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A mutation in the c-fos gene associated with congenital generalized lipodystrophy.

Knebel B, Kotzka J, Lehr S, Hartwig S, Avci H, Jacob S, Nitzgen U, Schiller M, März W, Hoffmann MM, Seemanova E, Haas J, Muller-Wieland D - Orphanet J Rare Dis (2013)

Bottom Line: Several genes (BSCL1-4) were found to be associated to the syndrome but not all CGL patients carry mutations in these genes.Cellular investigations demonstrate that the wild type c-fos promoter can reconstitute the signaling defect in the patient, excluding further upstream signaling alterations, and vice versa the investigations with the c-fos promoter containing the identified mutation generally reduce basal and inducible c-fos transcription activity.As a consequence of the identified point mutation gene expression including c-Fos targeted genes is significantly altered, shown exemplified in cells of the patient.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center at the Heinrich-Heine-University Duesseldorf, Leibniz Center for Diabetes Research, Duesseldorf, Germany.

ABSTRACT

Background: Congenital generalized lipodystrophy (CGL) or Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare genetic syndrome characterized by the absence of adipose tissue. As CGL is thought to be related to malfunctions in adipocyte development, genes involved in the mechanisms of adipocyte biology and maintenance or differentiation of adipocytes, especially transcription factors are candidates. Several genes (BSCL1-4) were found to be associated to the syndrome but not all CGL patients carry mutations in these genes.

Methods and results: In a patient with CGL and insulin resistance we investigated the known candidate genes but the patient did not carry a relevant mutation. Analyses of the insulin activated signal transduction pathways in isolated fibroblasts of the patient revealed a postreceptor defect altering expression of the immediate early gene c-fos. Sequence analyses revealed a novel homozygous point mutation (c.-439, T→A) in the patients' c-fos promoter. The point mutation was located upstream of the well characterized promoter elements in a region with no homology to any known cis-elements. The identified mutation was not detected in a total of n=319 non lipodystrophic probands. In vitro analyses revealed that the mutation facilitates the formation of a novel and specific protein/DNA complex. Using mass spectrometry we identified the proteins of this novel complex. Cellular investigations demonstrate that the wild type c-fos promoter can reconstitute the signaling defect in the patient, excluding further upstream signaling alterations, and vice versa the investigations with the c-fos promoter containing the identified mutation generally reduce basal and inducible c-fos transcription activity. As a consequence of the identified point mutation gene expression including c-Fos targeted genes is significantly altered, shown exemplified in cells of the patient.

Conclusion: The immediate-early gene c-fos is one essential transcription factor to initiate adipocyte differentiation. According to the role of c-fos in adipocyte differentiation our findings of a mutation that initiates a repression mechanism at c-fos promoter features the hypothesis that diminished c-fos expression might play a role in CGL by interfering with adipocyte development.

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Effect of the identified homozygous c-fos promoter point mutation on c-fos transcription. A) Basal and inducible c-fos promoter activity is dependent on wt c-fos promoter and abrogated by mutated c-fos promoter (pc-fos-c.–439 T→A) in patient and control cells. Data of replicate promoter reporter analyses (n=6) are given as mean (±S.D.; p< 0.05). General transcriptional impairment due to c-fos promoter (pc-fos-c.–439 T→A) mutation in B) preadipocytes (3T3L1), C) muscle cells (A7r5) and D) liver cells (HepG2). Data of promoter reporter analyses are given as mean of replicate experiments (n=6) (±S.D; p<0.05).
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Figure 4: Effect of the identified homozygous c-fos promoter point mutation on c-fos transcription. A) Basal and inducible c-fos promoter activity is dependent on wt c-fos promoter and abrogated by mutated c-fos promoter (pc-fos-c.–439 T→A) in patient and control cells. Data of replicate promoter reporter analyses (n=6) are given as mean (±S.D.; p< 0.05). General transcriptional impairment due to c-fos promoter (pc-fos-c.–439 T→A) mutation in B) preadipocytes (3T3L1), C) muscle cells (A7r5) and D) liver cells (HepG2). Data of promoter reporter analyses are given as mean of replicate experiments (n=6) (±S.D; p<0.05).

Mentions: To test the regulatory relevance of identified mutation in the c-fos promoter we performed promoter reporter gene analyses. Transfecting a wild type c-fos promoter into the patient’s cells revealed that the basal and inducible promoter activity is completely reconstituted (Figure 4A). Vice versa transfecting a c-fos promoter bearing the identified mutation into control cells revealed that the observed diminished expression and inducibility of c-fos was dependent from cellular environment (Figure 4A). These data exclude further proximal signaling defects and support a defect intrinsic to the c-fos promoter as the mutation identified. Further analyses demonstrated that this observation was not cell specific as in preadipocytes, muscle and liver cells c-fos promoter activity and activation was evenly inhibited by the point mutation (Figure 4B-D).


A mutation in the c-fos gene associated with congenital generalized lipodystrophy.

Knebel B, Kotzka J, Lehr S, Hartwig S, Avci H, Jacob S, Nitzgen U, Schiller M, März W, Hoffmann MM, Seemanova E, Haas J, Muller-Wieland D - Orphanet J Rare Dis (2013)

Effect of the identified homozygous c-fos promoter point mutation on c-fos transcription. A) Basal and inducible c-fos promoter activity is dependent on wt c-fos promoter and abrogated by mutated c-fos promoter (pc-fos-c.–439 T→A) in patient and control cells. Data of replicate promoter reporter analyses (n=6) are given as mean (±S.D.; p< 0.05). General transcriptional impairment due to c-fos promoter (pc-fos-c.–439 T→A) mutation in B) preadipocytes (3T3L1), C) muscle cells (A7r5) and D) liver cells (HepG2). Data of promoter reporter analyses are given as mean of replicate experiments (n=6) (±S.D; p<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750569&req=5

Figure 4: Effect of the identified homozygous c-fos promoter point mutation on c-fos transcription. A) Basal and inducible c-fos promoter activity is dependent on wt c-fos promoter and abrogated by mutated c-fos promoter (pc-fos-c.–439 T→A) in patient and control cells. Data of replicate promoter reporter analyses (n=6) are given as mean (±S.D.; p< 0.05). General transcriptional impairment due to c-fos promoter (pc-fos-c.–439 T→A) mutation in B) preadipocytes (3T3L1), C) muscle cells (A7r5) and D) liver cells (HepG2). Data of promoter reporter analyses are given as mean of replicate experiments (n=6) (±S.D; p<0.05).
Mentions: To test the regulatory relevance of identified mutation in the c-fos promoter we performed promoter reporter gene analyses. Transfecting a wild type c-fos promoter into the patient’s cells revealed that the basal and inducible promoter activity is completely reconstituted (Figure 4A). Vice versa transfecting a c-fos promoter bearing the identified mutation into control cells revealed that the observed diminished expression and inducibility of c-fos was dependent from cellular environment (Figure 4A). These data exclude further proximal signaling defects and support a defect intrinsic to the c-fos promoter as the mutation identified. Further analyses demonstrated that this observation was not cell specific as in preadipocytes, muscle and liver cells c-fos promoter activity and activation was evenly inhibited by the point mutation (Figure 4B-D).

Bottom Line: Several genes (BSCL1-4) were found to be associated to the syndrome but not all CGL patients carry mutations in these genes.Cellular investigations demonstrate that the wild type c-fos promoter can reconstitute the signaling defect in the patient, excluding further upstream signaling alterations, and vice versa the investigations with the c-fos promoter containing the identified mutation generally reduce basal and inducible c-fos transcription activity.As a consequence of the identified point mutation gene expression including c-Fos targeted genes is significantly altered, shown exemplified in cells of the patient.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Clinical Biochemistry and Pathobiochemistry, German Diabetes Center at the Heinrich-Heine-University Duesseldorf, Leibniz Center for Diabetes Research, Duesseldorf, Germany.

ABSTRACT

Background: Congenital generalized lipodystrophy (CGL) or Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare genetic syndrome characterized by the absence of adipose tissue. As CGL is thought to be related to malfunctions in adipocyte development, genes involved in the mechanisms of adipocyte biology and maintenance or differentiation of adipocytes, especially transcription factors are candidates. Several genes (BSCL1-4) were found to be associated to the syndrome but not all CGL patients carry mutations in these genes.

Methods and results: In a patient with CGL and insulin resistance we investigated the known candidate genes but the patient did not carry a relevant mutation. Analyses of the insulin activated signal transduction pathways in isolated fibroblasts of the patient revealed a postreceptor defect altering expression of the immediate early gene c-fos. Sequence analyses revealed a novel homozygous point mutation (c.-439, T→A) in the patients' c-fos promoter. The point mutation was located upstream of the well characterized promoter elements in a region with no homology to any known cis-elements. The identified mutation was not detected in a total of n=319 non lipodystrophic probands. In vitro analyses revealed that the mutation facilitates the formation of a novel and specific protein/DNA complex. Using mass spectrometry we identified the proteins of this novel complex. Cellular investigations demonstrate that the wild type c-fos promoter can reconstitute the signaling defect in the patient, excluding further upstream signaling alterations, and vice versa the investigations with the c-fos promoter containing the identified mutation generally reduce basal and inducible c-fos transcription activity. As a consequence of the identified point mutation gene expression including c-Fos targeted genes is significantly altered, shown exemplified in cells of the patient.

Conclusion: The immediate-early gene c-fos is one essential transcription factor to initiate adipocyte differentiation. According to the role of c-fos in adipocyte differentiation our findings of a mutation that initiates a repression mechanism at c-fos promoter features the hypothesis that diminished c-fos expression might play a role in CGL by interfering with adipocyte development.

Show MeSH
Related in: MedlinePlus