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

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.

Figure 1

Localization of the postreceptor defect to c-fos expression. A) Western blot analyses of Akt phosphorylation and abundance. B) Phosphorylation of MAPK was assayed by western blot analyses. Activity of MAPK was detected in in-gel kinase assays. C) Formation of the ternary complex at sre element of c-fos promoter in control and patient. The specific complex is indicated by an arrow (lane F: free probe, 1: no competition; competition: 2: 100x SRE, 3: 10x SRE, 4: 100x SP-1, 5: 10x SP-1). D) Activation of ternary complex factor Elk-1 following insulin and PDGF stimulation in patient and control cells. Results are given as means (±S.D.). *p< 0.05 vs basal control. E) Transcriptional activation of c-fos mRNA. The mRNA levels were normalized against 18S rRNA as internal control. Values are means (±S.D.) from four independent experiments, each performed in triplicate. *p< 0.05 vs basal control.

Figure 2

Identification and impact of a homozygous c-fos promoter point mutation in the patient. A) A homozygous point mutation in c-fos promoter (T → A) at position c.-439 was identified in the patient. B) The mutation was not identified in the patient’s father or mother or C) in a restriction based assay with 319 control subjects. Representative samples are shown (lane 1–5; p: patient; M: size standard).

Figure 3

Identification and characterization of a novel protein/DNA complex forming specifically at the mutation identified in the patient. A) Protein binding to c-fos promoter and mapping of nucleotides necessary for complex formation. Radiolabeled c-fos-wt or mutated pc-fos-c.–439 T→A DNA fragments were subjected to DNaseI protection assay with increasing amounts (◄: 4 μg, 8 μg, 20 μg, 40 μg) of liver cells (HepG2) nuclear protein extracts and digested with varying DNaseI concentrations (◄: 0.11U, 0.33U to 1.0U). A protein/DNA interaction does solely occur with c-fos–c.-439T>A. The sequence of protected areas P1 (nt −413 to −403 (CCCAGCCGCGG) P2 (nt −441 to −428 (CAATCTGCGCCGTT) and P3 (nt −458 to −455 (GTGC) indicated the mutation being located in P2. A typical result from 5 experiments is shown (lane GA: purine sequence ladder). B) EMSA with nuclear protein extracts from liver cells (HepG2) using c-fos-wt (−451 to −430) or mutated pc-fos-c.–439 T→A as probe. The specific complex is indicated by an arrow. Mutation specific complex formation was tested by cross competition with 100-fold excess of non radiolabeled fragments of either c-fos-wt or pc-fos-c.–439 T→A (lane F. free probe, 1: no competition; competition: 2: 50x, 3: 100x specific competitor, 4: 100x cross competition) C) Size fractionation of EMSA protein band on denaturing SDS PAGE. All resulting protein bands were subjected to mass spectrometry. Acquired data from each individual spot were used to search a human sub-set of Swiss-Prot (Sprot_2011; 20249 protein entries) for protein identification.

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).

Figure 5

Gene expression alterations due to diminished c-fos expression. A) Gene expression date were analyzed for statistically significant different gene expression (1.5-fold difference; p<0.05). Resulting transcript IDs were subjected to automated annotation for conserved AP-1 transcription factor binding sites. B) The pathways showing altered transcriptional regulation were deduced from differential gene expression identified. Colored genes (red and green) were identified differentially regulated by microarray analysis. Genes directly regulated by c-fos are indicated (blue line). Predicted transcription factors (white) involved in observed gene regulation were deduced using Ingenuity Pathway Analysis ((IPA System (http://www.ingenuity.com).

Figure 6

Can deminished c-fos transactivation be one cause of adipose tissue malformation? Postulated model how the identyfied c-fos promoter mutation affects signalling by cFos and AP-1 and interferes with adipocte differentiation. The BSCL genes (italic; seipin, AGPAT2, lamininA/C, caveolin, cavin) are included at the levels of functional interaction in c-fos signalling.