Microphthalmia transcription factor expression contributes to bone marrow failure in Fanconi anemia

Abstract

Fancd2-/and WT MEFs, but clearly lower in these cells than in Fanca-/or Fancc-/-MEFs (Figure 1F and data not shown). Together, our previous data robustly demonstrate that loss of function of key components of the FANC core complex is associated with high expression of MiTF at both the RNA and protein levels. SMAD and p38 signaling pathways are necessary but not sufficient to activate MiTF expression in FA MEFs. The increase in Mitf expression in FANC pathway-deficient MEFs with increasing time in the same culture medium (Figure 1, D and E) could be induced by cellto-cell contacts or in an autocrine manner by cell-secreted factor or factors. Consistent with the latter hypothesis, we observed that Mitf expression is robustly induced at both the protein and RNA levels by 24 hours after replating FANC pathway-deficient MEFs in a "conditioned" medium in which the same cells previously grew for 72 hours (Figure 2, A and B, and data not shown). Notably, WT-derived 72-hour-old conditioned medium was unable to significantly induce Mitf in Fanca-/or Fancc-/-MEFs. Similarly, Fanca-/-or Fancc-/-derived conditioned medium was unable to induce Mitf in WT MEFs (Figure 2, A and B, and data not shown). Thus, it is conceivable that Mitf is induced in response to the accumulation in the culture medium of factor or factors secreted by the Fanc-deficient cells themselves. FA cells appear able to secrete and respond to such factor(s), whereas WT cells are poor secretors and nonresponders. Next, we performed ELISA to determine the presence, if any, of known extracellular factors in the culture medium of MEFs. To better perform our analysis, 2 days after dilution, the complete culture medium was washed out, and the cells were fed 2 more days with medium without serum (a longer incubation affects cell morphology, indicating the presence of stress and survival). The serumfree medium was collected and filtered to eliminate detached cells and cellular debris to perform ELISA. We failed to detect TNF-α or IL-1β in FA-derived conditioned medium, but revealed 4 to 6 times higher concentrations of 2 TGF family members (43, 44), TGF-β and activin A (Figure 2, C and D). Finally, the conditioned medium was analyzed by mass spectrometry. Again, we identified high levels of both TGF-β and activin A in the culture medium isolated from FANC pathway-deficient MEFs, but did not distinguish other growth factors differentially expressed between WT and FA MEFs (data not shown). Thus, 2 alternative approaches validated that cultured FANC pathway-deficient MEFs secrete TGF-β and activin A. TGF-β and activin A determine cell fate and hematopoiesis by signaling mainly through the Smad and p38 pathways (8, 43-46), which were previously described as chronically activated in FA cells (24, 32). In Fanca-/or Fancc-/-MEFs maintained for 24 hours in 72-hour-old conditioned medium, we observed a modest but reproducible increase in Smad2/3 phosphorylation and extremely strong phosphorylation of p38 (Figure 2E). The culture of WT MEFs in conditioned medium (both from FA...

The underestimated role of the microphthalmia-associated transcription factor (MiTF) in normal and pathological haematopoiesis

“…Another example of how MiTF deregulation can impact BM homeostasis was reported in a recent study we published on Fanconi anaemia (FA) [ 121 ]. FA, a rare genetic syndrome presenting developmental abnormalities of the skeleton, BM failure, leukaemia predisposition and genetic instability, is due to the loss-of-function of at least one of more than 22 genes that encode proteins constituting a major nuclear pathway involved in DNA repair and replication safeguards and rescue [ 11 , 39 , 115 ].…”

“…More importantly, in contrast with the transient p38/MiTF activation observed during stress-induced haematopoiesis, we demonstrated that the p38/MITF axis is constitutionally active and associated with BMF in Fanca −/− mice. Supporting the notion that the unscheduled and constitutional activation of the p38/MiTF axis has pathological consequences, p38 inhibition or siRNA-mediated depletion of MiTF was sufficient to rescue HSCs defects in the Fanca −/− mice [ 121 ]. The previous observations shed light on two important physiologic aspects of the biology of both MiTF and FA.…”

Tipping the Scale: MYC Gains Weight in Fanconi Anemia Bone Marrow Failure Progression

“…These mutations can cause downstream DNA damage accumulation and increased chromosome breakage, resulting in BMF. BMF has been attributed to hematopoietic stem cell (HSC) exhaustion and is paradoxically associated with hyperproliferation of hematopoietic stem and progenitor cells (HSPCs) and overactivation of several growth inhibitory pathways, including the p53, TGFb, and TNFa signaling pathways (Briot et al, 2008;Ceccaldi et al, 2012;Oppezzo et al, 2020;Rosselli et al, 1994;Walter et al, 2015;Zhang et al, 2016).…”

“…How MYC-dependent transcriptomic landscapes participate in the replication fork regulation likewise remains to be elucidated. Interestingly, MYC belongs to the same family of transcription factors as MiTF, which is highly expressed in FA HSPCs (Oppezzo et al, 2020). It would be interesting to see not only how MiTF behaves in the reported gradient but also if it changes in response to (+)-JQ1.…”

Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis

“…However, FANCA is dispensable for optimal FANCD2/ FANCI monoubiquitination (12), and several BMF-associated mutations of FANCA do not lead to DNA damage hypersensitivity (13)(14)(15), suggesting that FANCA affects BM homeostasis by also participating in pathways other than the DNA repair network. Beyond the DNA repair/DDR, the FANC pathway has been associated with several other cellular and systemic biological activities (16), including proinflammatory cytokine production and responses (17)(18)(19)(20), transcription (21)(22)(23), mRNA splicing (24), and reactive oxygen species metabolism (25)(26)(27), and its failure extends further than BMF to developmental abnormalities, cancer predisposition, and chromosomal instability.…”