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Originally published In Press as doi:10.1074/jbc.M410010200 on September 16, 2004

J. Biol. Chem., Vol. 279, Issue 45, 47109-47114, November 5, 2004
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ATF4, the Osteoblast Accumulation of Which Is Determined Post-translationally, Can Induce Osteoblast-specific Gene Expression in Non-osteoblastic Cells*

Xiangli Yang and Gerard Karsenty{ddagger}

From the Department of Molecular and Human Genetics and Bone Disease Program of Texas, Baylor College of Medicine, Houston, Texas 77030

Received for publication, August 31, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Based on the analysis of a loss-of-function model, we recently showed that ATF4 regulates osteoblast terminal differentiation and function and is implicated in the pathophysiology of Coffin-Lowry syndrome. That study, however, did not address whether forced expression of Atf4 in non-osteoblastic cells would lead to osteoblast-specific gene expression, one of the most important features of a cell differentiation factor. To address this question we searched for cell lines that would not express Atf4. Contrasting with the restricted pattern of its protein accumulation, Atf4 mRNA was found in all cell lines and mouse tissues tested. Treatment of non-osteoblastic cells with MG115, a proteasome inhibitor, induced ATF4 accumulation and resulted in activation of an Osteocalcin promoter luciferase construct as well as expression of endogenous Osteocalcin, a molecular marker of differentiated osteoblasts and a target gene of ATF4. Eliminating the expression of {beta}-TrCP1, an ubiquitin-protein isopeptide ligase interacting with ATF4 by RNA interference, led to ATF4 accumulation and to endogenous Osteocalcin expression in fibroblasts. These results indicate that the absence of ATF4 in most cell types is determined, at least in part, by an ubiquitination-dependent process. To our knowledge ATF4 is the first cell-specific transcription factor in which cell-specific distribution is achieved post-translationally. This study also establishes that ATF4, like other osteoblast differentiation factors, such as Runx2 and Osterix, has the ability to induce osteoblast-specific gene expression in non-osteoblastic cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The osteoblast, a cell of mesenchymal origin, is the only cell type responsible for extracellular matrix deposition in bone, a process called bone formation. Bone formation is a physiological process involved in skeletal growth, bone remodeling, and fracture repair (1). That diseases affecting bone formation are common and often debilitating (2–5) underscores the biomedical importance of elucidating the molecular basis of osteoblast-specific gene expression and osteoblast differentiation. It is likely, as is the case in many cell lineages, that cell differentiation along the osteoblastic lineage is controlled, at least in part, by cell-specific transcription factors.

To search for osteoblast-specific transcription factors that could also act as cell differentiation factors, we systematically analyzed the regulation of one of the two mouse Osteocalcin genes, OG2, the expression of which is strictly osteoblast-specific. Analysis of the OG2 promoter revealed that a 147-bp fragment, which can confer osteoblast-specific expression to a reporter gene in cell culture, contains two osteoblast cis-acting elements, termed OSE1 and OSE2 (6). Two lines of evidence indicated that both cis-acting elements are equally important. First, mutation of either one of them decreased the activity of a reporter construct containing a 147-bp fragment of the OG2 promoter (pOG2-Luc) only 50%. Second, multimers of either OSE1 or OSE2 were able to confer osteoblast-specific activity to a heterologous promoter in cell cultures (6, 7). Subsequent studies revealed that the OSE2-binding protein was Runx2, a master regulator of osteoblast differentiation (8–11), thus validating the use of the OG2 promoter to search for osteoblast differentiation factors.

More recent biochemical, molecular, and genetic studies revealed that the OSE1-binding protein is ATF4 (12). ATF4, also known as cAMP-response element protein 2, RAXREB67, mTR67, or C/ATF (13–17), belongs to the subfamily of cAMP-response element-binding protein/ATF basic leucine zipper proteins. In accordance with the critical role of OSE1 in osteoblast gene expression, targeted disruption of Atf4 in mice causes perinatal lethality, dwarfism, and severe osteoporosis, which are abnormalities caused by a failure of osteoblasts to fully differentiate and to properly function. Moreover, ATF4 is the substrate in osteoblasts of RSK2, a gene encoding a kinase that is inactivated in Coffin-Lowry syndrome (12). Previous studies did not address, however, whether ATF4, like the two other known osteoblast-specific transcription factors, Runx2 and Osterix (1), can induce osteoblast-specific gene expression in non-osteoblastic cells, a hallmark of an osteoblast differentiation factor. In the course of addressing this question we uncovered a peculiar mechanism whereby a post-translational regulation of ATF4 controls its osteoblast-specific accumulation and thus its cell specificity. We also demonstrated that ATF4, like Runx2 and Osterix, has the ability to induce osteoblast-specific gene expression in non-osteoblastic cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Reagents—All cell lines except for 2T3 mouse osteoblasts used in this study were purchased from ATCC. COS1 monkey kidney cells and NIH3T3 mouse fibroblasts were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. C2C12 mouse myoblasts and S194 mouse lymphoblasts were grown in Dulbecco's modified Eagle's medium containing 10% horse serum and 1% penicillin/streptomycin. ROS17/2.8 rat osteoblasts were grown in Dulbecco's modified Eagle's medium/F-12 containing 10% fetal bovine serum and 1% penicillin/streptomycin. 2T3 cells were kindly provided by Dr. Mundy's laboratory and maintained in {alpha}-minimum Eagle's medium with 10% fetal bovine serum and 1% penicillin/streptomycin. MG115 was purchased from Peptide International Inc.

Gene Expression Analysis—Total RNA from different adult mouse tissues or mRNA from the indicated cell lines was isolated using TRIzol or the MessageMaker mRNA isolation system (Invitrogen) following the manufacturer's protocols. 10 µg of total RNA or 1 µg of mRNA of each sample was resolved in 1% agarose gel. After transferring the RNA samples onto a nylon membrane, the blot was then cross-linked by UV light and hybridized with either Atf4, Osteocalcin, or Gapdh, cDNA probes following standard protocols. For Western blot analysis, 25 µgof nuclear extracts isolated from the indicated cell lines, primary osteoblasts, and mouse tissues as described (18) were used. Antibody against ATF4 was raised as described (12), and Sp1 antibody was obtained from Santa Cruz Biotechnology Inc. For in situ hybridization analyses, E16 mouse embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 µm. A 271-bp 5'-untranslated region of Atf4 was cloned by reverse transcription-PCR and used to generate 35S-labeled antisense riboprobes.

Electrophoretic Mobility Shift Assays and DNA Transfection Assays—EMSA1 and DNA transfection assays were performed as described (6). Nuclear extracts (5 µg) of the indicated sources were incubated with 5 fmol of a radiolabeled double-stranded OSE1 oligonucleotide (6) at room temperature for 10 min. For supershift assays, anti-ATF4 antibody (2 µg, Santa Cruz Biotechnology Inc.) was incubated with nuclear extracts prior to addition of the binding reaction mix. For DNA transfection experiments, COS1 cells were plated at a density of 5x 104/well in 12-well plates and transfected with 0.5 µg of wild-type or mutant forms of pOG2-Luc (a construct containing a 147-bp fragment of the OG2 promoter upstream of the luciferase gene (6)) or wild-type or mutant p6OSE1-Luc (a vector containing 6 copies of OSE1, the binding site of ATF4 (6)) and 0.05 µg of pRSV-{beta}gal reporter vectors. MG115 (25 µM in Me2SO) was added 42 h post-transfection, and luciferase and {beta}-galactosidase assays were performed 48 h post-transfection. Data represent ratios of luciferase/{beta}-galactosidase activity of at least three different experiments each done in triplicate performed for each DNA sample.

RNA Interference Experiment—Double-stranded RNA molecules (siRNA) against N and C termini of {beta}-TrCP1 or CDC4 were custom synthesized and transfected into NIH3T3 cells using LipofectAMINE (Invitrogen). The siRNA target mRNA sequences were: {beta}-TrCP1N, AAA GGA GCU GUG UGU CAA GUA; {beta}-TrCP1C, AAC AAG UAA AGG GGU UUA CUG; CDC4N, AAA AAU ACA GAA AAU AUG GGU; and CDC4C, AAC AGG ACA GUG UUU ACA AAC. Total RNA and nuclear extracts were prepared 48 h post-transfection. A Northern blot was hybridized using a probe containing a partial {beta}-TrCP1 cDNA (1–516 bp), and Western blot analysis was performed using the ATF4 and Sp1 antibodies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Discrepancy between Atf4 Expression and ATF4 Accumulation—Osteocalcin is the most osteoblast-specific structural gene identified to date and is the latest molecular marker gene of the osteoblast phenotype (19). We showed recently that Osteocalcin is also a transcriptional target of ATF4 (12). However, we did not address in that study whether ATF4 has the ability to induce osteoblast-specific gene expression in non-osteoblastic cells. To answer this question, we intended to use as a readout the induction of Osteocalcin expression by ATF4 in cell lines of either mesenchymal origin such as NIH3T3 fibroblasts and C2C12 myoblasts or of non-mesenchymal origin such as COS1 kidney cells and S194 lymphoblasts. Prior to performing these experiments, we verified that ATF4 was absent in these cells. As expected, ATF4 could not be observed by either Western blot analysis or EMSA in any of these cell lines (Fig. 1A and data not shown). However, Atf4 mRNA was present in all cell types tested, and its abundance was similar to the one seen in osteoblastic cell lines (Fig. 1B). This observation was surprising given the restricted distribution of ATF4 and the limited number of tissues, i.e. bone, fetal liver, and eye, affected in Atf4-/- mice (12, 20–22). To understand this discrepancy we further examined Atf4 expression and ATF4 accumulation in vivo.



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  		FIG. 1.
ATF4 is an osteoblast-specific protein. A, ATF4 is detectable only in osteoblasts. Western blot analysis of nuclear extracts from indicated cell lines using anti-ATF4 antibody. Note that ATF4 is present only in osteoblastic cell lines. B, Atf4 mRNA is expressed in non-osteoblasts. Northern blot was carried out using total RNA from indicated cell lines and an Atf4 full-length cDNA as a probe. Gapdh was used here as loading control. C, ubiquitous expression of Atf4. Total RNA from indicated postnatal mouse tissues was analyzed by Northern blot hybridization using an Atf4 cDNA probe. Gapdh was used here as a loading control. D, ATF4 is an osteoblast-specific protein. Western blot analysis was performed as described in A using nuclear extracts from primary osteoblasts (POB) and various mouse tissues. Recombinant His-tagged ATF4 (His-ATF4) and purified OSE1-binding protein (OSE1-BP (7)) from osteoblasts were used here as positive controls. Sp1 antibody was used as a loading control.

 
When analyzed by Northern hybridization, Atf4 was ubiquitously expressed although its level of expression was higher in calvaria, brain, thymus, liver, and lung than in other tissues (Fig. 1C). In contrast, when we performed Western blot analysis to assess the distribution of the ATF4 protein, we were struck by the marked difference between the high amount of ATF4 protein in osteoblasts compared with its absence in other tissues (Fig. 1D). This pattern, which was very reproducible from one Western blot to another, was strikingly different from what was seen when studying Atf4 expression as bone was not the site of highest expression. This discrepancy also led us to analyze whether Atf4 has a more restricted expression pattern during development. Although Atf4 is highly expressed in the developing skeleton, it was also expressed, at a comparable level, in organs such as liver, lung, and intestine (Fig. 2). Thus Atf4 expression pattern during development and after birth contrasts with the limited distribution of the protein and the limited set of functions that have been identified to date through genetic means for this gene.



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  		FIG. 2.
Ubiquitous expression of Atf4 during embryonic development. In situ hybridization was performed using sections of E16 mouse embryos and a 5'-untranslated region of an Atf4 cDNA probe. Atf4 transcripts are detected in all tissues analyzed. Examples are ribs (a), liver (b), lung (c), and small intestine (d). Magnification, x50.

 
Inhibition of ATF4 Proteasomal Degradation Leads to Ectopic Osteoblast Gene Expression—A yeast two-hybrid system screen using {beta}-TrCP1, an E3 ubiquitin ligase, as a bait identified ATF4 as a {beta}-TrCP1-interacting protein (23). This observation led us to test whether the absence of ATF4 in non-osteoblastic cells could be explained, at least in part, by a proteasomal degradation mechanism. To test this hypothesis, we treated non-osteoblastic cell lines such as NIH3T3, C2C12, S194, and COS1 cells that have no detectable ATF4 protein with MG115. MG115 is a proteasome inhibitor that acts by blocking the N-terminal threonine in the active site of the {beta}-subunit of the 26 S proteasomal complex (24, 25). Following MG115 treatment for 5 h, nuclear extracts were isolated from these cells and used either in Western blot or in EMSA. As shown in Fig. 3A, MG115 treatment of all indicated non-osteoblastic cells led to the accumulation of ATF4 in their nuclei. Likewise, MG115 treatment led to the appearance of an OSE1 binding activity in nuclear extracts of these cells as determined by EMSA (Fig. 3B). That the OSE1 binding activity contains ATF4 was determined by a supershift experiment using an ATF4 antibody (Fig. 3B). These results suggested that ATF4 is degraded more readily in non-osteoblastic cells than in osteoblasts.



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  		FIG. 3.
Inhibition of ubiquitin-mediated protein degradation stabilizes ATF4 in non-osteoblasts. A, MG115 induces ATF4 accumulation in non-osteoblasts. Nuclear extracts of Me2SO-treated (-) or MG115-treated (+) indicated cell lines were analyzed by Western blot using an ATF4 antibody. Nuclear extracts of ROS17/2.8 (ROS) osteoblasts were included as a positive control, and Sp1 was used here as a loading control. B, MG115 induces ATF4 binding to OSE1 element of the OG2 promoter. EMSA was performed using the OSE1 oligonucleotide as a probe and nuclear extracts of indicated cell lines treated with Me2SO (-) or MG115 (+) as a source of proteins. ROS17/2.8 nuclear extracts were included here as a positive control. Note that the protein-OSE1 complex (solid arrow) was supershifted (empty arrow) by an ATF4 antibody. C, inhibition of ATF4 degradation by MG115 increases OG2 promoter activity. COS1 cells were transfected with wild-type or mutant forms of pOG2-Luc containing either a mutation in OSE1 that abolishes the binding of ATF4 to OSE1 (pOG2mOSE1-Luc) or a mutation in OSE2 that eliminates binding of Runx2 to OSE2 (pOG2mOSE2-Luc). Transfected cells were then treated with Me2SO (-) or MG115 (+) 5 h prior to luciferase and {beta}-galactosidase assays. Note that the mutation in OSE1 abolished the MG115-induced increase in pOG2-Luc activity. D, transfection of COS1 cells with reporter construct containing 6 copies of OSE1 (p6OSE1-Luc) or OSE2. Note that MG115 only induced the increase of luciferase activity of p6OSE1-Luc to which ATF4 binds. E, ATF4 accumulation induces endogenous Osteocalcin expression in non-osteoblasts. mRNA from cells treated with Me2SO (-) or MG115 (+) were analyzed by Northern blot using an Osteocalcin cDNA probe. Total RNA from mature osteoblasts (d10) was loaded in the same gel as a positive control. Gapdh was used as a loading control.

 
Taking into account this peculiar mechanism used by ATF4 to achieve osteoblast-specific accumulation, we then asked whether ATF4 could induce osteoblast-specific gene expression in cells other than osteoblasts. We first performed DNA transfection experiments using the activity of pOG2-Luc as a readout. This OG2 promoter fragment contains 1 copy of OSE1 (the ATF4 binding site) and 1 copy of OSE2 (the Runx2 binding site) and is virtually inactive in COS1 cells that do not express Osteocalcin (6). MG115 treatment of COS1 cells following transfection with pOG2-Luc resulted in a 5-fold increase in luciferase activity of this vector (Fig. 3C). This increase in reporter activity was secondary to accumulation of ATF4 and ATF4 binding to OSE1, as a mutation in OSE1 that prevents ATF4 from binding to DNA nearly abolished this increase in luciferase activity (Fig. 3C). In contrast, a mutation in OSE2 that prevents Runx2 from binding to DNA did not affect the MG115-induced increase in luciferase activity of pOG2-Luc following MG115 treatment (Fig. 3C). Likewise, the activity of p6OSE1-Luc, a vector containing 6 copies of OSE1, the cis-acting element to which ATF4 binds (12), was increased 12-fold following MG115 treatment of transfected COS1 cells. In contrast, MG115 treatment did not increase more than 3-fold the activity of p6OSE2-Luc, a vector containing 6 copies of the cis-acting element to which Runx2 binds (Fig. 3D). These results demonstrate that preventing ATF4 proteasomal degradation increases the activity of luciferase reporter constructs containing 1 or several copies of OSE1, the ATF4 binding site.

Next we asked whether MG115 treatment of non-osteoblastic cells could induce endogenous Osteocalcin expression. mRNA from cells treated with MG115 or vehicle was analyzed by Northern blot. Osteocalcin expression was detected in all tested non-osteoblastic cells, including C2C12 myoblasts, NIH3T3 fibroblasts, and even S194 lymphoblasts (Fig. 3E). Taken together, our data indicate first, that proteasomal degradation is one mechanism accounting for the absence of ATF4 in non-osteoblastic cells, and second, that ATF4 is an osteoblast differentiation transcription factor as defined by its ability to induce ectopic Osteocalcin expression.

Inactivation of {beta}-TrCP1 Leads to Osteoblast-specific Gene Expression in Non-osteoblastic Cells—Having established that ATF4 can induce ectopic osteoblast-specific gene expression in cell lines of different origins, we next used one of these cell types, NIH3T3 fibroblasts, to analyze the molecular pathway leading to ATF4 degradation.

In eukaryotes, one signature for protein degradation by the 26 S proteasome is attachment of polyubiquitin to a target protein, a process involving three sequential enzymatic events that include activation (E1), conjugation (E2), and ligation (E3). E3 determines the specificity of targets; hence it is the key to controlling individual target protein abundance. Two mammalian E3 ligase complexes, SCFskp2 and SCF{beta}TrCP1, have been identified to date (26). The observation that {beta}-TrCP1, a component of the E3 ubiquitin ligase, interacts with ATF4 and leads to its proteasomal degradation in HeLa cells (23) prompted us to test whether it was a molecular link explaining, at least in part, the absence of ATF4 in non-osteoblasts. To test this hypothesis we relied on RNA interference (siRNA) to decrease endogenous {beta}-TrCP1 expression. Introduction of siRNAs targeting {beta}-TrCP1 in NIH3T3 fibroblasts did result in a virtual absence of {beta}-TrCP1 transcripts. This effect was specific because targeting through siRNAs of a related gene, CDC4, which encodes another mouse E3 ligase, failed to completely abolish {beta}-TrCP1 expression (Fig. 4A). The decrease of {beta}-TrCP1 expression caused by disrupting CDC4 expression was not sufficient to affect ATF4 accumulation indicating that {beta}-TrCP1 was still active and able to trigger ATF4 ubiquitination.



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  		FIG. 4.
Accumulation of ATF4 in non-osteoblasts induces endogenous Osteocalcin expression. A and B, knock-down {beta}-TrCP1 by siRNA induces ATF4 accumulation in non-osteoblastic cell types. Total RNA and nuclear extracts were isolated from transfected NIH3T3 cells with indicated siRNA. Northern (A) and Western (B) blot analyses were performed. Gapdh and Sp1 were used as loading controls for Northern and Western blots, respectively. C, inactivation of {beta}-TrCP1 by siRNA induces endogenous Osteocalcin expression in NIH3T3 fibroblasts. Northern blot analysis was performed using mRNA samples isolated from siRNAs of {beta}-TrCP1- or CDC4-transfected NIH3T3 cells and Osteocalcin or Gapdh cDNA probes. D, ubiquitous expression of {beta}-TrCP1. mRNA from indicated tissues was used in Northern blot analysis. Gapdh was used as a loading control.

 
As a result of {beta}-TrCP1 inactivation, there was an accumulation of ATF4 in nuclear extracts of NIH3T3 fibroblasts as shown by Western blot (Fig. 4B) and expression of endogenous Osteocalcin in NIH3T3 fibroblasts (Fig. 4C), further demonstrating that ATF4 can induce ectopic osteoblast-specific gene expression and that removal of a specific E3 ligase is sufficient to achieve this result. This function of {beta}-TrCP1 was specific because decreasing CDC4 expression did not induce ATF4 accumulation or Osteocalcin expression in NIH3T3 fibroblasts (Fig. 4, A–C). Osteocalcin was the only tested gene for which expression was up-regulated in NIH3T3 cells following inactivation of {beta}-TrCP1. This is consistent with the fact that Osteocalcin is a known target gene of ATF4 (12).

These observations suggest that one mechanism explaining the accumulation of ATF4 in osteoblasts is the absence or decrease of its ubiquitination in this cell type. We next asked whether this was because of the absence of {beta}-TrCP1 expression in osteoblasts. Northern blot analysis of multiple tissues demonstrated that {beta}-TrCP1 is expressed in osteoblasts albeit at a slightly reduced level (Fig. 4D). This result indicates that the cell specificity of ATF4 accumulation is not because of the absence of {beta}-TrCP1 expression in osteoblasts.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study establishes that ATF4 has the ability to induce expression of at least one osteoblast-specific gene, Osteocalcin, in non-osteoblastic cell types. ATF4 shares this property with Runx2 and Osterix, the two known osteoblast differentiation factors to date (1). Whether ATF4, like Runx2, has the ability to induce differentiation of non-osteoblastic cell types in vivo may be more difficult to determine because of the unexpected finding reported in this study. This is, however, a question of critical importance given the critical role for ATF4 as a regulator of osteoblast terminal differentiation and function (12). The fact that Osteocalcin was the only osteoblast-specific gene the expression of which was up-regulated by the inactivation of {beta}-TrCP1 does not exclude this possibility. Indeed, ATF4 affects osteoblast function to a large extent by regulating amino acid imports and Type I collagen synthesis without affecting Type I collagen expression. Thus it is possible that, if it can be achieved, stabilization of ATF4 in non-osteoblastic cells in vivo may up-regulate Type I collagen synthesis by favoring amino acid import.

ATF4 accumulation contrasts strikingly with the near ubiquitous expression of the gene encoding it. This discrepancy is explained, at least in part, by the fact that ATF4 is degraded in most cells except osteoblasts and a few other cell types. Indeed, osteoblast is not the only cell type affected by Atf4 disruption. For instance Atf4-/- mice are blind because of a defect in eye lens fibers (20, 21). Likewise, ATF4 is required for fetal hematopoiesis (23) and the differentiation of the lamina propria layer of the vas deferens (27). Thus in this regard, ATF4 cannot be viewed as being solely an absolute osteoblast-specific transcription factor; however, the severity and complexity of the bone phenotype observed in the absence of ATF4 establishes the importance of this factor for bone formation. Moreover, Atf4 deletion has no overt phenotypic consequences in tissues where it is highly expressed, such as brain, kidney, and lungs or other organs. This discrepancy between Atf4 expression and ATF4 accumulation is unique among cell-specific transcription factors and could have several explanations that are not necessarily exclusive. It could be that in the cells where ATF4 does not appear to be critically important in vivo another transcription factor can contribute for its functions. Alternately and/or in addition, it may be that ATF4 is protected from degradation in osteoblasts and a few other cell types.

We show here that it is the case that ATF4 appears to be ubiquitinated in most of the cells except osteoblasts. This ubiquitination process requires {beta}-TrCP1 as an E3 ubiquitin ligase. The fact that {beta}-TrCP1 is expressed in osteoblasts suggests the existence of an additional molecular link to explain ATF4 accumulation. Presumably this molecular link could be the yet to be characterized kinase phosphorylating ATF4 at serine 219 prior to ubiquitination (23) that would be absent in osteoblasts or a deubiquitination mechanism specific to osteoblasts. Further study aimed at identifying this kinase or enzyme responsible for removing the polyubiquitin will allow us to address this question. Regardless of the complexity of these mechanisms, ATF4 is, to our knowledge, the first cell-specific transcription factor the distribution of which is controlled to a large extent post-translationally. In addition, our data propose a molecular basis supporting the existence of a control of bone formation by inhibition of the ubiquitin-dependent proteasomal degradation machinery as recently reported (28).


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant RO1 DE 11290 (to G. K.) and Fellowship F01.013/1/4 from the Children's Brittle Bone Foundation (to X. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 713-798-5488; Fax: 713-798-1465; E-mail: karsenty{at}bcm.tmc.edu.

1 The abbreviations used are: EMSA, electrophoretic mobility shift assay; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; siRNA, small interfering RNA. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Wade Harper and Sharon Plon for reagents and suggestions, Patricia Ducy for repeated reading of the manuscript, and Lingzhen Li for superb technical support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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