| Nucleic Acids Research | Pages |
Structural requirements for DNA binding of GCM proteins
Introduction
Materials And Methods
Plasmids and site-directed mutagenesis
Cell culture, transfection and extract preparation
DNA-protein binding assays
Results
Discussion
Acknowledgement
References
Structural requirements for DNA binding of GCM proteins
ABSTRACT
INTRODUCTION
Analysis of Drosophila melanogaster enhancer trap lines and chemically induced mutants has led to the identification of glial cells missing (gcm), a gene involved in the early determination events of neural precursor cells (1-3). Transient expression of gcm in neural precursors of both the peripheral and central nervous system is sufficient to direct these cells towards a glial cell fate, whereas the default neuronal fate is chosen in its absence. As a consequence, gcm-deficient flies produce a surplus of neurons at the expense of their glial counterparts, whereas gcm-overexpressing flies produce a surplus of glia at the expense of neurons. Therefore, gcm has been postulated to function as a binary genetic switch between neuronal and glial determination in Drosophila (4,5). A similar role for gcm has also been postulated outside the nervous system in the scavenger cell lineage of Drosophila (6).
The GCM protein did not show any structural homology to known proteins at the time of its identification. Structure-function analyses revealed, however, that GCM contained a DNA binding domain (7,8). This DNA binding domain is situated in the N-terminal region of GCM, spans ~150 amino acids and is responsible for binding to the 5[prime]-ATGCGGGT-3[prime] motif or its 5[prime]-ACCCGCAT-3[prime] complement. In addition to this DNA binding domain, GCM contains a powerful transactivation domain in its C-terminus, so that GCM functioned as abona fide transcriptional activator in cell culture systems (8).
GCM-like proteins also exist in mouse and man (7,9). At present it is unclear whether these mammalian proteins perform functions analogous to GCM from Drosophila. Sequence similarities between GCM and its mammalian relatives are highest within the N-terminal DNA binding domain, the so-called GCM box. To examine the DNA binding specificities of GCM-like proteins and to obtain a first characterization of the structurally novel GCM box, we here performed a detailed mutational analysis of the GCM box, its DNA recognition site and their interaction.
MATERIALS AND METHODS
Plasmids and site-directed mutagenesis
A fragment spanning the complete open reading frame of mGCMa (7) was isolated from cDNA of undifferentiated P19 cells and was inserted into pCMV5. The N-terminal 174 amino acids of mGCMa were amplified from this fragment by PCR and cloned between the EcoRI and SalI sites of pCMV5, yielding the eukaryotic expression vector for the mGCMa DNA binding domain. Using this plasmid as a template, alanine substitutions of single amino acids were generated by the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The DNA binding domain of Drosophila GCM (corresponding to amino acids 31-190) was obtained by PCR amplification from Gcm-N9delta (1) and inserted between the EcoRI and KpnI sites of pCMV5. The expression plasmid coding for Krox24/Egr-1 was as described (10).
Cell culture, transfection and extract preparation
COS cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum and transfected by the DEAE-dextran technique using a concentration of 500 µg/ml DEAE-dextran followed by chloroquine treatment (11). For extract preparation transiently transfected COS cells were lysed in the presence of 2 µg/µl leupeptin and aprotinin each in ice-cold 10 mM HEPES, pH 7.9, 0.2 mM EDTA, 2 mM DTT and 1% Nonidet-P40. Immediately after lysis, NaCl was added to a final concentration of 400 mM. After incubation for 15 min under constant rotation, cell debris was removed from the extract by centrifugation. To determine the content of GCM proteins present in each extract Western blot analysis was performed using a polyclonal antiserum against GCM (1:3000 dilution) (8), horseradish peroxidase-coupled protein A and the enhanced chemiluminescence detection system (Amersham). The volumes were adjusted so that the concentrations of GCM proteins were comparable between different extracts.
DNA-protein binding assays
Electrophoretic mobility shift assays were performed as described (12) with whole cell extracts from transiently transfected COS cells after equalizing GCM protein concentrations. Briefly, 0.5 ng 32P-labeled probe (sequences as shown in Fig. 2A) were incubated with whole cell extract for 20 min on ice in a 20 µl reaction mixture containing 10 mM HEPES, pH 8.0, 5% glycerol, 50 mM NaCl, 5 mM MgCl2, 2 mM DTT, 0.1 mM EDTA, 2 µg bovine serum albumin and 1 µg poly(dI·dC) as non-specific competitor. To test the redox dependency of GCM proteins, extracts were diluted 40-fold in reaction buffer that either contained 2 mM DTT or were devoid of DTT. Additionally, aliquots of diluted whole cell extracts were treated with 70 µM diazenedicarboxylic acid bis(N,N-dimethylamide) for 5 min at room temperature in reaction buffer without DTT, prior to incubation with the radiolabeled probe. In some experiments 1 mM 1,10-phenanthroline or 0.5 µl antiserum were added to the reaction mixture. Samples were loaded onto native 4% polyacrylamide gels and electrophoresed in 0.5× TBE (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.3).
Methylation interference analysis was carried out as described (13). An oligonucleotide containing the GCM consensus binding site was selectively 5[prime]-labeled on its upper or lower strand with T4 polynucleotide kinase, methylated with dimethyl sulfide and used as the probe in electrophoretic mobility shift assays. Protein-bound and free probe were separately isolated, cleaved at G residues by piperidine treatment and analyzed side by side (as equal c.p.m.) on an 8 M urea-15% polyacrylamide gel.
RESULTS
Using a degenerate RT-PCR strategy we have isolated a GCM-like protein from the mouse embryonal carcinoma cell line P19 (data not shown). Its sequence was almost identical to the published sequence of mGCMa over the entire length of the open reading frame (7). As previously shown and summarized in Figure 1, mGCMa and GCM from Drosophila melanogaster show a particularly high degree of amino acid identity within their respective GCM boxes, making it likely that all GCM-like proteins possess similar DNA binding specificities.
Figure 1. Sequence comparison of the DNA binding domains of Drosophila GCM and mGCMa. Conserved amino acids are shown by asterisks. The 28 amino acids of mGCMa substituted by alanine residues in this study are indicated by arrows. Figure 2. DNA binding requirements of Drosophila GCM and mGCMa. (A) Sequence of oligonucleotides (wt and m1-m8) used in electrophoretic mobility shift assays. (B) The DNA binding domains of Drosophila GCM (upper) and mGCMa (lower) were analyzed for their ability to bind to radiolabeled oligonucleotides containing either the consensus GCM recognition sequence (wt) or various mutants (m1-m8). (C) Summary of the results from (B). Base pairs in the consensus site with strong influence on GCM binding are marked by filled circles. The preferred recognition element for GCM from Drosophila had previously been determined in two independent binding site selection assays (7,8). To evaluate the contribution of each base pair within the octameric DNA recognition element for GCM binding we introduced substitutions into each of the eight positions (Fig. 2A). Substitutions were performed such that a given base pair was replaced by the alternate one and that the strand distribution of purines and pyrimidines was switched. When electrophoretic mobility shifts were performed with extracts from COS cells transfected with the DNA binding domain of Drosophila GCM we readily detected binding of GCM to the wild-type recognition element 5[prime]-ATGCGGGT-3[prime] (Fig. 2B). Binding of GCM to all mutant sites was decreased in comparison with the wild-type site. However, single mutations differed dramatically in their effect. Mutation of an A-T base pair to C-G at position 1 interfered only slightly with GCM binding, as is evident from the intensity of the obtained GCM-DNA complex. In contrast, mutation of T-A to G-C at position 2 or of G-C to T-A at position 3 abolished DNA binding completely. Mutation of position 4 from C-G to A-T or of position 5 from G-C to T-A, on the other hand, had a much less severe impact, such that GCM binding could readily be observed, albeit at reduced strength. Mutation of position 6 or 7 from G-C to T-A again had detrimental effects similar to mutations of positions 2 and 3. No GCM-DNA complexes were formed with sites carrying these mutations. A mutation of position 8 from T-A to G-C also interfered with GCM binding. However, contrary to mutations at positions 2, 3, 6 and 7, a residual complex could still be detected. When the same set of mutant GCM binding sites was used in electrophoretic mobility shift analyses with mammalian mGCMa we obtained a binding pattern that was indistinguishable from that of Drosophila GCM (Fig. 2B). Again, mutation of positions 2, 3, 6 and 7 had the strongest impact on mGCMa binding, followed by mutation of position 8 (Fig. 2C). These results indicate that Drosophila GCM and mGCMa have very similar, if not identical, DNA binding properties. In effect, we failed to detect any significant difference between the DNA binding characteristics of Drosophila GCM and mGCMa throughout our study. Therefore, only those data which we obtained for mGCMa will be presented in the following. Several of the positions identified as being important for GCM/mGCMa binding to its consensus recognition site carry G residues on either strand. Their influence on DNA binding was analyzed by methylation interference assay (Fig. 3). A striking difference in the effect of methylation was observed between the upper and lower strands. Methylation of any of the four G residues on the upper strand of the binding site strongly interfered with mGCMa binding. These included not only those G residues present at positions 3, 6 and 7 that had been found to be important for DNA binding in mutational analysis of the GCM consensus recognition site (Fig. 2), but also included the G at position 5, although its substitution by a T only caused a minor reduction in DNA binding. Figure 3. Methylation interference assay. Cleavage patterns of GCM-bound and free oligonucleotide probe were compared for both the upper (left) and lower strands (right). G residues which interfered with GCM binding in their methylated state are indicated by arrows in the autoradiograph and in the sequence at the bottom. In contrast to the upper strand, methylation of the single G present on the lower strand within the GCM consensus binding site did not interfere with mGCMa binding. Similarly, methylation of G residues in the flanking regions on either strand did not affect binding of mGCMa, indicating that specific contacts between GCM proteins and DNA are restricted to the core recognition element. Having characterized the requirements for GCM binding on the DNA level, we next turned to the protein. To determine whether GCM proteins bind DNA as monomers or dimers, extracts containing either the DNA binding domain of mGCMa or the full-length mGCMa protein were mixed before incubation with a radioactively labeled consensus recognition site. When electrophoretic mobility shift assays were performed with mixed extracts two complexes were observed with mobilities corresponding to those characteristic of full-length mGCMa or the isolated DNA binding domain (Fig. 4A). No intermediate band indicative of a heterodimer between both proteins was obtained, suggesting that GCM proteins bind DNA as monomers. Figure 4. GCM binding and requirement for zinc. (A) Equivalent amounts of the DNA binding domain of mGCMa (mGCMa) and of the full-length mGCMa (fl) were bound to a recognition site for GCM proteins either alone or in combination. (B) The DNA binding domain of mGCMa (left) and Krox-24/Egr1 (right) were tested for their ability to bind to their respective consensus sequences in the presence (+ 1,10-phen.) or absence (-1,10-phen) of 1,10-phenanthroline. [alpha]Krox24, antibody against Krox24/Egr1 added. As is evident from the sequence alignment in Figure 1, the DNA binding domain of GCM proteins contains a high percentage of conserved arginines, lysines, cysteines and histidines. The positively charged arginine and lysine residues have previously been shown in other DNA binding domains to be engaged in direct contact with DNA, whereas cysteines and histidines are often involved in coordination of zinc ions. Although the cysteines and histidines of GCM proteins are not arranged in a configuration that overtly resembles any of the known zinc-containing structures, it is still conceivable that these residues participate in a novel type of coordination center which is important for the DNA binding activity of GCM proteins. Using the high affinity zinc chelator 1,10-phenanthroline (14,15) we probed for the presence of zinc in the DNA binding domain of mGCMa. As shown in Figure 4B, preincubation with 1,10-phenanthroline prior to addition of radiolabeled DNA prevents binding of the zinc finger protein Krox-24/Egr-1 to its consensus recognition site. However, the same treatment did not influence binding of mGCMa to its recognition site (Fig. 4B). DNA binding of mGCMa was not only refractory to the presence of 1,10-phenanthroline, but also to the presence of EDTA, which chelates a wide spectrum of divalent metal ions, including Mg2+, Ca2+ and Zn2+ (data not shown). Thus, it seems unlikely that zinc ions play a critical role in determining the structural identity of the DNA binding domain of GCM proteins. Cysteine residues are not only known for their metal coordination capacity, but also for their ability to confer redox sensitivity to transcription factors such as Jun/Fos, CREB/ATF, NF-[kappa]B and c-Myb proteins (16-18). Given the large number of conserved cysteines within the DNA binding domain of GCM proteins, we asked whether DNA binding of mGCMa was dependent on its redox state. As shown in Figure 5A, mGCMa which was kept in the absence of DTT had a strongly diminished DNA binding activity compared with mGCMa which was continuously kept in the presence of DTT. Redox-dependent loss of DNA binding proved to be reversible, as re-addition of DTT to mGCMa-containing extracts led to recovery of DNA binding (data not shown). Figure 5. Redox sensitivity of GCM binding. Following dilution, whole cell extracts of COS cells transfected with wild-type mGCMa (A) or the alanine substitution mutants C101A, C86A and K103A (B) were assayed for their DNA binding activity under various conditions. +DTT, dilution and incubation with probe in the presence of DTT; -DTT, dilution and incubation with probe in the absence of DTT; +diamide, addition of diamide to DTT-free whole cell extract. Pretreatment of mGCMa with strong sulfhydryl oxidizing agents, such as diazenedicarboxylic acid bis(N,N-dimethylamide) (diamide), on the other hand, abolished DNA binding of mGCMa, thus clearly showing that DNA binding of mGCMa only occurs in the reduced state. Incubation of mGCMa with the radiolabeled GCM consensus recognition site prior to diamide treatment protected mGCMa from inactivation (data not shown). This suggests that the amino acids which mediate redox sensitivity are associated with DNA. To be able to determine the role of single amino acids in DNA binding, redox dependency and in the structural context of the DNA binding domain, we next performed a scanning mutagenesis study during which 28 amino acids of mGCMa were changed to alanine residues. The chosen residues were the arginine, lysine, cysteine and histidine residues conserved between GCM proteins. As indicated in Figure 1, these residues were evenly distributed throughout the GCM box. Each substitution mutant was expressed in transiently transfected COS cells and assayed for its DNA binding activity. Several mutants were poorly expressed (Table 1). These included the cysteine residues, except C101, as well as a cluster of amino acids in the C-terminal part of the GCM box (H152, H154 and R156). Table 1. The amounts of all alanine substitution mutants were adjusted in Western blots. For mutants with low expression rates ~6- to 11-fold the amount of COS extract was used as for mutants with high expression rates, as exemplified in Figure 6A. When tested in electrophoretic mobility shift analyses with wild-type and mutant GCM binding sites, most mutants exhibited unaltered DNA binding, as defined by strong binding to the consensus GCM binding site and a binding strength for each mutant site similar to that observed with wild-type mGCMa. Alanine substitutions of only three residues abrogated DNA binding (Table 1). These included the lysine at position 74 (K74) of mGCMa and the cysteines at positions 76 and 125 (C76 and C125). Two aspects are remarkable about the location of these residues: (i) K74 and C76 are in such close proximity to each other that it seems plausible to assume that this region is part of the DNA recognition interface of mGCMa; (ii) C76 and C125 are the two outer of seven conserved cysteines which are symmetrically spaced relative to the center cysteine at position 101. Their arrangement is summarized in the formula C76-X5-C82-X3- C86-X14-C101-X11-C113-X2-C116-X8-C125. Figure 6. Expression and DNA binding of mutant mGCMa proteins. (A) Normalization of mGCMa contents for wild-type protein and several alanine substitution mutants by Western blot. Note that different amounts of COS extracts were used for each mutant, as indicated below the lanes. (B) Binding of mGCMa and several alanine substitution mutants to the consensus DNA motif. -, no protein; mock, COS extract; C82A, C86A, C113A and C116A, single amino acid substitutions of mGCMa; C82A,C86A, C82A,C113A and C82A,C116A, double amino acid substitutions of mGCMa; mGCMa, wild-type mGCMa. A functional relevance of this structure is suggested by the observation that alanine substitution of all other of these seven cysteines also led to detectable alterations in the gel shift pattern (Table 1 and Fig. 6B). Mutation of either C82, C86, C113 or C116 did not significantly change the affinity of mGCMa for its binding sites, but led to protein-DNA complexes that exhibited slower mobility in electrophoretic mobility shift assays than the wild-type mGCMa or all other alanine substitution mutants. Thus, it seems likely that substitution of any of these residues alters the normal conformation of the DNA binding domain. The same altered mobility was retained in double alanine substitution mutants, in which the C82A mutation was combined with mutations in C113 or C116 respectively (Fig. 6B). Combination of C82A with C86A, in contrast, led to a strong reduction in DNA binding activity such that complex formation was barely detectable for the C82A,C86A double mutant. Alanine substitution of C101, the cysteine at the center of the above mentioned structure, showed another interesting feature. The C101A mutant was expressed well and bound to the consensus GCM binding site with considerable strength. However, when tested for redox dependency of DNA binding the C101A mutant exhibited a peculiar behavior. Similarly to wild-type mGCMa, most mutants bound to DNA only in a reducing environment, as exemplified in Figure 5B for the K103A and C86A substitutions. The C101A mutant, however, failed to show this redox dependency, as binding to DNA was observed even in the absence of the reducing agent DTT and in the presence of the sulfhydryl oxidizing agent diamide (Fig. 5B). Thus, it seems likely that C101 is involved in the redox regulation of mGCMa binding to DNA.
Construct
Expressiona
DNA binding
Affinityb
Remarksc
Wild-type
+++
+++
Mutation of arginines
R62A
+++
+++
R97A
+++
+++
R105A
+++
+++
R126A
+++
+++
R138A
+++
+++
R156A
+
+++
R166A
+++
+++
R167A
+++
+++
Mutation of lysines
K73A
+++
+++
K74A
+++
-
K103A
+++
+++
K107A
+++
+++
K111A
+++
+++
K149A
+++
+++
K160A
+++
+++
Mutation of histidines
H43A
+++
+++
H55A
+++
+++
H67A
+++
+++
H128A
+++
+++
H152A
+
+++
H154A
+
+++
Mutation of cysteines
C76A
+
-
C76S
+
-
C82A
+
+++
Altered mobility
C86A
+
+++
Altered mobility
C101A
+++
+++
Redox-independent
C113A
+
+++
Altered mobility
C116A
+
+++
Altered mobility
C125A
+
-
DISCUSSION
In this report we characterized the DNA binding properties of GCM proteins. Similarity between Drosophila GCM and its mammalian homologs is highest within the N-terminal part of the protein (7,9), which also harbors the DNA binding domain (7,8). It is an important finding of this study that the high degree of conservation between the DNA binding domains of GCM proteins translates into very similar, if not identical, DNA binding characteristics. Throughout this study we failed to detect any significant difference in DNA binding behavior or strength between GCM and mammalian mGCMa.
An octameric sequence 5[prime]-ATGCGGGT-3[prime] (or its complement 5[prime]-ACCCGCAT-3[prime]) had previously been defined by us and others as the optimal DNA recognition site (7,8). This site does not possess the palindromic structure that is indicative of recognition by protein dimers. In agreement, our analysis shows that GCM proteins bind DNA as monomers.
One of the main objectives of this study was to analyze by site-directed mutagenesis the contribution of each single base pair to GCM binding. Although all base pair substitutions decreased binding of GCM, there was substantial variability in the extent to which single base pair mutations were detrimental. Presence of the optimal base pair was most important at positions 2, 3, 6 and 7 of the octameric recognition site, and least important at position 1. The influence of base pair mutations at positions 4, 5 and 8 was intermediate, with mutation of base pair 8 having more severe effects than mutations of either base pair 4 or 5. These studies should prove helpful in the search for GCM binding sites in the regulatory regions of potential GCM target genes. Deviations from the consensus GCM recognition site at positions 1, 4 or 5 should be much more tolerable than alterations at position 8. Positions 2, 3, 6 and 7 are expected to be invariant. Recently identified GCM binding sites in the 5[prime]-flanking region of the repo gene, a potential target for Drosophila GCM, fit very well with these predicitions (7).
In general, results from methylation interference experiments were in good agreement with results from binding site mutagenesis. Methylation of four G residues interfered with binding of GCM. Three of them were located at positions 3, 6 and 7, all of which had been identified as important determinants of GCM binding. Methylation of the fourth G at position 5 also interfered with GCM binding, despite the fact that this position had been found to be of lesser importance for GCM binding. However, as position 5 lies in the center of the GCM recognition site it is conceivable that base methylation at this position causes sufficient steric hindrance to prevent GCM binding even in the absence of direct contacts between this position and the protein. It is intriguing that all interfering bases were found on the upper strand.
To be able to better understand the structural requirements for DNA binding of GCM proteins we performed an elaborate alanine scanning mutagenesis of the DNA binding domain of mGCMa. Among all of the 15 positively charged arginine and lysine residues analyzed by alanine substitution only K74 was sufficiently important for DNA binding to cause inactivation of mGCMa upon mutation (Fig. 7). Even more surprising, mutation of the six conserved histidine residues did not interfere with DNA binding of GCM proteins. Our analysis, however, pointed to the importance of the seven conserved cysteine residues for DNA binding of GCM proteins. Mutation of two cysteines abolished DNA binding completely, whereas mutation of four other cysteine residues altered mobility of the mGCMa-DNA complex (Fig. 7). Furthermore, mutation of the remaining cysteine led to loss of the intrinsic redox sensitivity of mGCMa. Thus, cysteines within the DNA binding domain of GCM proteins are involved in shaping the overall conformation of the domain, in the process of DNA binding and in the redox regulation of DNA binding. At present it is unclear how these cysteine residues perform their function. We failed to obtain evidence for a role of these cysteines in coordination of zinc or other divalent metal ions. Furthermore, the arrangement of these residues did not exhibit any structural resemblance to other cysteine-containing structures, including metal coordination centers. Given the highly symmetrical arrangement of the cysteines within the DNA binding domain of GCM proteins (Fig. 7) it is tempting to speculate that these cysteines are the backbone of a higher order structure. The exact nature of this structure will have to await crystallographic and NMR spectroscopic analyses.
Figure 7. Summary of the alanine scanning mutagenesis. Residues with influence on DNA binding of GCM proteins are boxed. The structure of the symmetrically arranged cysteine residues is highlighted. Finally, it is highly remarkable that DNA binding of GCM proteins is subject to redox control. Redox sensitivity of DNA binding has previously been observed in a number of other transcription factors, such as Jun/Fos, CREB/ATF, NF-[kappa]B, c-Myb, p53, NF-Y and HIF-1 (16-22). Most of these transcription factors are stimulated transiently in response to extracellular signals and have to be tightly regulated in their action. GCM proteins are under a similarly tight control, as evidenced by transient induction of Drosophila GCM during neural development (1,2). This transience has been attributed in part to the presence of an instability signal in the mRNA and to a PEST sequence in the GCM protein itself (1,2). The PEST signal has been conserved in mammalian mGCMa (7,9). We propose that the redox sensitivity, which we have observed in this study for the DNA binding ability of GCM proteins, provides an additional means of fine tuning the action of this group of transcription factors. Thus, it should be possible to control GCM proteins not only by regulating their rate of synthesis and turnover, but also by regulating their activity through changes in cellular redox potential.
ACKNOWLEDGEMENT
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 444) to M.W.
REFERENCES
*To whom correspondence should be addressed. Tel: +49 40 4717 6274; Fax: +49 40 4717 6602; Email: wegner@plexus.uke.uni-hamburg.de
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 13 May 1998
Copyright©Oxford University Press, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Mannstadt, G. Bertrand, M. Muresan, G. Weryha, B. Leheup, S. R. Pulusani, B. Grandchamp, H. Juppner, and C. Silve Dominant-Negative GCMB Mutations Cause an Autosomal Dominant Form of Hypoparathyroidism J. Clin. Endocrinol. Metab., September 1, 2008; 93(9): 3568 - 3576. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. W. Schubert, A. Abendroth, K. Kilian, T. Vogler, B. Mayr, I. Knerr, and S. Hashemolhosseini bZIP-Type transcription factors CREB and OASIS bind and stimulate the promoter of the mammalian transcription factor GCMa/Gcm1 in trophoblast cells Nucleic Acids Res., June 1, 2008; 36(11): 3834 - 3846. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Chang, D. Mukherjea, R. M. Gobble, K. A. Groesch, R. J. Torry, and D. S. Torry Glial Cell Missing 1 Regulates Placental Growth Factor (PGF) Gene Transcription in Human Trophoblast Biol Reprod, May 1, 2008; 78(5): 841 - 851. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. W. Schubert, N. Lamoureux, K. Kilian, L. Klein-Hitpass, and S. Hashemolhosseini Identification of Integrin-{alpha}4, Rb1, and Syncytin A as Murine Placental Target Genes of the Transcription Factor GCMa/Gcm1 J. Biol. Chem., February 29, 2008; 283(9): 5460 - 5465. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-C. Chou, C. Chang, J.-H. Liu, L.-F. Chen, C.-D. Hsiao, and H. Chen Small Ubiquitin-like Modifier Modification Regulates the DNA Binding Activity of Glial Cell Missing Drosophila Homolog a J. Biol. Chem., September 14, 2007; 282(37): 27239 - 27249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-W. Chang, H.-C. Chuang, C. Yu, T.-P. Yao, and H. Chen Stimulation of GCMa Transcriptional Activity by Cyclic AMP/Protein Kinase A Signaling Is Attributed to CBP-Mediated Acetylation of GCMa Mol. Cell. Biol., October 1, 2005; 25(19): 8401 - 8414. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Thomee, S. W. Schubert, J. Parma, P. Q. Le, S. Hashemolhosseini, M. Wegner, and M. J. Abramowicz GCMB Mutation in Familial Isolated Hypoparathyroidism with Residual Secretion of Parathyroid Hormone J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2487 - 2492. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L Baumber, C Tufarelli, S Patel, P King, C A Johnson, E R Maher, and R C Trembath Identification of a novel mutation disrupting the DNA binding activity of GCM2 in autosomal recessive familial isolated hypoparathyroidism J. Med. Genet., May 1, 2005; 42(5): 443 - 448. [Full Text] [PDF]
|
|
|
|
 |
|
 H. Westers, P. G. Braun, L. Westers, H. Antelmann, M. Hecker, J. D. H. Jongbloed, H. Yoshikawa, T. Tanaka, J. M. van Dijl, and W. J. Quax Genes Involved in SkfA Killing Factor Production Protect a Bacillus subtilis Lipase against Proteolysis Appl. Envir. Microbiol., April 1, 2005; 71(4): 1899 - 1908. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-S. Yang, C. Yu, H.-C. Chuang, C.-W. Chang, G.-D. Chang, T.-P. Yao, and H. Chen FBW2 Targets GCMa to the Ubiquitin-Proteasome Degradation System J. Biol. Chem., March 18, 2005; 280(11): 10083 - 10090. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Hashemolhosseini and M. Wegner Impacts of a new transcription factor family: mammalian GCM proteins in health and disease J. Cell Biol., September 13, 2004; 166(6): 765 - 768. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Iwasaki, T. Hosoya, H. Takebayashi, Y. Ogawa, Y. Hotta, and K. Ikenaka The potential to induce glial differentiation is conserved between Drosophila and mammalian glial cells missing genes Development, December 15, 2003; 130(24): 6027 - 6035. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Shimizu, H. Hiroaki, D. Kohda, T. Hosoya, Y. Akiyama-Oda, Y. Hotta, E. H. Morita, and K. Morikawa NMR and ICP spectroscopic analysis of the DNA-binding domain of the Drosophila GCM protein reveals a novel Zn2+-binding motif Protein Eng. Des. Sel., April 1, 2003; 16(4): 247 - 254. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. W. Hamoen, W. K. Smits, A. d. Jong, S. Holsappel, and O. P. Kuipers Improving the predictive value of the competence transcription factor (ComK) binding site in Bacillus subtilis using a genomic approach Nucleic Acids Res., December 15, 2002; 30(24): 5517 - 5528. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Yu, K. Shen, M. Lin, P. Chen, C. Lin, G.-D. Chang, and H. Chen GCMa Regulates the Syncytin-mediated Trophoblastic Fusion J. Biol. Chem., December 13, 2002; 277(51): 50062 - 50068. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Cross, L. Anson-Cartwright, and I. C. Scott Transcription Factors Underlying the Development and Endocrine Functions of the Placenta Recent Prog. Horm. Res., January 1, 2002; 57(1): 221 - 234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Schreiber, E. Riethmacher-Sonnenberg, D. Riethmacher, E. E. Tuerk, J. Enderich, M. R. Bösl, and M. Wegner Placental Failure in Mice Lacking the Mammalian Homolog of Glial Cells Missing, GCMa Mol. Cell. Biol., April 1, 2000; 20(7): 2466 - 2474. [Abstract] [Full Text]
|
|
|
|
|
|
E. E. Tuerk, J. Schreiber, and M. Wegner Protein Stability and Domain Topology Determine the Transcriptional Activity of the Mammalian Glial Cells Missing Homolog, GCMb J. Biol. Chem., February 18, 2000; 275(7): 4774 - 4782. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kim, B. W. Jones, C. Zock, Z. Chen, H. Wang, C. S. Goodman, and D. J. Anderson Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing PNAS, October 13, 1998; 95(21): 12364 - 12369. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||