Gli2 Acetylation at Lysine 757 Regulates Hedgehog-Dependent Transcriptional Output by Preventing Its Promoter Occupancy

Abstract

The morphogenic Hedgehog (Hh) signaling regulates postnatal cerebellar development and its aberrant activation leads to medulloblastoma. The transcription factors Gli1 and Gli2 are the activators of Hh pathway and their function is finely controlled by different covalent modifications, such as phosphorylation and ubiquitination. We show here that Gli2 is endogenously acetylated and that this modification represents a key regulatory step for Hedgehog signaling. The histone acetyltransferase (HAT) coactivator p300, but not other HATs, acetylates Gli2 at the conserved lysine K757 thus inhibiting Hh target gene expression. By generating a specific anti acetyl-Gli2(Lys757) antisera we demonstrated that Gli2 acetylation is readily detectable at endogenous levels and is attenuated by Hh agonists. Moreover, Gli2 K757R mutant activity is higher than wild type Gli2 and is no longer enhanced by Hh agonists, indicating that acetylation represents an additional level of control for signal dependent activation. Consistently, in sections of developing mouse cerebella Gli2 acetylation correlates with the activation status of Hedgehog signaling. Mechanistically, acetylation at K757 prevents Gli2 entry into chromatin. Together, these data illustrate a novel mechanism of regulation of the Hh signaling whereby, in concert with Gli1, Gli2 acetylation functions as a key transcriptional checkpoint in the control of morphogen-dependent processes.

Combined inhibition of atypical PKC and histone deacetylase 1 is cooperative in basal cell carcinoma treatment

“…We recently identified overexpression of aPKC as a powerful mechanism of drug resistance in advanced BCCs Tables 5 and 6). This observation, coupled with previous data demonstrating that inhibition of aPKC (23) or HDAC1 (8,11) results in an abolishment of GLI1's chromatin affinity, as assayed by ChIP without perturbing its nuclear-cytoplasmic trafficking, led us to the hypothesis that aPKC and HDAC1 work in concert to modulate GLI1's transcriptional status.…”

“…HDAC1 is itself a transcriptional target of GLI, creating a third positive feedback loop of HH signaling. Of particular interest, HDAC inhibition has been proposed for the treatment of many HH-driven cancers (8)(9)(10)(11). HDAC inhibitors block growth and promote apoptosis by altering the histone-DNA complex and by altering the acetylation status of nonhistone proteins (12).…”

Gli1/DNA interaction is a druggable target for Hedgehog-dependent tumors

“…Furthermore, Y859, Y872 and S1060 of Gli1 are cooperatively bound by the E3 ligase Itch (Di Marcotullio et al, 2006a, while D C degrons (residues 462-467) bind bTrCP, all leading to protein ubiquitination and degradation (Huntzicker et al, 2006). Finally, K518 (Gli1) and K757 (Gli2) are instead acetylatable residues preventing transcriptional activity Coni et al, 2013b), whereas T374 of the Gli1ZF is a major PKA site contrasting Gli1 localization into the nucleus (Sheng et al, 2006). Once phosphorylated by aPKCι/k, S243 and T304 of the GliZF confer to the protein an enhanced ability to form a complex with DNA (Atwood et al, 2013), possibly through a conformational change or binding stabilization or the recruitment of additional co-regulatory proteins, as these amino acids are located in ZF-1 and ZF-3 that mainly establish a few contacts only with the phosphate backbone.…”

Druggable glycolytic requirement for Hedgehog-dependent neuronal and medulloblastoma growth

“…30,31 Cell pellets were lysed in TRI reagent solution (Ambion) and total RNA was purified. cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen) and transcript levels were quantified on an Applied Biosystems ViiA 7 Real-Time PCR System instrument using Power Sybr Green PCR Master Mix (Applied Biosystems).…”

“…31 The following antibodies and conditions were used: Ki67 (1:200, Leica Biosystems) was diluted in 1% serum PBS-T. Caspase 3 (1:500, Cell Signaling) was diluted in 5% serum PBS-T. Antibodies were incubated for one hour at room temperature. Nuclei were counterstained with hematoxylin in accordance with standard procedures.…”