 2 Expt 3 Average STDEV Std Error 1 E-box only 557 834 998 796 223 129 2 E-box + E12 593431 726604 825843 715293 116618 67329 3 E-box + E12 + EGFP-GRIPE (x1) 520084 661253 884097 688478 183527 105959 4 E-box + E12 + EGFP-GRIPE (x2) 223529 224691 405791 284670 104895 60561 5 E-box + E12 + EGFP-GRIPE (x4) 40819 57821 131673 76771 48301 27886 6 E-box + E12 + EGFP (x1) 1098473 757101 697164 850913 216478 124984 7 E-box + E12 + EGFP (x2) 991041 866927 1088300 982089 110957 64061 Table 10.5. Measurement of luciferase activity
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Minerva Access is the Institutional Repository of The University of Melbourne
Author/s: Heng, Julian Ik Tsen
Title: Cloning and characterisation of gripe: a novel interacting partner of e12 during brain development
Date: 2002-10
Citation: Heng, J. I. T. (2002). Cloning and characterisation of gripe, a novel interacting partner of e12 during brain development. PhD thesis, Howard Florey Institute of Experimental Physiology and Medicine & Department of Anatomy and Cell Biology, The University of Melbourne.
Publication Status: Unpublished
Persistent Link: http://hdl.handle.net/11343/39471
File Description: p.65-137
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